Compositions and methods for promoting homology directed repair

Abstract

This application provides improved methods of genome editing. Cas9 molecules can be used to create a break in a genomic region of interest. To increase the likelihood that the break is repaired by HDR (homology-directed repair), the cell can be contacted with molecules that bring a template nucleic acid in close proximity to the break, under conditions that allow the cell to repair the break using the template nucleic acid.

Description

BACKGROUND

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.

Recently, the CRISPR/Cas system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double strand breaks (DSBs) enables target nucleic acid alteration through homology-directed repair (HDR).

SUMMARY

This disclosure provides systems and methods for editing a genome, e.g., by correcting a mutation. Using the methods and compositions disclosed herein, a Cas9 fusion molecule or Cas9 system may be used to mediate a break or nick near a target position, e.g., a chromosome position, that one desires to edit. The cell then utilizes one of several repair pathways, e.g., HDR, to repair the break or nick. Provided herein is a Cas9 repair system comprising a Cas9 fusion molecule, a nucleic acid template system, and optionally, at least one gRNA molecule. While not being bound by theory, it is believed that by contacting a cell, or population of cells, with the Cas9 repair systems disclosed herein, the proximity of the template nucleic acid used by the cell to repair a Cas9-mediated cleavage event can be increased, and the frequency of use of a particular DNA repair pathway, e.g., HDR, can be modulated.

In one aspect, described herein is a Cas9 fusion molecule comprising a Cas9 molecule linked to a template binding domain.

In one embodiment, the template binding domain comprises a specific affinity for a template binding domain partner. In one embodiment, the template binding domain does not comprise substantial affinity for a nucleic acid from one or more of a human, a non-human animal, a mammal, a eukaryote, a plant, or a pathogen.

In one embodiment, the template binding domain is not a nuclease, a transcription factor, an antibody or other complementarity determining region (CDR)-based molecule, a protein that alters chromatin, a protein that binds chromatin, a protein that modifies DNA, a DNA methylase, a protein that cleaves DNA, a protein that unwinds DNA, or any combination thereof. In one embodiment, the nuclease is an endonuclease or an exonuclease. In one embodiment, the chromatin comprises a histone.

In one embodiment, the template binding domain does not comprise substantial affinity for a nucleic acid from a preselected species. In one embodiment, the preselected species is a human, a non-human animal, a mammal, a eukaryote, a plant, a pathogen, or any combination thereof. In one embodiment, the preselected species is human.

In one embodiment, the Cas9 molecule is covalently linked to the template binding domain. In another embodiment, the Cas9 molecule is non-covalently linked to the template binding domain.

In one embodiment, the template binding domain is linked to the N-terminus of the Cas9 molecule. In one embodiment, the template binding domain is covalently linked to the N-terminus of the Cas9 molecule. In another embodiment, the template binding domain is non-covalently linked to the N-terminus of the Cas9 molecule.

In one embodiment, the template binding domain is linked to the C-terminus of the Cas9 molecule. In one embodiment, the template binding domain is covalently linked to the C-terminus of the Cas9 molecule. In another embodiment, the template binding domain is non-covalently linked to the C-terminus of the Cas9 molecule.

In one embodiment, the Cas9 fusion molecule comprises at least two template binding domains.

In one embodiment, the template binding domain comprises a protein, a nucleic acid, or a small molecule. In one embodiment, the nucleic acid is a DNA or an RNA. In one embodiment, the template binding domain comprises a protein, wherein the protein comprises a DNA binding domain. In one embodiment, the protein comprises a repressor protein, or a fragment of a repressor protein. In one embodiment, the repressor protein, or the fragment of the repressor protein, is a TetR repressor, or a fragment of the TetR repressor; a LacI repressor, or a fragment of the LacI repressor; a Gal4 repressor, or a fragment of the Gal4 repressor; or a repressor protein C1, or a fragment of the repressor protein C1.

In one embodiment, the Cas9 fusion protein comprises a linker between the Cas9 molecule and the template binding domain. In one embodiment, the linker is sufficiently long to allow the Cas9 molecule to bind to a target nucleic acid and the template binding domain to bind to a template binding domain partner without steric interference. In one embodiment, the linker is at least 6, but no longer than 60 amino acids in length. In another embodiment, the linker is at least 18, but no longer than 180 nucleotides in length. In one embodiment, the linker is at least 10, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 Angstroms in length. In one embodiment, the linker is no more than 10, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 Angstroms in length. In one embodiment, the linker comprises a polypeptide. In one embodiment, the linker comprises serine, glycine, or glycine and serine. In one embodiment, the linker comprises a sequence of Table 2 or Table 3.

In yet another embodiment, the Cas9 molecule is a Cas9 molecule selected from Table 100. In one embodiment, the Cas9 molecule is a Cas9 molecule selected from Table 600. In one embodiment, the Cas9 molecule is not a wild-type Cas9 molecule.

In one embodiment, the Cas9 molecule is an eaCas9 molecule. In one embodiment, the eaCas9 molecule forms a double stranded break in a target nucleic acid. In one embodiment, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity and HNH-like domain cleavage activity. In another embodiment, the eaCas9 molecule forms a single stranded break in a target nucleic acid. In one embodiment, the eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity. In one embodiment, the eaCas9 molecule is an HNH-like domain nickase. In one embodiment, the eaCas9 molecule comprises a mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9. In yet another embodiment, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity but has no, or no significant, HNH-like domain cleavage activity. In one embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase. In one embodiment, the eaCas9 molecule comprises a mutation at an amino acid position corresponding to amino acid position H840 or N863 of S. pyogenes Cas9.

In another embodiment, the Cas9 molecule is an eiCas9 molecule. In some embodiments, the Cas9 molecule comprises a mutation, e.g., a point mutation, that causes the Cas9 molecule to be inactive, e.g., a mutation that eliminates the Cas9 molecule cleavage activity.

In one embodiment, the Cas9 molecule comprises a REC2 deletion, REC1_CT deletion, or a REC1_SUB deletion, or any combination thereof.

In one embodiment, the Cas9 molecule comprises an altered PI domain.

In another embodiment, the Cas9 molecule is less than about 1300 amino acids in length. In one embodiment, the Cas9 molecule is less than about 1200, 1100, 1000, 900, or 800 amino acids in length. In one embodiment, the Cas9 molecule is between about 800 to 1300, 900 to 1200, 900 to 1100, or 900 to 1000 amino acids in length.

In one embodiment, the Cas9 fusion molecule is substantially purified.

In another aspect, described herein is a Cas9 system comprising a Cas9 fusion molecule described herein, and a nucleic acid template system comprising a template binding domain partner and a template nucleic acid.

In one embodiment, the template binding domain of the Cas9 fusion molecule is bound to the template binding domain partner. In one embodiment, the template binding domain of the Cas9 fusion molecule is covalently bound to the template binding domain partner. In another embodiment, the template binding domain of the Cas9 fusion molecule is non-covalently bound to the template binding domain partner.

In one embodiment, the template binding domain partner is linked to the template nucleic acid. In one embodiment, the template binding domain partner is covalently linked to the template nucleic acid. In another embodiment, the template binding domain partner is non-covalently linked to the template nucleic acid.

In one embodiment, the template binding domain partner comprises a protein, a nucleic acid, or a small molecule. In one embodiment, the nucleic acid is a DNA or an RNA. In one embodiment, the template binding domain comprises a protein and the template binding domain partner comprises a protein.

In one embodiment, the template binding domain comprises a protein and the template binding domain partner comprises a nucleic acid. In one embodiment, the nucleic acid is a DNA. In one embodiment, the DNA is a double-stranded DNA or a single-stranded DNA. In one embodiment, the nucleic acid is an RNA.

In one embodiment, the template binding domain comprises a protein and the template binding domain partner comprises a small molecule.

In another embodiment, the template binding domain comprises a small molecule, and the template binding domain partner comprises a protein.

In one embodiment, the template binding domain comprises a small molecule, and the template binding domain partner comprises a small molecule.

In yet another embodiment, the template binding domain comprises a small molecule, and the template binding domain partner comprises a nucleic acid. In one embodiment, the template binding domain comprises a nucleic acid, and the template binding domain partner comprises a protein. In another embodiment, the template binding domain comprises a nucleic acid, and the template binding domain partner comprises a nucleic acid. In one embodiment, the template binding domain comprises a nucleic acid, and the template binding domain partner comprises a small molecule. In one embodiment, the nucleic acid is an RNA. In another embodiment, the nucleic acid is a DNA. In one embodiment, the DNA is a double-stranded DNA or a single-stranded DNA.

In one embodiment, the template binding domain partner is a DNA sequence recognized by a DNA binding protein. In one embodiment, the DNA sequence recognized by the DNA binding protein is selected from a Tet-O sequence, a Lac operon 01 sequence, a UAS sequence, or an Operator L and R sequence. In yet another embodiment, the template binding domain partner comprises a repressor-binding DNA sequence from a bacterial operon. In one embodiment, the protein comprises a TetR repressor, or a fragment of the TetR repressor, and the DNA comprises at least one Tet-O sequence. In one embodiment, the DNA comprises at least two, three, four, five, or six Tet-O sequences. In one embodiment, the DNA comprises a spacer sequence between the Tet-O sequences. In one embodiment, the spacer sequence is at least 17 nucleotides in length. In another embodiment, the spacer sequence is at least 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides in length. In one embodiment, the DNA does not comprise a spacer sequence between the Tet-O sequences.

In one embodiment, the template binding domain partner comprises a protein that is not a nuclease, a transcription factor, an antibody or other CDR-based molecule, a protein that alters chromatin, a protein that binds chromatin, a protein that modifies DNA, a DNA methylase, a protein that cleaves DNA, a protein that unwinds DNA, or any combination thereof. In one embodiment, the nuclease is an endonuclease or an exonuclease. In one embodiment, the chromatin comprises a histone.

In one embodiment, the Cas9 system comprises at least one additional template binding domain and at least one additional template binding domain partner.

In one embodiment, the template binding domain partner comprises a linear nucleic acid. In another embodiment, the template binding domain partner comprises a nucleic acid sequence located on a circular nucleic acid.

In one embodiment, the template binding domain partner comprises a double stranded nucleic acid sequence. In one embodiment, the template binding domain partner comprises a single stranded nucleic acid sequence.

In one embodiment, the template nucleic acid comprises a double stranded nucleic acid sequence or a single stranded nucleic acid sequence.

In one embodiment, the nucleic acid template system comprises a double stranded nucleic acid sequence or a single stranded nucleic acid sequence.

In one embodiment, the template nucleic acid comprises a linear nucleic acid. In another embodiment, the template nucleic acid is a nucleic acid located on a circular nucleic acid.

In one embodiment, the template binding domain partner comprises a double stranded nucleic acid; and the template nucleic acid comprises a double stranded nucleic acid.

In one embodiment, the template binding domain partner comprises a double stranded nucleic acid; and the template nucleic acid comprises a single stranded nucleic acid.

In one embodiment, the nucleic acid template system is a circular nucleic acid. In another embodiment, the nucleic acid template system is a linear nucleic acid.

In one embodiment, the template binding domain partner comprises a single stranded nucleic acid; and the template nucleic acid comprises a single stranded nucleic acid.

In another embodiment, the template binding domain partner comprises a single stranded nucleic acid; and the template nucleic acid comprises a double stranded nucleic acid.

In one embodiment, the nucleic acid template system is a single stranded nucleic acid, and the template binding domain partner is 5′ of the template nucleic acid. In another embodiment, the nucleic acid template system is a single stranded nucleic acid, and the template binding domain partner is 3′ of the template nucleic acid.

In one embodiment, the nucleic acid template system is a double stranded nucleic acid, and the template binding domain partner is 5′ of the template nucleic acid. In another embodiment, the nucleic acid template system is a double stranded nucleic acid, and the template binding domain partner is 3′ of the template nucleic acid.

In one embodiment, the template nucleic acid comprises about 50-500 nucleotides of homology with a target nucleic acid. In one embodiment, the template nucleic acid comprises about 100-200 nucleotides of homology with a target nucleic acid. In one embodiment, the template nucleic acid comprises about 500-2000 nucleotides of homology with a target nucleic acid.

In one embodiment, the template nucleic acid comprises a human nucleic acid sequence. In one embodiment, the template nucleic acid comprises a wild-type human nucleic acid sequence.

In yet another embodiment, the Cas9 system further comprises at least one additional template binding domain partner.

In one embodiment, the template nucleic acid lacks repeated elements. In one embodiment, the repeated element is an Alu element or a LINE element.

In one embodiment, the Cas9 system further comprises a gRNA. In one embodiment, the gRNA comprises a targeting domain, first and second complementary domains, and a proximal domain. In one embodiment, the gRNA is a chimeric gRNA.

In one embodiment, the Cas9 system further comprises at least one additional Cas9 molecule. In some embodiment, the Cas9 molecule is an eaCas9 molecule. In some embodiments, the Cas9 molecule is an eiCas9 molecule. In one embodiment, Cas9 system comprises a Cas9 fusion molecule, comprising an eiCas9 molecule linked to a template binding domain, and a Cas9 molecule. In one embodiment, Cas9 system comprises a Cas9 fusion molecule, comprising an eiCas9 molecule linked to a template binding domain, and an eaCas9 molecule.

In another aspect, described herein is a cell, or a population of cells, comprising a Cas9 system described herein.

In yet another aspect, described herein is a cell, or a population of cells, comprising a Cas9 fusion molecule described herein.

In another aspect, described herein is a nucleic acid encoding a Cas9 fusion molecule described herein. In yet another aspect, described herein is a vector comprising said nucleic acid. In one embodiment, the vector is an AAV vector. In one aspect, described herein is a cell, or a population of cells, comprising said nucleic acid.

In another aspect, described herein is a method of altering a nucleic acid at a target position in a cell, or a population of cells, the method comprising contacting the cell with the Cas9 system described herein, wherein the gRNA molecule and Cas9 fusion molecule interact with the nucleic acid, resulting in a cleavage event, wherein the cleavage event is repaired by at least one DNA repair pathway, and wherein the sequence of the nucleic acid after the cleavage event is different than the sequence of the nucleic acid prior to the cleavage event, thereby altering the nucleic acid at the target position in the cell, or in the population of cells.

In one embodiment, the method, further comprises contacting the cell, or the population of cells, with a second gRNA molecule, wherein the second gRNA molecule and the Cas9 fusion molecule interact with the nucleic acid, resulting in a second cleavage event.

In one embodiment, the at least one DNA repair pathway is selected from the group consisting of resection, mismatch repair (MMR), nucleotide excision repair (NER), base excision repair (BER), canonical non-homologous end joining (canonical NHEJ), alternative non-homologous end joining (ALT-NHEJ), canonical homology directed-repair (canonical HDR), alternative homology directed repair (ALT-HDR), microhomology-mediated end joining (MMEJ), Blunt End Joining, Synthesis Dependent Microhomology Mediated End Joining, single strand annealing (SSA), Holliday junction model or double strand break repair (DSBR), synthesis-dependent strand annealing (SDSA), single strand break repair (SSBR), translesion synthesis repair (TLS), and interstrand crosslink repair (ICL), and DNA/RNA processing.

In one embodiment, the at least one DNA repair pathway is canonical homology directed-repair (canonical HDR) or alternative homology directed repair (ALT-HDR).

In one embodiment, the cleavage event comprises one or more single strand breaks, one or more double strand breaks, or a combination of single strand breaks and double strand breaks.

In one embodiment, the gRNA molecule positions one cleavage event on each strand of the nucleic acid.

In yet another embodiment, the gRNA molecule positions the cleavage event on a strand of the nucleic acid that binds to the gRNA molecule.

In one embodiment, the second gRNA molecule positions the second cleavage event on a strand of the nucleic acid that binds to the second gRNA molecule.

In one embodiment, the Cas9 fusion molecule makes a single strand break in the nucleic acid. In another embodiment, the Cas9 fusion molecule makes a double strand break in the nucleic acid. In one embodiment, the single strand break is made in the strand of the nucleic acid to which a targeting domain of the gRNA molecule is complementary. In another embodiment, the single strand break is made in the strand of the nucleic acid other than the strand to which a targeting domain of the gRNA is complementary.

In one embodiment, the target position is in an HBB gene.

In one embodiment, the cell, or the population of cells, is a eukaryotic cell, or a population of eukaryotic cells. In one embodiment, the cell, or the population of cells, is a plant cell, or a population of plant cells. In one embodiment, the plant cell, or the population of plant cells, is a monocot plant cell, a dicot plant cell, a population of monocot plant cells, or a population of dicot plant cells.

In another embodiment, the cell, or the population of cells, is a mammalian cell, or a population of mammalian cells. In one embodiment, the cell, or the population of cells, is a human cell, or a population of human cells.

In one embodiment, the cell, or the population of cells, is a vertebrate, mammalian, rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, or human cell or population of cells.

In one embodiment, the cell, or the population of cells, is a somatic cell, a germ cell, or a prenatal cell or population of cells.

In another embodiment, the cell, or the population of cells, is a zygotic cell, a blastocyst, an embryonic cell, a stem cell, a mitotically competent cell, a meiotically competent cell or population of cells.

In one embodiment, the cell, or the population of cells, is a T cell, a CD8+ T cell, a CD8+ naïve T cell, a central memory T cell, an effector memory T cell, a CD4+ T cell, a stem cell memory T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a Hematopoietic Stem Cell, a long term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a monocytic precursor cell, an endocrine precursor cell, an exocrine cell, a fibroblast, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epithelial cell, a pulmonary epithelial progenitor cell, a striated muscle cell, a cardiac muscle cell, a muscle satellite cell, a myocyte, a neuron, a neuronal stem cell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, an embryonic stem cell, a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g. a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell, a gastrointestinal epithelial cell, a biliary epithelial cell, a pancreatic ductal epithelial cell, an intestinal stem cell, a hepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, an osteoclast, an adipocyte, a preadipocyte, a pancreatic precursor cell, a pancreatic islet cell, a pancreatic beta cell, a pancreatic alpha cell, a pancreatic delta cell, a pancreatic exocrine cell, a Schwann cell, or an oligodendrocyte, or population of such cells.

In one embodiment, the cell, or population of cells, is from a subject suffering from a disease or disorder.

In one embodiment, the disease is a blood disease, an immune disease, a neurological disease, a cancer, an infectious disease, a genetic disease, a disorder caused by aberrant mtDNA, a metabolic disease, a disorder caused by aberrant cell cycle, a disorder caused by aberrant angiogenesis, a disorder cause by aberrant DNA damage repair, or a pain disorder.

In one embodiment, the cell, or population of cells, is from a subject having at least one mutation at the target position.

In one embodiment, the method further comprises isolating the cell, or population of cells, from the subject prior to contacting the cell, or population of cells, with the Cas9 system described herein.

In one embodiment, the method further comprises introducing the cell, or the population of cells, into a subject after contacting the cell, or the population of cells, with the Cas9 system described herein.

In one embodiment, the contacting the cell, or the population of cells, with the Cas9 system described herein is performed ex vivo. In another embodiment, the contacting the cell, or the population of cells, with the Cas9 system described herein is performed in vivo. In one embodiment, the contacting the cell, or the population of cells, with the Cas9 system described herein is performed in vitro.

In one embodiment, the method further comprises sequencing the nucleic acid, or a portion of the nucleic acid, prior to contacting the cell, or the population of cells, with the Cas9 system described herein.

In one embodiment, the method further comprises sequencing the nucleic acid, or a portion of the nucleic acid, after the cleavage event.

In one embodiment, the cell, or the population of cells, is contacted with the gRNA molecule and the Cas9 fusion molecule as a pre-formed complex.

In one aspect, described herein is a cell, or a population of cells, altered by the methods described herein. In another aspect, described herein is a pharmaceutical composition comprising said cell, or said population of cells.

In yet another aspect, described herein is a pharmaceutical composition comprising a Cas9 system described herein.

In one aspect, described herein is a method of treating a subject comprising administering to the subject the cell, or the population of cells, altered by the methods described herein, or a pharmaceutical composition comprising said cell, or said population of cells.

In another aspect, described herein is a method of treating a subject suffering from a disease or disorder, the method comprising contacting a cell, or a population of cells, from the subject with the Cas9 system described herein, wherein the gRNA molecule and the Cas9 fusion molecule interact with a nucleic acid at a target position, resulting in a cleavage event, wherein the cleavage event is repaired by at least one DNA repair pathway, and wherein the sequence of the nucleic acid after the cleavage event is different than the sequence of the nucleic acid prior to the cleavage event, thereby treating the subject suffering from the disease or disorder.

In one embodiment, the method further comprises contacting the cell from the subject with a second gRNA molecule, wherein the second gRNA molecule and the Cas9 fusion molecule interact with the nucleic acid, resulting in a second cleavage event.

In one embodiment, the contacting the cell is performed ex vivo. In another embodiment, the contacting the cell is performed in vivo.

In certain aspects, the present disclosure provides a cell comprising:

• • a Cas9 molecule and a template binding domain, wherein optionally the Cas9 molecule is linked to the template binding domain;

wherein said template binding domain comprises specific affinity for a template binding domain partner and lacks substantial affinity for:

• • (i) the endogenous nucleic acid of said cell;
  • (ii) the endogenous chromosomal nucleic acid of said cell; or
  • (iii) the endogenous organellar nucleic acid, e.g., mitochondrial, chloroplast, or both, of said cell.

In certain aspects, the present disclosure also provides a cell comprising:

• • (a) a Cas9 molecule and a template binding domain; wherein optionally the Cas9 molecule is linked to the template binding domain; and
  • (b) a gRNA molecule having a targeting domain complementary with a target sequence;

wherein said template binding domain comprises specific affinity for a template binding domain partner and lacks substantial affinity for a nucleic acid sequence within 100, 500, 1000, 2000, 5,000, or 10,000 nucleotides of said target sequence.

In certain aspects, the present disclosure also provides a kit comprising packaging and at least three of

• • (a) a Cas9 fusion molecule,
  • (b) a template binding domain,
  • (c) a template binding domain partner, and
  • (d) a template nucleic acid.

In the kit, in some embodiments, (a) and (b) form part of a single composition; (a) and (c) form part of a single composition; (a) and (d) form part of a single composition; (b) and (c) form part of a single composition; (b) and (d) form part of a single composition; (c) and (d) form part of a single composition; (a), (b), and (c) form part of a single composition; (a), (b), and (d) form part of a single composition; (a), (c), and (d) form part of a single composition; (b), (c), and (d) form part of a single composition; or (a), (b), (c), and (d) form part of a single composition.

In certain aspects, the present disclosure also provides a reaction mixture comprising the cell and a solution.

In certain aspects, the present disclosure also provides a reaction mixture wherein the solution is a cell growth medium.

In certain aspects, the present disclosure provides a method of altering the structure of a cell, e.g., altering the sequence, of a target nucleic acid of the cell, comprising contacting the cell with the Cas9 system as described herein under conditions that allow for alteration of the structure of the cell, thereby altering the structure of a cell, e.g., altering the sequence of a target nucleic acid.

In some embodiments the method, comprises bringing a template nucleic acid in proximity with a target nucleic acid in a cell, e.g., sufficient proximity that the efficiency of altering the cell, e.g., a target nucleic acid of the cell, is increased over the level seen with a non bound template nucleic acid.

In some embodiments, the target nucleic acid is cleaved at a position that is:

• • i) at a target position;
  • ii) away from a target position;
  • iii) within 200 nucleotides of the target position;
  • iv) at least 10 nucleotides away from the target position; or
  • v) 10 to 200 nucleotides away from the target position.

In some embodiments, the target nucleic acid is cleaved at two positions that are:

• • within 55 nucleotides of one another;
  • at least 25 nucleotides apart; or within 25-55 nucleotides of one another.

Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates embodiments where a Cas9 molecule is engineered to selectively associate with a template nucleic acid, and therefore increase the proximity of the template nucleic acid to a target nucleic acid. The Cas9 molecule (which can be, e.g., wild-type or a nickase) is shown associated with a target nucleic acid, e.g., a mutant gene. The Cas9 molecule is associated with a gRNA that targets the Cas9 molecule to the target nucleic acid. The target nucleic acid comprises a target sequence which is recognized by a gRNA, as well as a PAM sequence that is recognized by the Cas9 molecule. The Cas9 molecule is shown linked to a template binding domain (illustrated as a trapezoid) using a linker region (illustrated as a line connecting the Cas9 molecule and the template binding domain). The template binding domain (which can be a DNA binding domain) has affinity for a template binding domain partner (which can be a DNA sequence specifically recognized by the DNA binding domain) which is shown as a shaded rectangle. In this figure, the template binding domain partner is part of a circular double stranded nucleic acid molecule. The circular nucleic acid molecule also comprises a template nucleic acid which is capable of participating in homology-dependent repair of a break in the target nucleic acid. Together, the template nucleic acid and the template binding domain partner form a nucleic acid template system. The figure also shows alternative nucleic acid template systems, one which is a double stranded linear nucleic acid, and one which is a single stranded linear nucleic acid.

FIG. 2 depicts exemplary nucleic acid template systems. The template binding domain partner (e.g., a stretch of DNA that a DNA-binding domain can bind) is shown as a shaded box. The template nucleic acid is illustrated as a single line which represents single-stranded DNA, or a double line which represents double stranded DNA. The figure illustrates that the template nucleic acid system can be a circular double stranded DNA molecule like a plasmid; a linear double stranded DNA molecule, or a linear DNA molecule that is single stranded in the template nucleic acid region and double stranded in the template binding domain partner region. When the DNA is linear, the template binding domain partner can be, e.g., on either end of the molecule.

FIG. 3 depicts exemplary arrangements of linear nucleic acid template systems. The target nucleic acid (a long double stranded DNA segment like a gene or chromosome) is depicted as a double stranded shaded line with a break. The regions of the target nucleic acid that are homologous to a template nucleic acid are shown as thick solid or dotted lines. The Cas9 molecule is depicted as a shaded circle positioned over the break, and the template binding domain extends from the Cas9 molecule. The nucleic acid template systems are shown below the target nucleic acids, with the template binding domain partner shown as a shaded box and the template nucleic acid as a dotted or solid line. In this figure, the nucleic acid template system can be double stranded, single stranded, or have a single stranded portion and a double stranded portion (e.g., where the template binding domain partner is double stranded and the template nucleic acid is single stranded). The top row shows embodiments where the template nucleic acid has homology on either side of the cut, e.g., approximately 50 to 100 bp or nucleotides both on the left and on the right side of the cut. The homology regions to the left are shown as solid lines and the homology regions to the right are shown as dotted lines. The central row shows embodiments where the template nucleic acid has the majority of the homology on the right side of the cut, e.g., approximately 150-200 bp or nucleotides of homology. The bottom row shows embodiments where the template nucleic acid has the majority of the homology on the left side of the cut, e.g., approximately 150-200 bp or nucleotides of homology. In all the rows, the template binding domain partner can be placed at either end of the nucleic acid template system; in the left column the template binding domain partner is placed at the right of the nucleic acid template system, and in the right column the template binding domain partner is placed at the left of the nucleic acid template system.

FIG. 4 depicts exemplary arrangements of circular nucleic acid template systems. The target nucleic acid (a long double stranded DNA segment like a gene or chromosome) is depicted as a double stranded shaded line with a break. The regions of the target nucleic acid that are homologous to a template nucleic acid are shown as thick solid or dotted lines. The Cas9 molecule is depicted as a shaded circle positioned over the break, and the template binding domain extends from the Cas9 molecule. The nucleic acid template systems are shown below the target nucleic acids, with the template binding domain partner is shown as a shaded box and the template nucleic acid as a dotted or solid line. In this figure, the circular nucleic acid template systems are double stranded DNA, but it is understood that the disclosure also contemplates circular molecules that are partly or whole single stranded. The top row shows embodiments where the template nucleic acid has a homology region to the right of the template binding domain partner. The center row shows embodiments where the template nucleic acid has a homology region to the left of the template binding domain partner. The bottom row shows embodiments where the template nucleic acid has a homology region on the opposite side of the circular DNA molecule from the template binding domain partner. In all nine arrangements, the homology region on the template nucleic acid can corresponds to a region on the target nucleic acid, where a solid line is homologous with a solid line and a dotted line is homologous with a dotted line. In the left column, the template nucleic acid has homology to the target nucleic acid on either side of the break. In the central column, the template nucleic acid has homology to the target nucleic acid on the left side of the break. In the right column, the template nucleic acid has homology to the target nucleic acid on the right side of the break. The homology region can be, e.g., 500 to 2000 bp.

FIGS. 5A and 5B are schematic representations of the domain organization of S. pyogenes Cas9. FIG. 5A shows the organization of the Cas9 domains, including amino acid positions, in reference to the two lobes of Cas9 (recognition (REC) and nuclease (NUC) lobes). FIG. 5B shows the percent homology of each domain across 83 Cas9 orthologs.

FIG. 6 is a schematic drawing of a Cas9 fusion molecule, comprising a Cas9 molecule fused to a template binding domain, bound to a target nucleic acid, and complexed with a nucleic acid template system comprising a template binding domain partner and a template nucleic acid.

FIG. 7 is a schematic drawing of a Cas9 system comprising an eaCas9 molecule bound to a target nucleic acid, and a Cas9 fusion molecule, comprising an eiCas9 molecule fused to a template binding domain, bound to a target nucleic acid, and complexed with a nucleic acid template system comprising a template binding domain partner and a template nucleic acid. The Cas9 fusion molecule positions the nucleic acid template system in close proximity to the eaCas9 molecule.

FIG. 8A illustrates four exemplary ssDNA nucleic acid template systems comprising a template binding domain partner, comprising three Tet Operator sequences, and a template nucleic acid. V1.1 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising three Tet Operator sequences separated by two 17 nucleotide spacers, N terminal of a 109 nucleotide template nucleic acid. V1.2 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising three Tet Operator sequences separated by two 17 nucleotide spacers, C terminal of a 109 nucleotide template nucleic acid. V2 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising three Tet Operator sequences separated by two 8 nucleotide spacers, N terminal of a 109 nucleotide template nucleic acid. V3 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising three Tet Operator sequences adjacent to one another without intervening spacers, N terminal of a 109 nucleotide template nucleic acid.

FIG. 8B shows Tet Repressor protein binding activity for four exemplary ssDNA nucleic acid template systems comprising a template binding domain partner, comprising three Tet Operator sequences, and a template nucleic acid (i.e., V1.1, V1.2, V2, and V3) and two control DNA templates (i.e., ssODN 179, and ssODN109. An electrophoretic mobility shift assay using a 10% polyacrylamide gel was performed using 500 nM of recombinant Tet Repressor protein 50 nM of ssDNA nucleic acid template system or control DNA template.

FIG. 9 illustrates four exemplary ssDNA nucleic acid template systems comprising a template binding domain partner, comprising three or four Tet Operator sequences, and a template nucleic acid. V4.1 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising three Tet Operator sequences separated by three 8 nucleotide spacers, N terminal of a 119 nucleotide template nucleic acid. V4.2 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising three Tet Operator sequences separated by three 8 nucleotide spacers, C terminal of a 119 nucleotide template nucleic acid. V5.1 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising four Tet Operator sequences separated by three 8 nucleotide spacers, N terminal of a 100 nucleotide template nucleic acid. V5.2 is an exemplary ssDNA nucleic acid template system comprising a template binding domain partner, comprising four Tet Operator sequences separated by three 8 nucleotide spacers, C terminal of a 100 nucleotide template nucleic acid.

DETAILED DESCRIPTION

Definitions

“Amino acids” as used herein encompasses the canonical amino acids as well as analogs thereof.

“Amino acid residues that flank a deletion”, as that phrase is used herein, refers to the amino acid residue that immediately precedes the deletion and the amino acid residue that immediately follows the deletion. By way of example, in a sequence _CT1-_CT2-_CT3-_CT7-_CT8-_CT9, wherein _CT4-_CT5-_CT6 is deleted, the flanking amino acid residues are, _CT3 and _CT7.

“Cas9 core domain”, as that term is used herein, refers to a polypeptide that does not include a functional PI domain, e.g., a polypeptide not having an endogenous PI domain, e.g., wherein the endogenous PI domain is deleted (deleted, as used in this context, refers merely to a sequence difference or the absence of amino acid residues and implies no process or origin limitation), or generally, a Cas9 molecule lacking a PI domain. In an embodiment, a Cas9 core domain comprises a REC1 domain, a REC2 domain, a BH domain, a RuvC domain, and an HNH domain. A Cas9 core domain, together with an altered PI domain, comprises a functional Cas9 molecule.

“Cas9 fusion molecule”, “Cas9 fusion protein”, or “Cas9 fusion”, as used herein, refers to a chimeric protein comprising a Cas9 molecule, e.g., Cas9 protein or Cas9 polypeptide, or a fragment thereof, linked to a template binding domain. In some embodiments, the template binding domain is a protein or polypeptide. In some embodiments, the template binding domain is a nucleic acid, e.g., DNA or RNA. In some embodiments, the template binding domain is a small molecule. In some embodiments, the Cas9 fusion molecule comprises a Cas9 molecule covalently linked to the template binding domain. For example, the Cas9 fusion protein may be a chimeric protein comprising one or more Cas9 protein domains and a DNA binding domain from a protein disclosed herein. In some embodiments, the Cas9 fusion molecule comprises a Cas9 molecule noncovalently linked to the template binding domain.

In an embodiment, a species X Cas9 core domain has at least 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with the corresponding sequence of a reference sequence, e.g., a naturally occurring species X Cas9 core domain, e.g., from a Cas9 core domain from Table 100. In an embodiment, each of a REC1 domain, a REC2 domain, a BH domain, a RuvC domain, and/or an HNH domain of a species X Cas9 core domain has, independently, at least 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with the corresponding sequence of a reference sequence, e.g., a naturally occurring species X Cas9 core domain, e.g., from a Cas9 core domain from Table 100.

“Cas9 molecule” or “Cas9 polypeptide”, as that term is used herein, refers to a polypeptide that can bind (1) a PAM (a protospacer adjacent motif) in a nucleic acid, and (2) a guide RNA (gRNA) molecule. In an embodiment, in concert with the gRNA molecule, a Cas9 molecule or Cas9 polypeptide can localize to a site which comprises a target domain.

Cas9 may be a nuclease (an enzyme that cleaves both strands of a double-stranded nucleic acid), a nickase (an enzyme that cleaves one strand of a double-stranded nucleic acid), or an enzymatically inactive (or dead) molecule. A Cas9 molecule having nuclease or nickase activity is referred to as an enzymatically active Cas9 molecule (an eaCas9 molecule). A Cas9 molecule lacking the ability to cleave target nucleic acid is referred to as an enzymatically inactive Cas9 molecule (an eiCas9 molecule). A Cas9 molecule can have the amino acid sequence of a naturally occurring Cas9 molecule or can be an altered, engineered or modified Cas9 molecule, which differs by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule, e.g., a Cas9 molecule from Table 100. (The terms altered, engineered or modified, as used in this context, refers merely to a difference from a reference or naturally occurring sequence, and impose no specific process or origin limitations.) For example, an altered, engineered or modified Cas9 molecule can comprise one or more point mutations which alter (e.g., increase, decrease and/or eliminate), one or more Cas9 molecule activities, e.g., a nuclease activity.

In an embodiment, a Cas9 molecule meets one or both of the following criteria:

• • it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with, or

it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 100 herein.

In one embodiment, the Cas9 molecule may be a Cas9 deletion, e.g., the Cas9 may comprise a deletion in one or more of the following domains: a REC2, REC1_CT, or REC1_SUB domain. Except for any REC deletion, a Cas9 molecule meets one or both of the following criteria:

• • it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with, or
  • it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 100 herein. Homology except for any REC deletion is determined as follows: a sequence having a deletion is altered by replacing the deleted sequence with the corresponding sequence from the reference sequence, and the altered sequence is compared with the reference sequence.

In another embodiment, the Cas9 molecule may be a Cas9 variant, e.g., the Cas9 may comprise an altered PI domain, or other modified amino acid sequence, or the Cas9 may comprise a linker. In an alternate embodiment, except for an altered PI domain or other modified amino acid sequence, a Cas9 molecule meets one or both of the following criteria:

• • it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with, or
  • it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 100 herein. Homology except for an altered PI domain, or other modified amino acid sequence is determined as follows: a sequence having an altered PI domain (or other modified amino acid sequence) is altered by restoring the altered PI domain (or other modified amino acid sequence) to the naturally occurring PI domain (or other naturally occurring sequence) from the reference sequence, and the thus altered sequence is compared with the reference sequence.

In an alternate embodiment, except for a linker, a Cas9 molecule meets one or both of the following criteria:

• • it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with, or
  • it differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400, amino acid residues from, the amino acid sequence of a reference sequences, e.g., naturally occurring Cas9 molecule, e.g., a Cas9 molecule described in Table 100 herein. Homology except for a linker is determined as follows: a sequence having a linker is altered by omitting the linker sequence, and the thus altered sequence is compared with the reference sequence.

In another embodiment, each domain of the Cas9 molecule (e.g., the domains named in FIG. 5 or elsewhere herein), including any remaining portion of a REC2, REC1_CT, or REC1_SUB domain having a deletion or an unaltered portion of a PI domain, will, independently have:

• • at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with such a domain described herein, e.g., in a species of Table 100. In an embodiment at least 1, 2, 3, 4, 5, of 6 domains will have, independently, at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with a corresponding domain, while any remaining domains will be absent, or have less homology to their corresponding naturally occurring domains.

“PI domain”, as that term is used herein, refers to the region of a Cas9 molecule that interacts with the PAM sequence of a target nucleic acid.

“Altered PI domain”, as that term is used herein, refers to a PI domain other than the native or endogenous PI domain associated with the naturally occurring Cas9 molecule. For example, a Cas9 molecule comprises an altered PI domain if its PI domain is other than the PI domain naturally associated with the Cas9 core domain of the Cas9 molecule, or if its PI domain is not a naturally occurring PI domain associated with any Cas9 molecule. (Derived, as used in this sense, is not limited to physical derivation or even derivation from a specific source, and does not require a process limitation, but in some embodiments, includes mere structural similarity). An altered PI domain may have less than 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 70, 60, 50, 30, 40, 30, 20, or 10% homology with the native or endogenous PI domain of a subject naturally occurring Cas9 molecule from which the Cas9 core domain is derived. An altered PI domain may have a different RKR motif (the PAM recognition sequence) than that of the native or endogenous PI domain of the Cas9 species that supplies the Cas9 core domain. The RKR motif of an altered PI domain may differ from the RKR motif of the native or endogenous PI domain of the Cas9 core domain by 1, 2, or 3 residues. The RKR motif of the altered PI differs at the first position, the second position, the third position, the first and second positions, the first and third positions, the second and third positions, or all three positions, from the RKR motif of the PI endogenous to or naturally associated with the Cas9 core domain. In an embodiment, an altered PI domain is one having greater homology with the PI domain of a reference or donor naturally occurring Cas9 molecule (a heterologous Cas9) that with the native PI domain of a subject Cas9.

The terms “homology” or “identity”, as used interchangeably herein, refer to sequence identity between two amino acid sequences or two nucleic acid sequences, with identity being a more strict comparison. The phrases “percent identity or homology” and “% identity or homology” refer to the percentage of sequence identity found in a comparison of two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be anywhere from 0-100% identical, or any value there between. Identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison to a reference sequence. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of homology of amino acid sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

Calculations of homology or sequence identity between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

“Linker”, as that term is used herein, refers to a molecular entity that may link a Cas9 molecule, or a fragment thereof, and a template binding domain, or may link a template binding domain partner to a template nucleic acid, or may link a Cas9 molecule fragment or domain to another Cas9 molecule fragment or domain. In one embodiment, the linker is a nucleic acid, e.g. an oligonucleotide. In another embodiment, the linker is a small molecule. In a further embodiment, the linker is a polypeptide comprising at least one amino acid. For example, a polypeptide linker may be disposed between sequences or domains of a Cas9 molecule. In an embodiment, the linker is disposed between the amino acid residues that flank a deletion. In an embodiment, the linker is disposed between the amino acid residues of a Cas9 core domain and an altered PI domain. By way of example, in a sequence _CT1-_CT2-_CT3-_CT7-_CT8-_CT9, wherein _CT4-_CT5-_CT6 is deleted, the linker is located immediately C-terminal to the amino acid residue _CT3 and immediately N-terminal to the amino acid residue _CT7. Preferably, the linker is selected such that the Cas9 molecule exhibits a tertiary structure or folded conformation similar to that of the corresponding naturally occurring Cas9 molecule, such that some Cas9 activity is retained. Suitable linkers are described herein. In some embodiments, the linker comprises a combination of Gly and Ser residues, e.g., (GS)_x(SEQ ID NO: 128) or (GGS)_x(SEQ ID NO: 1), where x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other embodiments, the linker comprises a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 2), referred to herein as XTEN linker or XTEN. Alternative linkers include (GSAGSAAGSGEF)_x, wherein x is 1, 2, 3 or 4 (SEQ ID NO: 3) and (SIVAQLSRPDPA)_x, wherein x is 1, 2, 3 or 4 (SEQ ID NO: 4). Linkers also include a combination of linkers described herein or known in the art.

“REC deletion”, as that term is used herein, refers to a REC2 deletion, a REC1_CT deletion, or a REC1_SUB deletion.

“REC2 deletion”, as that term is used herein, refers to a deletion of at least 10% of the amino acid residues of the REC2 domain.

“REC2 domain”, as that term is used herein, refers to a region, in the N terminal half of a naturally occurring Cas9 molecule that is not needed for cleavage or gRNA-mediated targeting. Its length and boundaries differ between Cas9 molecules from various species. In the case of S. aureus, the REC2 domain is about 41 amino acid residues in length and corresponds, approximately, to residues 126 to 166, of S. aureus Cas9. In the case of S. pyogenes, the REC2 domain is about 139 amino acid residues in length and corresponds, approximately, to residues 176 to 314 of S. pyogenes Cas9. In the case of C. jejuni, the REC2 domain is about 45 amino acid residues in length and corresponds, approximately, to residues 137 to 181 of C. jejuni Cas9. These, and the approximate sizes and boundaries of REC2 domains from other species, are provided in Table 100.

“REC1_CT deletion”, as that term is used herein, refers to a deletion of at least 10% of the amino acid residues of the REC1_CT domain.

“REC1_CT domain”, as that term is used herein, refers to a region, C terminal of the REC1 domain, of a naturally occurring Cas9 polypeptide that is not needed for cleavage or gRNA-mediated targeting. Its length and boundaries differ between Cas9 proteins from various species. In the case of S. aureus, the REC1_CT domain is about 146 amino acid residues in length and corresponds, approximately, to residues 288 to 166, of S. aureus Cas9. In the case of S. pyogenes, the REC1_CT domain is about 219 amino acid residues in length and corresponds, approximately, to residues 500 to 718 of S. pyogenes Cas9. In the case of C. jejuni, the REC1_CT domain is about 134 amino acid residues in length and corresponds, approximately, to residues 305 to 438 of C. jejuni Cas9. These, and the approximate sizes and boundaries of REC1 _CT domains from other species, are provided in Table 100.

“REC1 _SUB deletion”, as that term is used herein, refers to a deletion of at least 10% of the amino acid residues of the REC1_SUB domain.

“REC1 _SUB domain”, as that term is used herein, refers to a region, located within the REC1 _CT domain, of a naturally occurring Cas9 polypeptide that is not needed for cleavage or gRNA-mediated targeting. Its length and boundaries differ between Cas9 proteins from various species. In the case of S. aureus, the REC1_Sub domain is about 57 amino acid residues in length and corresponds, approximately, to residues 296 to 352, of S. aureus Cas9. In the case of S. pyogenes, the REC1_Sub domain is about 82 amino acid residues in length and corresponds, approximately, to residues 511 to 592 of S. pyogenes Cas9. In the case of C. jejuni, the REC1_Sub domain is about 45 amino acid residues in length and corresponds, approximately, to residues 316 to 360 of C. jejuni Cas9. These, and the approximate sizes and boundaries of REC1_Sub domains from other species, are provided in Table 100.

“n” as used herein in the context of proteins or Cas9 molecules described herein, refers to the number of amino acid residues that are deleted in a REC2, REC1_CT, or REC1_SUB deletion, unless otherwise specified.

“X” as used herein in the context of an amino acid sequence of a linker sequence, refers to any number of repeating units unless otherwise specified.

A disorder “caused by” a mutation, as used herein, refers to a disorder that is made more likely or severe by the presence of the mutation, compared to a subject that does not have the mutation. The mutation need not be the only cause of a disorder, i.e., the disorder can still be caused by the mutation even if other causes, such as environmental factors or lifestyle factors, contribute causally to the disorder. In some embodiments, the disorder is caused by the mutation if the mutation is a medically recognized risk factor for developing the disorder, and/or if a study has found that the mutation correlates with development of the disorder.

“Derived from”, as used herein, refers to the source or origin of a molecular entity, e.g., a nucleic acid or protein. The source of a molecular entity may be naturally-occurring, recombinant, unpurified, or a purified molecular entity. For example, a polypeptide that is derived from a second polypeptide comprises an amino acid sequence that is identical or substantially similar, e.g., is more than 50% homologous to, the amino acid sequence of the second protein. The derived molecular entity, e.g., a nucleic acid or protein, can comprise one or more modifications, e.g., one or more amino acid or nucleotide changes.

“Domain”, as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.

As used herein, “HDR”, or homology-directed repair, refers to the process of repairing DNA damage using a homologous nucleic acid (e.g., a sister chromatid or an exogenous nucleic acid). In a normal cell, HDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation.

“Large molecule”, as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kDa. Large molecules include proteins, polypeptides, nucleic acids, biologics, and carbohydrates.

“Polypeptide”, as used herein, refers to a polymer of amino acids.

“Reference molecule”, e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule or a subject gRNA molecule, e.g., a modified or candidate Cas9 molecule, is compared. For example, a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. aureus, S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared. In an embodiment, the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made.

“Replacement”, or “replaced”, as used herein with reference to a modification of a molecule does not require a process limitation but merely indicates that the replacement entity is present.

“Small molecule”, as used herein, refers to a compound having a molecular weight less than about 2 kDa, e.g., less than about 2 kDa, less than about 1.5 kDa, less than about 1 kDa, or less than about 0.75 kDa.

“Steric interference” or “steric hindrance”, as used herein, refers to the restriction or prevention of the binding or interaction of one molecular entity (e.g., a protein or a protein fragment) with another molecular entity (e.g., a nucleic acid or a protein).

“Subject”, as used herein, may mean either a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats. In an embodiment, the subject is a human. In other embodiments, the subject is poultry.

“Sufficiently long”, as the term is used herein to refer to linkers, refers to a linker length that does not prevent or restrict the binding folding, conformation, activity, and/or interaction of one molecular entity (e.g., a protein) with another molecular entity (e.g., a nucleic acid). In one embodiment, the linker is at least 6, but no longer than 60 amino acids in length. In another embodiment, the linker is at least 18, but no longer than 180 nucleotides in length. In one embodiment, the linker is at least 10, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 Angstroms in length. In one embodiment, the linker is no more than 10, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 Angstroms in length.

“Treat”, “treating” and “treatment”, as used herein, mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; and (c) curing the disease.

“Prevent,” “preventing” and “prevention,” as used herein, means the prevention of a disease in a subject, e.g., a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (2) affecting the predisposition toward the disease, e.g., preventing at least one symptom of the disease or to delay onset of at least one symptom of the disease.

“Specific affinity”, or “specifically binds”, or “specific binding”, as used herein, refer to a binding interaction between two or more molecular entities, e.g., a template binding domain and a template binding domain partner, such as, for example, a DNA-binding polypeptide and a DNA molecule, wherein one molecular entity preferentially binds to another molecular entity, but does not substantially bind to other molecular entities present in a molecular milieu, e.g., a heterologous molecular milieu, in a cell or in solution. In some embodiments, the term “specific affinity” refers to a DNA-binding protein or polypeptide that binds to a specific sequence of a nucleic acid molecule. Exemplary protein-protein pairs that specifically bind to each other are provided in Table V.6. Exemplary protein-small molecule pairs that specifically bind to each other are provided in Table V.7. Exemplary protein-DNA sequence pairs that specifically bind to each other are provided in Table V.1.

“Substantial affinity”, as the term is used herein, refers to a binding interaction between two or more molecular entities, e.g., a template binding domain and a template binding domain partner, such as, for example, a DNA-binding polypeptide and a DNA molecule, wherein the binding event induces a significant event, change or alteration in the molecular entity being bound, or a significant phenotypic change in a cell in which the binding event occurs.

“Target position” or “target nucleic acid” as used herein, refers to a site on a nucleic acid (e.g., a region of a chromosome) that is modified by a Cas9 molecule-dependent process. For example, the target position can be modified by a Cas9 molecule-mediated cleavage of the nucleic acid using a template nucleic acid. In an embodiment, a target position can be a site between two nucleotides, e.g., adjacent nucleotides, on the target nucleic acid into which one or more nucleotides is added. The target position may comprise one or more nucleotides that are altered, e.g., corrected, by a template nucleic acid. In an embodiment, the target position is within a “target sequence” (e.g., the sequence to which the gRNA binds). In an embodiment, a target position is upstream or downstream of a target sequence (e.g., the sequence to which the gRNA binds).

The “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid.

A “template binding domain partner” as used herein, is a molecule with specific affinity for a template binding domain. The template binding domain partner may be, e.g., DNA, protein, or a small molecule.

A “template nucleic acid”, as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with a Cas9 molecule and a gRNA molecule to alter the structure of a target position. “Template nucleic acid” is used interchangeably with “donor nucleic acid” and “swap nucleic acid” herein. In an embodiment, the target nucleic acid is modified to have some or all of the sequence of the template nucleic acid, typically at or near cleavage site(s). In an embodiment, the template nucleic acid is single stranded. In an alternate embodiment, the template nucleic acid is double stranded. In an embodiment, the template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the template nucleic acid is single stranded DNA. In an embodiment, the template nucleic acid is encoded on the same vector backbone, e.g. AAV genome, plasmid DNA, as the Cas9 and gRNA. In an embodiment, the template nucleic acid is excised from a vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences.

“Wild type”, as used herein, refers to a gene or polypeptide which has the characteristics, e.g., the nucleotide or amino acid sequence, of a gene or polypeptide from a naturally-occurring source. The term “wild type” typically includes the most frequent observation of a particular gene or polypeptide in a population of organisms found in nature.

“X” as used herein in the context of an amino acid sequence, refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.

A “template binding domain,” as that term is used herein, refers to an entity which, by virtue of its specific affinity for a template binding domain partner, mediates the association of a template nucleic acid with a Cas9 with which the template binding domain is associated. The template binding domain associates with, e.g., by non-covalent or covalent interactions, with a template binding domain partner. The template binding domain partner is associated with, e.g., covalently or non-covalently bound to, the template nucleic acid. In an embodiment, the template binding domain comprises a polypeptide and the template binding domain partner comprises a nucleic acid. Typically, the template binding domain does not cleave nucleic acid.

I. GRNA Molecules

A gRNA molecule, as that term is used herein, refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid. Typically, the nucleic acid will incorporate the functions or structure of both crRNA and tracrRNA, e.g., the functions of processed or mature crRNA and of processed or mature tracrRNA. gRNA molecules can be unimolecular (having a single nucleic acid molecule, e.g., which incorporates both crRNA function or structure and the tracrRNA function or structure), sometimes referred to herein as “chimeric” gRNAs, or modular (comprising more than one, and typically two, separate nucleic acid molecules, e.g., where one incorporates the crRNA function or structure and the other incorporates the tracrRNA function or structure). A gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below. Additional details on gRNAs are provided in Section I entitled “gRNA molecules” of PCT Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference.

In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′: a targeting domain (which is complementary to a target nucleic acid, and which is sometimes referred to as a spacer); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain. In an embodiment, the targeting domain, and first complementarity domain correspond functionally or structurally to elements of a crRNA, e.g., a mature or processed crRNA. In an embodiment, the second complementarity domain, proximal domain, and tail domain correspond functionally or structurally to elements of a tracrRNA, e.g., a processed or mature tracrRNA.

In an embodiment, a modular gRNA comprises: a first strand (which corresponds to a crRNA) comprising, preferably from 5′ to 3′; a targeting domain (which is complementary to a target nucleic acid); and a first complementarity domain; and a second strand (which corresponds to a tracrRNA), comprising preferably from 5′ to 3′: optionally, a 5′ extension domain; a second complementarity domain; a proximal domain; and optionally, a tail domain.

The domains are discussed briefly below.

The Targeting Domain

The targeting domain (which can also be referred to as a “spacer”) comprises a nucleotide sequence that is complementary, e.g., at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in an embodiment, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the target domain itself comprises in the 5′ to 3′ direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence. In an embodiment, the targeting domain is 5 to 50 nucleotides in length, e.g., 10 to 30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the targeting domain can have a modification, e.g., a modification found in Section XI herein.

In an embodiment, the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides.

Targeting domains are discussed in more detail below.

The First Complementarity Domain

The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first complementarity domain is 5 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 22 nucleotides in length. In an embodiment, the first complementary domain is 7 to 18 nucleotides in length. In an embodiment, the first complementary domain is 7 to 15 nucleotides in length. In an embodiment, the first complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

In an embodiment, the first complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length. In an embodiment, the 3′ subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section XI herein.

First complementarity domains are discussed in more detail below.

The Linking Domain

A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain covalently couples the first and second complementarity domains. In an embodiment, the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain. Typically the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

In modular gRNA molecules the two molecules are associated by virtue of the hybridization of the complementarity domains.

A wide variety of linking domains are suitable for use in unimolecular gRNA molecules. Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length. In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5′ to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section XI herein.

Linking domains are discussed in more detail below.

The 5′ Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain, referred to herein as the 5′ extension domain. In an embodiment, the 5′ extension domain is, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4, nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

The Second Complementarity Domain

The second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.

In an embodiment, the second complementarity domain is 5 to 27 nucleotides in length. In an embodiment, it is longer than the first complementarity region. In an embodiment the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the second complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In an embodiment, the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3′ subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.

In an embodiment, the 5′ subdomain and the 3′ subdomain of the first complementarity domain, are respectively, complementary, e.g., fully complementary, with the 3′ subdomain and the 5′ subdomain of the second complementarity domain.

The second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section XI herein.

A Proximal Domain

In an embodiment, the proximal domain is 5 to 20 nucleotides in length. In an embodiment, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, proximal domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section XI herein.

A Tail Domain

A broad spectrum of tail domains are suitable for use in gRNA molecules. In an embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In embodiment, the tail domain nucleotides are from or share homology with sequence from the 5′ end of a naturally occurring tail domain. In an embodiment, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.

In an embodiment, the tail domain is absent or is 1 to 50 nucleotides in length. In an embodiment, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, tail domain. In an embodiment, the tail domain includes nucleotides at the 3′ end that are related to the method of in vitro or in vivo transcription. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3′ end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. When alternate pol-III promoters are used, these nucleotides may be various numbers or uracil bases or may include alternate bases.

The domains of gRNA molecules are described in more detail below.

The Targeting Domain

The “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid. The strfnd of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the gRNA is referred to herein as the “complementary strand” of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al. (2014) NAT. BIOTECHNOL. 32: 279-84 (doi: 10.1038/nbt.2808) and Sternberg S H et al. (2014) NATURE 507: 62-7 (doi: 10.1038/nature13011).

In an embodiment, the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides.

In an embodiment, the targeting domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.

In an embodiment, the targeting domain is 20+/−5 nucleotides in length.

In an embodiment, the targeting domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.

In an embodiment, the targeting domain is 30+/−10 nucleotides in length.

In an embodiment, the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.

In another embodiment, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

Typically the targeting domain has full complementarity with the target sequence. In an embodiment the targeting domain has or includes 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain.

In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.

In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.

In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 fusion molecule to the target nucleic acid.

In an embodiment, the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain (“non-complementary nucleotides”), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, no two consecutive nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.

In an embodiment, there are no non-complementary nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section XI. However, in an embodiment, the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the targeting domain can be modified with a phosphorothioate, or other modification from Section XI. In an embodiment, a nucleotide of the targeting domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section XI.

In an embodiment, the targeting domain includes 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the targeting domain includes 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the targeting domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.

In an embodiment, the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

Modifications in the targeting domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII gRNAs having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system in Section VII. The candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain. In another embodiment, 1, 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.

In an embodiment, the targeting domain comprises, preferably in the 5′→3′ direction: a secondary domain and a core domain. These domains are discussed in more detail below.

The Core Domain and Secondary Domain of the Targeting Domain

The “core domain” of the targeting domain is complementary to the “core domain target” on the target nucleic acid. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3′ end of the targeting domain (e.g., the most 3′ 8 to 13 nucleotides of the targeting domain).

In an embodiment, the core domain of the targeting domain and core domain target, are independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, or 16+−2, nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target, are independently, 10+/−2 nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target, are independently, 10+/−4 nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target are independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length. In an embodiment, the core domain of the targeting domain and core domain target are independently 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20 10 to 20 or 15 to 20 nucleotides in length.

In an embodiment, the core domain of the targeting domain and core domain target are independently 3 to 15, e.g., 6 to 15, 7 to 14, 7 to 13, 6 to 12, 7 to 12, 7 to 11, 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10 or 8 to 9 nucleotides in length.

The core domain of the targeting domain is complementary with the core domain target. Typically the core domain has exact complementarity with the core domain target. In an embodiment, the core domain of the targeting domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain target. In an embodiment, the degree of complementarity, together with other properties of the gRNA molecule, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

The “secondary domain” of the targeting domain of the gRNA is complementary to the “secondary domain target” of the target nucleic acid.

In an embodiment, the secondary domain is positioned 5′ to the core domain.

In an embodiment, the secondary domain is absent or optional.

In an embodiment, if the targeting domain is 26 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 13 to 18 nucleotides in length.

In an embodiment, if the targeting domain is 25 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.

In an embodiment, if the targeting domain is 24 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 11 to 16 nucleotides in length.

In an embodiment, if the targeting domain is 23 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 10 to 15 nucleotides in length.

In an embodiment, if the targeting domain is 22 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 9 to 14 nucleotides in length.

In an embodiment, if the targeting domain is 21 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 8 to 13 nucleotides in length.

In an embodiment, if the targeting domain is 20 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 7 to 12 nucleotides in length.

In an embodiment, if the targeting domain is 19 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 6 to 11 nucleotides in length.

In an embodiment, if the targeting domain is 18 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 5 to 10 nucleotides in length.

In an embodiment, if the targeting domain is 17 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 4 to 9 nucleotides in length.

In an embodiment, if the targeting domain is 16 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 3 to 8 nucleotides in length.

In an embodiment, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length.

The secondary domain of the targeting domain is complementary with the secondary domain target. Typically the secondary domain of the targeting domain has exact complementarity with the secondary domain target. In an embodiment the secondary domain of the targeting domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the secondary domain target. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 fusion molecule to the target nucleic acid.

In an embodiment, the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section XI. However, in an embodiment, the core domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the core domain can be modified with a phosphorothioate, or other modification(s) from Section XI. In an embodiment a nucleotide of the core domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section XI. Typically, a core domain will contain no more than 1, 2, or 3 modifications.

Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII gRNAs having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section VII. The candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section XI. However, in an embodiment, the secondary domain comprises one or more modifications, e.g., modifications that render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the secondary domain can be modified with a phosphorothioate, or other modification(s) from Section XI. In an embodiment a nucleotide of the secondary domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification from Section XI. Typically, a secondary domain will contain no more than 1, 2, or 3 modifications.

Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII. gRNAs having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section VII. The candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, (1) the degree of complementarity between the core domain of the targeting domain and its target (i.e., the core domain target), and (2) the degree of complementarity between the secondary domain of the targeting domain and its target (i.e., the secondary domain target), may differ. In an embodiment, (1) may be greater than (2). In an embodiment, (1) may be less than (2). In an embodiment, (1) and (2) are the same, e.g., each may be completely complementary with its target.

In an embodiment, (1) the number of modifications (e.g., modifications from Section XI) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section XI) of the nucleotides of the secondary domain, may differ. In an embodiment, (1) may be less than (2). In an embodiment, (1) may be greater than (2). In an embodiment, (1) and (2) may be the same, e.g., each may be free of modifications.

The First and Second Complementarity Domains

The first complementarity domain is complementary with the second complementarity domain.

Typically the first domain does not have exact complementarity with the second complementarity domain. In an embodiment, the first complementarity domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain. In an embodiment, 1, 2, 3, 4, 5 or 6, e.g., 3 nucleotides, will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region. In an embodiment, an unpaired, or loop-out, region, e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain. In an embodiment, the unpaired region begins 1, 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5′ end of the second complementarity domain.

In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

In an embodiment, the first and second complementarity domains are:

independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2, 21+/−2, 22+/−2, 23+/−2, or 24+/−2 nucleotides in length;

independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, nucleotides in length; or

independently, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides in length.

In an embodiment, the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.

In an embodiment, the first and second complementary domains, independently, do not comprise modifications, e.g., modifications of the type provided in Section XI.

In an embodiment, the first and second complementary domains, independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate, or other modification(s) from Section XI. In an embodiment a nucleotide of the domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section XI.

In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the first and second complementary domains, independently, include as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.

In an embodiment, the first and second complementary domains, independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no nucleotide that is modified within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.

Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII. gRNAs having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section VII. The candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the first complementarity domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain, or a first complementarity domain described herein.

In an embodiment, the second complementarity domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, second complementarity domain, or a second complementarity domain described herein.

The duplexed region formed by first and second complementarity domains is typically 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).

In an embodiment, the first and second complementarity domains, when duplexed, comprise 11 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 5)
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.

In an embodiment, the first and second complementarity domains, when duplexed, comprise 15 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 90)
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGCAUAGCAA
GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG
GUGC.

In an embodiment the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 91)
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACAGCAUAGC
AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU
CGGUGC.

In an embodiment the first and second complementarity domains, when duplexed, comprise 21 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

(SEQ ID NO: 92)
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGGAAACAAA
ACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU
GGCACCGAGUCGGUGC.

In an embodiment, nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):

(SEQ ID NO: 93)
NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAGUUAAUAU
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

(SEQ ID NO: 94)
NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAGUUUAAAU
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;
or
(SEQ ID NO: 95)
NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGGAAACAAU
ACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU
GGCACCGAGUCGGUGC.

The 5′ Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain. In an embodiment, the 5′ extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

In an embodiment, the 5′ extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section XI. However, in an embodiment, the 5′ extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the 5′ extension domain can be modified with a phosphorothioate, or other modification(s) from Section XI. In an embodiment, a nucleotide of the 5′ extension domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section XI.

In an embodiment, the 5′ extension domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.

In an embodiment, the 5′ extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain.

Modifications in the 5′ extension domain can be selected so as to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII. gRNAs having a candidate 5′ extension domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section VII. The candidate 5′ extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the 5′ extension domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference 5′ extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, 5′ extension domain, or a 5′ extension domain described herein.

The Linking Domain

In a unimolecular gRNA molecule the linking domain is disposed between the first and second complementarity domains. In a modular gRNA molecule, the two molecules are associated with one another by the complementarity domains.

In an embodiment, the linking domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.

In an embodiment, the linking domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.

In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.

In another embodiment, the linking domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

In an embodiment, the linking domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.

In and embodiment, the linking domain is a covalent bond.

In an embodiment, the linking domain comprises a duplexed region, typically adjacent to or within 1, 2, or 3 nucleotides of the 3′ end of the first complementarity domain and/or the 5-end of the second complementarity domain. In an embodiment, the duplexed region can be 20+/−10 base pairs in length. In an embodiment, the duplexed region can be 10+/−5, 15+/−5, 20+/−5, or 30+/−5 base pairs in length. In an embodiment, the duplexed region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length.

Typically the sequences forming the duplexed region have exact complementarity with one another, though in an embodiment as many as 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.

In an embodiment, the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section XI. However, in an embodiment, the linking domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the linking domain can be modified with a phosphorothioate, or other modification(s) from Section XI. In an embodiment a nucleotide of the linking domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section XI. In an embodiment, the linking domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.

Modifications in a linking domain can be selected so as to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII. gRNAs having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated a system described in Section VII. A candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the linking domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein.

The Proximal Domain

In an embodiment, the proximal domain is 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 14+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2 nucleotides in length.

In an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.

In an embodiment, the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section XI. However, in an embodiment, the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the proximal domain can be modified with a phosphorothioate, or other modification(s) from Section XI. In an embodiment a nucleotide of the proximal domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section XI.

In an embodiment, the proximal domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.

In an embodiment, the proximal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.

Modifications in the proximal domain can be selected so as to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII. gRNAs having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section VII. The candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the proximal domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, proximal domain, or a proximal domain described herein.

The Tail Domain

In an embodiment, the tail domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.

In an embodiment, the tail domain is 20+/−5 nucleotides in length.

In an embodiment, the tail domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length. In an embodiment, the tail domain is 25+/−10 nucleotides in length.

In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.

In another embodiment, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

In an embodiment, the tail domain is 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides in length.

In an embodiment, the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section XI. However, in an embodiment, the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the tail domain can be modified with a phosphorothioate, or other modification(s) from Section XI. In an embodiment a nucleotide of the tail domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section XI.

In an embodiment, the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.

In an embodiment, the tail domain comprises a tail duplex domain, which can form a tail duplexed region. In an embodiment, the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In an embodiment, a further single stranded domain, exists 3′ to the tail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment it is 4 to 6 nucleotides in length.

In an embodiment, the tail domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, tail domain, or a tail domain described herein.

In an embodiment, the proximal and tail domain, taken together comprise the following sequences:

(SEQ ID NO: 96)
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU,
or
(SEQ ID NO: 97)
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC,
or
(SEQ ID NO: 98)
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAU
C,
or
(SEQ ID NO: 99)
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG,
or
(SEQ ID NO: 100)
AAGGCUAGUCCGUUAUCA,
or
(SEQ ID NO: 101)
AAGGCUAGUCCG.

In an embodiment, the tail domain comprises the 3′ sequence UUUUUU, e.g., if a U6 promoter is used for transcription.

In an embodiment, the tail domain comprises the 3′ sequence UUUU, e.g., if an H1 promoter is used for transcription.

In an embodiment, tail domain comprises variable numbers of 3′ Us depending, e.g., on the termination signal of the pol-III promoter used.

In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template if a T7 promoter is used.

In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.

In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if a promoter is used to drive transcription.

Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section VII gRNAs having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section VII. The candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.

In an embodiment a gRNA has the following structure:

• • 5′ [targeting domain]-[first complementarity domain]-[linking domain]-[second complementarity domain]-[proximal domain]-[tail domain]-3′
  • wherein, the targeting domain comprises a core domain and optionally a secondary domain, and is 10 to 50 nucleotides in length;
  • the first complementarity domain is 5 to 25 nucleotides in length and, In an embodiment has at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with a reference first complementarity domain disclosed herein;
  • the linking domain is 1 to 5 nucleotides in length;
  • the second complementarity domain is 5 to 27 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with a reference second complementarity domain disclosed herein;
  • the proximal domain is 5 to 20 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with a reference proximal domain disclosed herein; and
  • the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in an embodiment has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with a reference tail domain disclosed herein.Exemplary Chimeric gRNAs

In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′:

• • a targeting domain (which is complementary to a target nucleic acid);
  • a first complementarity domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides;
  • a linking domain;
  • a second complementarity domain (which is complementary to the first complementarity domain);
  • a proximal domain; and
  • a tail domain,
  • wherein,
  • (a) the proximal and tail domain, when taken together, comprise
  • at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;
  • (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain; or
  • (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number:

(SEQ ID NO: 102)
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
UU.

In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. pyogenes gRNA molecule.

In some embodiments, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number:

(SEQ ID NO: 103)
NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUA
AAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU
UU.

In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. aureus gRNA molecule.Exemplary Modular gRNAs

In an embodiment, a modular gRNA comprises:

• • a first strand comprising, preferably from 5′ to 3′;
    • a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides;
    • a first complementarity domain; and
    • a second strand, comprising, preferably from 5′ to 3′:
    • optionally a 5′ extension domain;
    • a second complementarity domain;
    • a proximal domain; and
    • a tail domain,
  • wherein:
  • (a) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;
  • (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain; or
  • (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 5 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In another aspect, methods and compositions discussed herein provide methods and compositions for gene editing by using a gRNA molecule which comprises a polyA tail. In one embodiment, a polyA tail of undefined length ranging from 1 to 1000 nucleotide is added enzymatically using a polymerase such as E. coli polyA polymerase (E-PAP). In one embodiment, the polyA tail of a specified length (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 100, or 150 nucleotides) is encoded on a DNA template and transcribed with the gRNA via an RNA polymerase (e.g., T7 RNA polymerase). In one embodiment, a polyA tail of defined length (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 100, or 150 nucleotides) is synthesized as a synthetic oligonucleotide and ligated on the 3′ end of the gRNA with either an RNA ligase or a DNA ligase with our without a splinted DNA oligonucleotide complementary to the guide RNA and the polyA oligonucleotide. In one embodiment, the entire gRNA including a defined length of polyA tail is made synthetically, in one or several pieces, and ligated together by either an RNA ligase or a DNA ligase with or without a splinted DNA oligonucleotide.

Additional exemplary gRNAs for use in the present invention are disclosed in International Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference

II. Methods for Designing gRNAS

Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein. Targeting Domains discussed herein can be incorporated into the gRNAs described herein.

Methods for selection and validation of target sequences as well as off-target analyses are described, e.g., in Mali et al., 2013 SCIENCE 339(6121): 823-826; Hsu et al. NAT BIOTECHNOL, 31(9): 827-32; Fu et al., 2014 NAT BIOTECHNOL, doi: 10.1038/nbt.2808. PubMed PMID: 24463574; Heigwer et al., 2014 NAT METHODS 11(2):122-3. doi: 10.1038/nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 BIOINFORMATICS PubMed PMID: 24463181; Xiao A et al., 2014 BIOINFORMATICS PubMed PMID: 24389662. Additional considerations for designing gRNAs are discussed in the section entitled “gRNA Design” in PCT Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference.

For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For each possible gRNA choice using S. pyogenes Cas9, the tool can identify all off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA is then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for CRISPR construction, primer design for the on-target Surveyor assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-gen sequencing, can also be included in the tool. Candidate gRNA molecules can be evaluated by art-known methods or as described in Section VII herein.

The targeting domains discussed herein can be incorporated into the gRNAs described herein.

As an example, three strategies are utilized to identify gRNAs for use with S. pyogenes, S. aureus and N. meningitidis Cas9 enzymes.

Guide RNAs (gRNAs) for use with S. pyogenes, S. aureus and N. meningitidis Cas9 molecules are identified using a DNA sequence searching algorithm. Guide RNA design is carried out using a custom guide RNA design software based on the public tool cas-offinder (reference:Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases, Bioinformatics. 2014 Feb. 17. Bae S, Park J, Kim J S. PMID:24463181). Said custom guide RNA design software scores guides after calculating their genome-wide off-target propensity. Typically matches ranging from perfect matches to 7 mismatches are considered for guides ranging in length from 17 to 24. Once the off-target sites are computationally determined, an aggregate score is calculated for each guide and summarized in a tabular output using a web-interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. Genomic DNA sequence for each gene was obtained from the UCSC Genome browser and sequences were screened for repeat elements using the publically available RepeatMasker program. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence.

Following identification, gRNAs are ranked into tiers based on their distance to the target site, their orthogonality and presence of a 5′ G (based on identification of close matches in the human genome containing a relevant PAM (e.g., in the case of S. pyogenes, a NGG PAM, in the case of S. aureus, a NNGRRT (SEQ ID NO: 104) or NNGRRV (SEQ ID NO: 105) PAM, and in the case of N. meningitidis, a NNNNGATT (SEQ ID NO: 106) or NNNNGCTT (SEQ ID NO: 107) PAM). Orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A “high level of orthogonality” or “good orthogonality” may, for example, refer to 20-mer gRNAs that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.

gRNAs are identified for both single-gRNA nuclease cleavage and for a dual-gRNA paired “nickase” strategy. Criteria for selecting gRNAs and the determination for which gRNAs can be used for the dual-gRNA paired “nickase” strategy is based on two considerations:

• • 1. gRNA pairs should be oriented on the DNA such that PAMs are facing out and cutting with the D10A Cas9 nickase will result in 5′ overhangs.
  • 2. An assumption that cleaving with dual nickase pairs will result in deletion of the entire intervening sequence at a reasonable frequency. However, cleaving with dual nickase pairs can also result in indel mutations at the site of only one of the gRNAs. Candidate pair members can be tested for how efficiently they remove the entire sequence versus causing indel mutations at the site of one gRNA.

The targeting domains discussed herein can be incorporated into the gRNAs described herein.

In an embodiment, two or more (e.g., three or four) gRNA molecules are used with one Cas9 fusion molecule. In another embodiment, when two or more (e.g., three or four) gRNAs are used with two or more Cas9 fusion molecules, at least one Cas9 molecule is from a different species than the other Cas9 molecule(s). For example, when two gRNA molecules are used with two Cas9 fusion molecules, one Cas9 molecule can be from one species and the other Cas9 molecule can be from a different species. Both Cas9 species are used to generate a single or double-strand break, as desired.

Any of the targeting domains in the tables described herein can be used with a Cas9 nickase molecule to generate a single strand break.

Any of the targeting domains in the tables described herein can be used with a Cas9 nuclease molecule to generate a double strand break.

When two gRNAs designed for use to target two Cas9 molecules, one Cas9 can be one species, the second Cas9 can be from a different species. Both Cas9 species are used to generate a single or double-strand break, as desired.

It is contemplated herein that any upstream gRNA described herein may be paired with any downstream gRNA described herein. When an upstream gRNA designed for use with one species of Cas9 is paired with a downstream gRNA designed for use from a different species of Cas9, both Cas9 species are used to generate a single or double-strand break, as desired.

III. Cas9 Molecules

Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes, S. aureus, and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, e.g., Staphylococcus aureus and Neisseria meningitidis Cas9 molecules. Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumonias, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

A Cas9 molecule, or Cas9 polypeptide, as the term is used herein, refers to a molecule or a polypeptide that can interact with a guide RNA (gRNA) molecule) and, in concert with the gRNA molecule, localizes to a site which comprises a target domain, and in some embodiments, a PAM sequence. Cas9 molecule and Cas9 polypeptide, as those terms are used herein, refer to naturally occurring Cas9 molecules and to engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence of Table 100.

Cas9 Domains

Crystal structures have been determined for two different naturally occurring bacterial Cas9 molecules (Jinek et al., SCIENCE, 343(6176):1247997, 2014) and for S. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., CELL, 156:935-949, 2014; and Anders et al., NATURE, 2014, doi: 10.1038/nature13579).

A naturally occurring Cas9 molecule comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprise domains described herein. The REC lobe comprises the arginine-rich bridge helix (BH), the REC1 domain, and the REC2 domain. The REC lobe does not share structural similarity with other known proteins, indicating that it is a Cas9-specific functional domain. The BH domain is a long a helix and arginine rich region and comprises amino acids 60-93 of the sequence of S. pyogenes Cas9. The REC1 domain is important for recognition of the repeat:anti-repeat duplex, e.g., of a gRNA or a tracrRNA, and is therefore critical for Cas9 activity by recognizing the target sequence. The REC1 domain comprises two REC1 motifs at amino acids 94 to 179 and 308 to 717 of the sequence of S. pyogenes Cas9. These two REC1 domains, though separated by the REC2 domain in the linear primary structure, assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or parts thereof, may also play a role in the recognition of the repeat:anti-repeat duplex. The REC2 domain comprises amino acids 180-307 of the sequence of S. pyogenes Cas9.

The NUC lobe comprises the RuvC domain, the HNH domain, and the PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The RuvC domain is assembled from the three split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referred to in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098, respectively, of the sequence of S. pyogenes Cas9. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, however in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, e.g., the complementary strand of the target nucleic acid molecule. The HNH domain lies between the RuvC II-III motifs and comprises amino acids 775-908 of the sequence of S. pyogenes Cas9. The PI domain interacts with the PAM of the target nucleic acid molecule, and comprises amino acids 1099-1368 of the sequence of S. pyogenes Cas9.

A RuvC-Like Domain and an HNH-Like Domain

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises an HNH-like domain and a RuvC-like domain. In an embodiment, cleavage activity is dependent on a RuvC-like domain and an HNH-like domain. A Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In an embodiment, a Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide and the eaCas9 molecule or eaCas9 polypeptide comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below.

RuvC-Like Domains

In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The Cas9 molecule or Cas9 polypeptide can include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains). In an embodiment, a RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 18, 17, 16 or 15 amino acids in length. In an embodiment, the Cas9 molecule or Cas9 polypeptide comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.

N-Terminal RuvC-Like Domains

Some naturally occurring Cas9 molecules comprise more than one RuvC-like domain with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules or Cas9 polypeptide can comprise an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9  (SEQ ID NO: 108),

• • wherein,
  • X1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
  • X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
  • X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
  • X4 is selected from S, Y, N and F (e.g., S);
  • X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
  • X6 is selected from W, F, V, Y, S and L (e.g., W);
  • X7 is selected from A, S, C, V and G (e.g., selected from A and S);
  • X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
  • X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R, or, e.g., selected from T, V, I, L and Δ).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 108, by as many as 1 but no more than 2, 3, 4, or 5 residues.

In embodiment, the N-terminal RuvC-like domain is cleavage competent.

In embodiment, the N-terminal RuvC-like domain is cleavage incompetent.

In an embodiment, a eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II:D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9,  (SEQ ID NO: 109),

• • wherein
  • X1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
  • X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
  • X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
  • X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
  • X6 is selected from W, F, V, Y, S and L (e.g., W);
  • X7 is selected from A, S, C, V and G (e.g., selected from A and S);
  • X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
  • X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L and Δ).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 109 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:D-I-G-X2-X3-S-V-G-W-A-X8-X9  (SEQ ID NO: 110),

• • wherein
  • X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
  • X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
  • X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
  • X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L and Δ).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:110 by as many as 1 but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:D-I-G-T-N-S-V-G-W-A-V-X  (SEQ ID NO: 111),

• • wherein
  • X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X is selected from V, I, L and T.

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 111 by as many as 1 but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-terminal RuvC like domain disclosed herein, as many as 1 but no more than 2, 3, 4, or 5 residues.

Additional RuvC-Like Domains

In addition to the N-terminal RuvC-like domain, the Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more additional RuvC-like domains. In an embodiment, the Cas9 molecule or Cas9 polypeptide can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to 10 amino acids in length, e.g., 8 amino acids in length.

An additional RuvC-like domain can comprise an amino acid sequence:I-X1-X2-E-X3-A-R-E  (SEQ ID NO: 112), wherein

• • X1 is V or H,
  • X2 is I, L or V (e.g., I or V); and
  • X3 is M or T.

In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:I-V-X2-E-M-A-R-E  (SEQ ID NO: 113), wherein

• • X2 is I, L or V (e.g., I or V).

An additional RuvC-like domain can comprise an amino acid sequence: H-H-A-X1-D-A-X2-X3 (SEQ ID NO: 114), wherein

• • X1 is H or L;
  • X2 is R or V; and
  • X3 is E or V.

In an embodiment, the additional RuvC-like domain comprises the amino acid sequence: H-H-A-H-D-A-Y-L (SEQ ID NO:115).

In an embodiment, the additional RuvC-like domain differs from a sequence of SEQ ID NO: 112, 113, 114, 115 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In some embodiments, the sequence flanking the N-terminal RuvC-like domain is a sequences of formula V:K-X1′-Y-X2′-X3′-X4′-Z-T-D-X9′-Y,  (SEQ ID NO: 116),

• • wherein
  • X1′ is selected from K and P,
  • X2′ is selected from V, L, I, and F (e.g., V, I and L);
  • X3′ is selected from G, A and S (e.g., G),
  • X4′ is selected from L, I, V and F (e.g., L);
  • X9′ is selected from D, E, N and Q; and
  • Z is an N-terminal RuvC-like domain, e.g., as described above.HNH-Like Domains

In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. In an embodiment, an HNH-like domain is at least 15, 20, 25 amino acids in length but not more than 40, 35 or 30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VI:X1-X2-X3-H-X4-X5-P-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-N-X16-X17-X18-X19-X20-X21-X22-X23-N(SEQ ID NO: 117), wherein

• • X1 is selected from D, E, Q and N (e.g., D and E);
  • X2 is selected from L, I, R, Q, V, M and K;
  • X3 is selected from D and E;
  • X4 is selected from I, V, T, A and L (e.g., A, I and V);
  • X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
  • X6 is selected from Q, H, R, K, Y, I, L, F and W;
  • X7 is selected from S, A, D, T and K (e.g., S and A);
  • X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
  • X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
  • X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
  • X11 is selected from D, S, N, R, L and T (e.g., D);
  • X12 is selected from D, N and S;
  • X13 is selected from S, A, T, G and R (e.g., S);
  • X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
  • X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
  • X16 is selected from K, L, R, M, T and F (e.g., L, R and K);
  • X17 is selected from V, L, I, A and T;
  • X18 is selected from L, I, V and A (e.g., L and I);
  • X19 is selected from T, V, C, E, S and A (e.g., T and V);
  • X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
  • X21 is selected from S, P, R, K, N, A, H, Q, G and L;
  • X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and
  • X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, a HNH-like domain differs from a sequence of SEQ ID NO: 117 by at least one but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain is cleavage competent.

In an embodiment, the HNH-like domain is cleavage incompetent.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:X1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-K-V-L-X19-X20-X21-X22-X23-N  (SEQ ID NO: 118),

• • wherein
  • X1 is selected from D and E;
  • X2 is selected from L, I, R, Q, V, M and K;
  • X3 is selected from D and E;
  • X4 is selected from I, V, T, A and L (e.g., A, I and V);
  • X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
  • X6 is selected from Q, H, R, K, Y, I, L, F and W;
  • X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
  • X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
  • X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
  • X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
  • X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
  • X19 is selected from T, V, C, E, S and A (e.g., T and V);
  • X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
  • X21 is selected from S, P, R, K, N, A, H, Q, G and L;
  • X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and
  • X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 118 by 1, 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:X1-V-X3-H-I-V-P X6-S-X8-X9-X10-D-D-S-X14-X15-N-K-V-L-T-X20-X21-X22-X23-N  (SEQ ID NO: 119),

• • wherein
  • X1 is selected from D and E;
  • X3 is selected from D and E;
  • X6 is selected from Q, H, R, K, Y, I, L and W;
  • X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
  • X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
  • X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
  • X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
  • X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
  • X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
  • X21 is selected from S, P, R, K, N, A, H, Q, G and L;
  • X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and
  • X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 119 by 1, 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VIII:D-X2-D-I-X5-P-Q-X7-F-X9-X10-D-X12-S-I-D-N-X16-V-L-X19-X20-S-X22-X23-N  (SEQ ID NO: 118),

• • wherein
  • X2 is selected from I and V;
  • X5 is selected from I and V;
  • X7 is selected from A and S;
  • X9 is selected from I and L;
  • X10 is selected from K and T;
  • X12 is selected from D and N;
  • X16 is selected from R, K and L; X19 is selected from T and V;
  • X20 is selected from S and R;
  • X22 is selected from K, D and A; and
  • X23 is selected from E, K, G and N (e.g., the eaCas9 molecule or eaCas9 polypeptide can comprise an HNH-like domain as described herein).

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 120 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises the amino acid sequence of formula IX:L-Y-Y-L-Q-N-G-X1′-D-M-Y-X2′-X3′-X4′-X5′-L-D-I-X6′-X7′-L-S-X8′-Y-Z-N-R-X9′-K-X10′-D-X11′-V-P  (SEQ ID NO: 121),

• • wherein
  • X1′ is selected from K and R;
  • X2′ is selected from V and T;
  • X3′ is selected from G and D;
  • X4′ is selected from E, Q and D;
  • X5′ is selected from E and D;
  • X6′ is selected from D, N and H;
  • X7′ is selected from Y, R and N;
  • X8′ is selected from Q, D and N; X9′ is selected from G and E;
  • X10′ is selected from S and G;
  • X11′ is selected from D and N; and
  • Z is an HNH-like domain, e.g., as described above.

In an embodiment, the eaCas9 molecule or eaCas9 polypeptide comprises an amino acid sequence that differs from a sequence of SEQ ID NO: 121 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, by as many as 1 but no more than 2, 3, 4, or 5 residues.

Cas9 Activities

Nuclease and Helicase Activities

In an embodiment, the Cas9 molecule or Cas9 polypeptide is capable of cleaving a target nucleic acid molecule. Typically wild type Cas9 molecules cleave both strands of a target nucleic acid molecule. Cas9 molecules and Cas9 polypeptides can be engineered to alter nuclease cleavage (or other properties), e.g., to provide a Cas9 molecule or Cas9 polypeptide which is a nickase, or which lacks the ability to cleave target nucleic acid. A Cas9 molecule or Cas9 polypeptide that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 (an enzymatically active Cas9) molecule or eaCas9 polypeptide.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises one or more of the following enzymatic activities:

• • a nickase activity, i.e., the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule;
  • a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
  • an endonuclease activity;
  • an exonuclease activity; and
  • a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.

In an embodiment, an enzymatically active or an eaCas9 molecule or eaCas9 polypeptide cleaves both DNA strands and results in a double stranded break. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with a RuvC domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH domain and cleavage activity associated with a RuvC domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an active, or cleavage competent, HNH domain and an inactive, or cleavage incompetent, RuvC domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, RuvC domain.

Some Cas9 molecules or Cas9 polypeptides have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule localize to a core target domain, but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates. Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 molecule or eiCas9 polypeptide. For example, an eiCas9 molecule or eiCas9 polypeptide can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule or eiCas9 polypeptide, as measured by an assay described herein.

Targeting and PAMs

A Cas9 molecule or Cas9 polypeptide, is a polypeptide that can interact with a guide RNA (gRNA) molecule and, in concert with the gRNA molecule, localizes to a site which comprises a target domain, and in an embodiment, a PAM sequence.

In an embodiment, the ability of an eaCas9 molecule or eaCas9 polypeptide to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In an embodiment, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In an embodiment, an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Mali et al., SCIENCE (2013) 339(6121): 823-826. In an embodiment, an eaCas9 molecule of S. thermophilus recognizes the sequence motif NGGNG (SEQ ID NO.: 122) and/or NNAGAAW (W=A or T) (SEQ ID NO.: 123) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from these sequences. See, e.g., Horvath et al., SCIENCE (2010); 327(5962):167-170, and Deveau et al., J. BACTERIOL. 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. mutans recognizes the sequence motif NGG and/or NAAR (R=A or G) (SEQ ID NO.: 124) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R=A or G) (SEQ ID NO.: 125) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRN (R=A or G)(SEQ ID NO: 126) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRT (R=A or G)(SEQ ID NO: 104) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRV (R=A or G) (SEQ ID NO.: 105) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT (SEQ ID NO.: 106) or NNNGCTT (R=A or G) (SEQ ID NO: 127) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al. (2013) PROC. NAT'L. ACAD. SCI. USA 110(39):15644-15649. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al. (2012) SCIENCE 337:816. In the aforementioned embodiments, N can be any nucleotide residue, e.g., any of A, G, C or T.

As is discussed herein, Cas9 molecules can be engineered to alter the PAM specificity of the Cas9 molecule.

Exemplary naturally occurring Cas9 molecules are described in Chylinski et al. (2013) RNA BIOLOGY 10:5, 727-737. Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27 bacterial family, a cluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30 bacterial family, a cluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34 bacterial family, a cluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37 bacterial family, a cluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40 bacterial family, a cluster 41 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family, a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 51 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family.

Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family. Examples include a Cas9 molecule of S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g., strain LIVID-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC11558), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S. agalactiae (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L. innocua, e.g., strain Clip11262), Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231,408). Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitidis (Hou et al., PNAS Early Edition 2013, 1-6 and a S. aureus cas9 molecule.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence:

• • having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with;
  • differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues when compared with;
  • differs by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids, but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or
  • is identical to any Cas9 molecule sequence described herein, or a naturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al. (2013) RNA BIOLOGY 10:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6. In an embodiment, the Cas9 molecule or Cas9 polypeptide comprises one or more of the following activities: a nickase activity; a double stranded cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the ability, together with a gRNA molecule, to localize to a target nucleic acid.Engineered or Altered Cas9 Molecules and Cas9 Polypeptides

Cas9 molecules and Cas9 polypeptides described herein, e.g., naturally occurring Cas9 molecules, can possess any of a number of properties, including: nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In an embodiment, a Cas9 molecule or Cas9 polypeptide can include all or a subset of these properties. In a typical embodiment, a Cas9 molecule or Cas9 polypeptide has the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules and Cas9 polypeptides.

Cas9 molecules include engineered Cas9 molecules and engineered Cas9 polypeptides (engineered, as used in this context, means merely that the Cas9 molecule or Cas9 polypeptide differs from a reference sequences, and implies no process or origin limitation). An engineered Cas9 molecule or Cas9 polypeptide can comprise altered enzymatic properties, e.g., altered nuclease activity (as compared with a naturally occurring or other reference Cas9 molecule) or altered helicase activity. As discussed herein, an engineered Cas9 molecule or Cas9 polypeptide can have nickase activity (as opposed to double strand nuclease activity). In an embodiment an engineered Cas9 molecule or Cas9 polypeptide can have an alteration that alters its size, e.g., a deletion of amino acid sequence that reduces its size, e.g., without significant effect on one or more, or any Cas9 activity. In an embodiment, an engineered Cas9 molecule or Cas9 polypeptide can comprise an alteration that affects PAM recognition. For example, an engineered Cas9 molecule can be altered to recognize a PAM sequence other than that recognized by the endogenous wild-type PI domain. In an embodiment a Cas9 molecule or Cas9 polypeptide can differ in sequence from a naturally occurring Cas9 molecule but not have significant alteration in one or more Cas9 activities.

Cas9 molecules or Cas9 polypeptides with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring Cas9 molecule or Cas9 polypeptide, to provide an altered Cas9 molecule or Cas9 polypeptide having a desired property. For example, one or more mutations or differences relative to a parental Cas9 molecule, e.g., a naturally occurring or engineered Cas9 molecule, can be introduced. Such mutations and differences comprise: substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions. In an embodiment, a Cas9 molecule or Cas9 polypeptide can comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to a reference, e.g., a parental Cas9 molecule.

In an embodiment, a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, a mutation or mutations have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein.

Non-Cleaving and Modified-Cleavage Cas9 Molecules and Cas9 Polypeptides

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule or Cas9 polypeptide can differ from a naturally occurring Cas9 molecule, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded nucleic acid (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.

Alterations in the Ability to Cleave One or Both Strands of a Target Nucleic Acid

In an embodiment, exemplary Cas9 activities comprise one or more of PAM specificity, cleavage activity, and helicase activity. A mutation(s) can be present, e.g., in: one or more RuvC domains, e.g., an N-terminal RuvC domain; an HNH domain; a region outside the RuvC domains and the HNH domain. In an embodiment, a mutation(s) is present in a RuvC domain. In an embodiment, a mutation(s) is present in an HNH domain. In an embodiment, mutations are present in both a RuvC domain and an HNH domain.

Exemplary mutations that may be made in the RuvC domain or HNH domain with reference to the S. pyogenes Cas9 sequence include: D10A, E762A, H840A, N854A, N863A and/or D986A. Exemplary mutations that may be made in the RuvC domain with reference to the S. aureus Cas9 sequence include: N580A.

In an embodiment, a Cas9 molecule is an eiCas9 molecule comprising one or more differences in a RuvC domain and/or in an HNH domain as compared to a reference Cas9 molecule, and the eiCas9 molecule does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wild type, e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1% of a reference Cas9 molecule, as measured by an assay described herein.

Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc., can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative. In an embodiment, a “non-essential” amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an “essential” amino acid residue results in a substantial loss of activity (e.g., cleavage activity).

In an embodiment, a Cas9 molecule comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. aureus, S. pyogenes, or C. jejuni as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. aureus, S. pyogenes, or C. jejuni); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. aureus, S. pyogenes, or C. jejuni); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.

In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising one or more of the following activities: cleavage activity associated with a RuvC domain; cleavage activity associated with an HNH domain; cleavage activity associated with an HNH domain and cleavage activity associated with a RuvC domain.

In an embodiment, the altered Cas9 molecule is an eiCas9 molecule which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni or N. meningitidis. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. In an embodiment, the eiCas9 molecule lacks substantial cleavage activity associated with a RuvC domain and cleavage activity associated with an HNH domain.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can be a fusion, e.g., of two of more different Cas9 molecules, e.g., of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of a Cas9 molecule of S. pyogenes comprising an N-terminal RuvC-like domain can be fused to a fragment of Cas9 molecule of a species other than S. pyogenes (e.g., S. thermophilus) comprising an HNH-like domain.

Cas9 Molecules with Altered PAM Recognition or No PAM Recognition

Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example the PAM recognition sequences described above for, e.g., S. pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.

In an embodiment, a Cas9 molecule or Cas9 polypeptide has the same PAM specificities as a naturally occurring Cas9 molecule. In an embodiment, a Cas9 molecule or Cas9 polypeptide has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule or Cas9 polypeptide recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule or Cas9 polypeptide can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to high level of identity (e.g., 98%, 99% or 100% match between gRNA and a PAM sequence), e.g., to decrease off target sites and increase specificity. In an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. In an embodiment, the Cas9 specificity requires at least 90%, 95%, 96%, 97%, 98%, 99% or more homology between the gRNA and the PAM sequence. Cas9 molecules or Cas9 polypeptides that recognize different PAM sequences and/or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt et al. (2011) NATURE 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section VII.

Alterations of the PI domain, which mediates PAM recognition are discussed below.

Synthetic Cas9 Molecules and Cas9 Polypeptides with Altered PI Domains

Current genome-editing methods are limited in the diversity of target sequences that can be targeted by the PAM sequence that is recognized by the Cas9 molecule utilized. A synthetic Cas9 molecule (or Syn-Cas9 molecule), or synthetic Cas9 polypeptide (or syn-Cas9 polypeptide), as that term is used herein, refers to a Cas9 molecule or Cas9 polypeptide that comprises a Cas9 core domain from one bacterial species and a functional altered PI domain, i.e., a PI domain other than that naturally associated with the Cas9 core domain, e.g., from a different bacterial species.

In an embodiment, the altered PI domain recognizes a PAM sequence that is different from the PAM sequence recognized by the naturally-occurring Cas9 from which the Cas9 core domain is derived. In an embodiment, the altered PI domain recognizes the same PAM sequence recognized by the naturally-occurring Cas9 from which the Cas9 core domain is derived, but with different affinity or specificity. A Syn-Cas9 molecule or Syn-Cas9 polypeptide can be, respectively, a Syn-eaCas9 molecule or Syn-eaCas9 polypeptide or a Syn-eiCas9 molecule Syn-eiCas9 polypeptide.

An exemplary Syn-Cas9 molecule Syn-Cas9 polypeptide comprises:

• • a) a Cas9 core domain, e.g., a Cas9 core domain from Table 100 or 200, e.g., a S. aureus, S. pyogenes, or C. jejuni Cas9 core domain; and
  • b) an altered PI domain from a species X Cas9 sequence selected from Tables 400 and 500.

In an embodiment, the RKR motif (the PAM binding motif) of said altered PI domain comprises: differences at 1, 2, or 3 amino acid residues; a difference in amino acid sequence at the first, second, or third position; differences in amino acid sequence at the first and second positions, the first and third positions, or the second and third positions; as compared with the sequence of the RKR motif of the native or endogenous PI domain associated with the Cas9 core domain.

In an embodiment, the Cas9 core domain comprises the Cas9 core domain from a species X Cas9 from Table 100 and said altered PI domain comprises a PI domain from a species Y Cas9 from Table 100.

In an embodiment, the RKR motif of the species X Cas9 is other than the RKR motif of the species Y Cas9.

In an embodiment, the RKR motif of the altered PI domain is selected from XXY, XNG, and XNQ.

In an embodiment, the altered PI domain has at least 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology with the amino acid sequence of a naturally occurring PI domain of said species Y from Table 100.

In an embodiment, the altered PI domain differs by no more than 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue from the amino acid sequence of a naturally occurring PI domain of said second species from Table 100.

In an embodiment, the Cas9 core domain comprises a S. aureus core domain and altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 400 or Table 500.

In an embodiment, the Cas9 core domain comprises a S. pyogenes core domain and the altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 400 or Table 500.

In an embodiment, the Cas9 core domain comprises a C. jejuni core domain and the altered PI domain comprises: an A. denitrificans PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 400 or Table 500.

In an embodiment, the Cas9 molecule further comprises a linker disposed between said Cas9 core domain and said altered PI domain.

In an embodiment, the linker comprises: a linker described elsewhere herein disposed between the Cas9 core domain and the heterologous PI domain.

Exemplary altered PI domains for use in Syn-Cas9 molecules are described in Tables 400 and 500. The sequences for the 83 Cas9 orthologs referenced in Tables 400 and 500 are provided in Table 100. Table 250 provides the Cas9 orthologs with known PAM sequences and the corresponding RKR motif.

In an embodiment, a Syn-Cas9 molecule may also be size-optimized, e.g., the Syn-Cas9 molecule comprises one or more deletions, and optionally one or more linkers disposed between the amino acid residues flanking the deletions. In an embodiment, a Syn-Cas9 molecule comprises a REC deletion.

Size-Optimized Cas9 Molecules

Engineered Cas9 molecules and engineered Cas9 polypeptides, as described herein, include a Cas9 molecule or Cas9 polypeptide comprising a deletion that reduces the size of the molecule while still retaining desired Cas9 properties, e.g., essentially native conformation, Cas9 nuclease activity, and/or target nucleic acid molecule recognition. Provided herein are Cas9 molecules or Cas9 polypeptides comprising one or more deletions, and optionally one or more linkers, wherein a linker is disposed between the amino acid residues that flank the deletion. Methods for identifying suitable deletions in a reference Cas9 molecule, methods for generating Cas9 molecules with a deletion and a linker, and methods for using such Cas9 molecules will be apparent to one of ordinary skill in the art upon review of this document.

A Cas9 molecule, e.g., a S. aureus, S. pyogenes, or C. jejuni, Cas9 molecule, having a deletion is smaller, e.g., has reduced number of amino acids, than the corresponding naturally-occurring Cas9 molecule. The smaller size of the Cas9 molecules allows increased flexibility for delivery methods, and thereby increases utility for genome-editing. A Cas9 molecule can comprise one or more deletions that do not substantially affect or decrease the activity of the resultant Cas9 molecules described herein. Activities that are retained in the Cas9 molecules comprising a deletion as described herein include one or more of the following:

• • a nickase activity, i.e., the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule; a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
  • an endonuclease activity;
  • an exonuclease activity;
  • a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid;
  • and recognition activity of a nucleic acid molecule, e.g., a target nucleic acid or a gRNA.

Activity of the Cas9 molecules described herein can be assessed using the activity assays described herein or in the art.

Identifying Regions Suitable for Deletion

Suitable regions of Cas9 molecules for deletion can be identified by a variety of methods. Naturally-occurring orthologous Cas9 molecules from various bacterial species, e.g., any one of those listed in Table 100, can be modeled onto the crystal structure of S. pyogenes Cas9 (Nishimasu et al. (2014) CELL, 156: 935-949) to examine the level of conservation across the selected Cas9 orthologs with respect to the three-dimensional conformation of the protein. Less conserved or unconserved regions that are located spatially distant from regions involved in Cas9 activity, e.g., the interface with a target nucleic acid molecule and/or gRNA, represent regions or domains that are candidates for deletion without substantially affecting or decreasing Cas9 activity.

REC-Optimized Cas9 Molecules

A REC-optimized Cas9 molecule, as that term is used herein, refers to a Cas9 molecule that comprises a deletion in one or both of the REC2 domain and the RE1_CT domain (collectively a REC deletion), wherein the deletion comprises at least 10% of the amino acid residues in the cognate domain. A REC-optimized Cas9 molecule can be an eaCas9 molecule or an eiCas9 molecule. An exemplary REC-optimized Cas9 molecule comprises:

• • a) a deletion selected from:
    • i) a REC2 deletion;
    • ii) a REC1_CT deletion; or
    • iii) a REC1_SUB deletion.

Optionally, a linker is disposed between the amino acid residues that flank the deletion. In an embodiment a Cas9 molecule includes only one deletion, or only two deletions. A Cas9 molecule can comprise a REC2 deletion and a REC1_CT deletion. A Cas9 molecule can comprise a REC2 deletion and a REC1_SUB deletion.

Generally, the deletion will contain at least 10% of the amino acids in the cognate domain, e.g., a REC2 deletion will include at least 10% of the amino acids in the REC2 domain. A deletion can comprise: at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the amino acid residues of its cognate domain; all of the amino acid residues of its cognate domain; an amino acid residue outside its cognate domain; a plurality of amino acid residues outside its cognate domain; the amino acid residue immediately N terminal to its cognate domain; the amino acid residue immediately C terminal to its cognate domain; the amino acid residue immediately N terminal to its cognate and the amino acid residue immediately C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain; a plurality of e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain and a plurality of e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain.

In an embodiment, a deletion does not extend beyond: its cognate domain; the N terminal amino acid residue of its cognate domain; the C terminal amino acid residue of its cognate domain.

A REC-optimized Cas9 molecule can include a linker disposed between the amino acid residues that flank the deletion. Linkers for use in generating recombinant proteins, e.g., multi-domain proteins, are known in the art (Chen et al. (2013) ADV. DRUG DELIVERY REV. 65:1357-69). Any linkers known in the art that maintain the conformation or native fold of the Cas9 molecule (thereby retaining Cas9 activity) can be used. Several properties of linkers, such as length, hydrophobicity, intrinsic properties of the amino acids residues themselves, and secondary structure should be considered in the context of the goal to maintain native conformation and functional activity of Cas9. Any linkers known in the art that maintain the conformation or native fold of the Cas9 molecule (thereby retaining Cas9 activity) can be used. Several properties of linkers, such as length, hydrophobicity, intrinsic properties of the amino acids residues themselves, and secondary structure should be considered in the context of the goal to maintain native conformation and functional activity of Cas9.

A flexible linker can be utilized in the Cas9 molecules described herein. Flexible linkers allow a certain degree of movement and/or interaction within and between the joined domains or regions of the protein. Generally, flexible linkers are composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows mobility of the connected domains or regions. Furthermore, the incorporation of Ser or Thr can help maintain the stability of the linker in aqueous solutions by hydrogen bonding with the water molecules, thereby reducing unfavorable interactions between the linker and the other protein moieties. Commonly used flexible linkers are comprised of sequences that primarily consist of Gly and Ser residues. Often, these flexible linkers consist of repeating units of a combination of Gly and Ser residues, e.g., (GGS)_x, where the number of repeating units, e.g., x, can be optimized to achieve the appropriate separation of other domains or regions of the protein.

In some cases, a rigid linker may be preferred if there is significant distance between the joined domains or regions, or to maintain a fixed distance between the joined domains or regions of a protein and independent functions of the domains/regions. Rigid linkers often have defined secondary structure, e.g., alpha helix, or other stabilizing interactions, e.g., salt bridges and disulfide bonds. Rigid linkers commonly contain multiple Pro residues, or repeating combinations of Glu-Pro or Lys-Pro because Pro imposes a strong conformation constraint due to its structure.

The linker can comprise an amino acid residue, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. Typically, the linker will comprises less than 10, 20 or 30 amino acid residues. Typically, the linker is less than 50, 40, 30, 20, 10, or 5% of the length of the deleted sequence. Suitable linkers include: [Gly-Ser]_x, wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 128); [Gly-Gly-Ser]_x, wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 129); [Gly-Gly-Ser]; [Gly-Ser-Gly-Ser]_x, wherein x is 1, 2, 3, 4, or 5 (SEQ ID NO: 130); [Gly-Ser-Gly-Ser] (SEQ ID NO: 131); (GSAGSAAGSGFF)_x, wherein x is 1, 2, 3 or 4 (SEQ ID NO: 132); (SIVAQLSRPDPA)_x, wherein x is 1, 2, 3 or 4 (SEQ ID NO: 133); or an XTEN sequence, e.g., the XTEN sequence of SEQ ID NO: #______, or a sequence that differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In an embodiment linker comprises an amino acid sequence other than a sequence within REC2.

In an embodiment, a REC-optimized Cas9 molecule comprises an amino acid sequence that, other than any REC deletion and associated linker, has at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology with the amino acid sequence of a naturally occurring Cas9, e.g., a Cas9 molecule described in Table 100, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

In an embodiment, a REC-optimized Cas9 molecule comprises an amino acid sequence that, other than any REC deletion and associated linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues from the amino acid sequence of a naturally occurring Cas9, e.g., a Cas9 molecule described in Table 100, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

In an embodiment, a REC-optimized Cas9 molecule comprises an amino acid sequence that, other than any REC deletion and associate linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% of the amino acid residues from the amino acid sequence of a naturally occurring Cas9, e.g., a Cas9 molecule described in Table 100, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) ADV. APPL. MATH. 2: 482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. MOL. BIOL. 48:443, by the search for similarity method of Pearson and Lipman, (1988) PROC. NAT'L. ACAD. SCI. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) NUC. ACIDS RES. 25:3389-3402; and Altschul et al. (1990) J. MOL. BIOL. 215: 403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (1988) COMPUT. APPL. BIOSCI. 4:11-17, which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. MOL. BIOL. 48:444-453 algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Sequence information for exemplary REC deletions are provided for 83 naturally-occurring Cas9 orthologs in Table 100.

The amino acid sequences of exemplary Cas9 molecules from different bacterial species are shown below.

TABLE 100
Amino Acid Sequence of Cas9 Orthologs
		REC2	REC1CT	Recsub
Species/		start	stop	# AA	start	stop	# AA	start	stop	# AA
Composite		(AA	(AA	deleted	(AA	(AA	deleted	(AA	(AA	deleted
ID	Amino acid sequence	pos)	pos)	(n)	pos)	pos)	(n)	pos)	pos)	(n)
Staphylococcus	MKRNYILGLDIGITSVGYGIID	126	166	41	296	352	57	296	352	57
aureus	YETRDVIDAGVRLFKEANVENN
tr|J7RUA5|	EGRRSKRGARRLKRRRRHRIQR
J7RUA5_STAAU	VKKLLFDYNLLTDHSELSGINP
	YEARVKGLSQKLSEEEFSAALL
	HLAKRRGVHNVNEVEEDTGNEL
	STKEQISRNSKALEEKYVAELQ
	LERLKKDGEVRGSINRFKTSDY
	VKEAKQLLKVQKAYHQLDQSFI
	DTYIDLLETRRTYYEGPGEGSP
	FGWKDIKEWYEMLMGHCTYFPE
	ELRSVKYAYNADLYNALNDLNN
	LVITRDENEKLEYYEKFQIIEN
	VFKQKKKPTLKQIAKEILVNEE
	DIKGYRVTSTGKPEFTNLKVYH
	DIKDITARKEIIENAELLDQIA
	KILTIYQSSEDIQEELTNLNSE
	LTQEEIEQISNLKGYTGTHNLS
	LKAINLILDELWHTNDNQIAIF
	NRLKLVPKKVDLSQQKEIPTTL
	VDDFILSPVVKRSFIQSIKVIN
	AIIKKYGLPNDIIIELAREKNS
	KDAQKMINEMQKRNRQTNERIE
	EIIRTTGKENAKYLIEKIKLHD
	MQEGKCLYSLEAIPLEDLLNNP
	FNYEVDHIIPRSVSFDNSFNNK
	VLVKQEENSKKGNRTPFQYLSS
	SDSKISYETFKKHILNLAKGKG
	RISKTKKEYLLEERDINRFSVQ
	KDFINRNLVDTRYATRGLMNLL
	RSYFRVNNLDVKVKSINGGFTS
	FLRRKWKFKKERNKGYKHHAED
	ALIIANADFIFKEWKKLDKAKK
	VMENQMFEEKQAESMPEIETEQ
	EYKEIFITPHQIKHIKDFKDYK
	YSHRVDKKPNRELINDTLYSTR
	KDDKGNTLIVNNLNGLYDKDND
	KLKKLINKSPEKLLMYHHDPQT
	YQKLKLIMEQYGDEKNPLYKYY
	EETGNYLTKYSKKDNGPVIKKI
	KYYGNKLNAHLDITDDYPNSRN
	KVVKLSLKPYRFDVYLDNGVYK
	FVTVKNLDVIKKENYYEVNSKC
	YEEAKKLKKISNQAEFIASFYN
	NDLIKINGELYRVIGVNNDLLN
	RIEVNMIDITYREYLENMNDKR
	PPRIIKTIASKTQSIKKYSTDI
	LGNLYEVKSKKHPQIIKKG
	(SEQ ID NO: 6)
Streptococcus	MDKKYSIGLDIGTNSVGWAVIT	176	314	139	511	592	82	511	592	82
pyogenes	DEYKVPSKKFKVLGNTDRHSIK
sp|Q99ZW2|	KNLIGALLFDSGETAEATRLKR
CAS9_STRP1	TARRRYTRRKNRICYLQEIFSN
	EMAKVDDSFFHRLEESFLVEED
	KKHERHPIFGNIVDEVAYHEKY
	PTIYHLRKKLVDSTDKADLRLI
	YLALAHMIKFRGHFLIEGDLNP
	DNSDVDKLFIQLVQTYNQLFEE
	NPINASGVDAKAILSARLSKSR
	RLENLIAQLPGEKKNGLFGNLI
	ALSLGLTPNFKSNFDLAEDAKL
	QLSKDTYDDDLDNLLAQIGDQY
	ADLFLAAKNLSDAILLSDILRV
	NTEITKAPLSASMIKRYDEHHQ
	DLTLLKALVRQQLPEKYKEIFF
	DQSKNGYAGYIDGGASQEEFYK
	FIKPILEKMDGTEELLVKLNRE
	DLLRKQRTFDNGSIPHQIHLGE
	LHAILRRQEDFYPFLKDNREKI
	EKILTFRIPYYVGPLARGNSRF
	AWMTRKSEETITPWNFEEVVDK
	GASAQSFIERMTNFDKNLPNEK
	VLPKHSLLYEYFTVYNELTKVK
	YVTEGMRKPAFLSGEQKKAIVD
	LLFKTNRKVTVKQLKEDYFKKI
	ECFDSVEISGVEDRFNASLGTY
	HDLLKIIKDKDFLDNEENEDIL
	EDIVLTLTLFEDREMIEERLKT
	YAHLFDDKVMKQLKRRRYTGWG
	RLSRKLINGIRDKQSGKTILDF
	LKSDGFANRNFMQLIHDDSLTF
	KEDIQKAQVSGQGDSLHEHIAN
	LAGSPAIKKGILQTVKVVDELV
	KVMGRHKPENIVIEMARENQTT
	QKGQKNSRERMKRIEEGIKELG
	SQILKEHPVENTQLQNEKLYLY
	YLQNGRDMYVDQELDINRLSDY
	DVDHIVPQSFLKDDSIDNKVLT
	RSDKNRGKSDNVPSEEVVKKMK
	NYWRQLLNAKLITQRKFDNLTK
	AERGGLSELDKAGFIKRQLVET
	RQITKHVAQILDSRMNTKYDEN
	DKLIREVKVITLKSKLVSDFRK
	DFQFYKVREINNYHHAHDAYLN
	AVVGTALIKKYPKLESEFVYGD
	YKVYDVRKMIAKSEQEIGKATA
	KYFFYSNIMNFFKTEITLANGE
	IRKRPLIETNGETGEIVWDKGR
	DFATVRKVLSMPQVNIVKKTEV
	QTGGFSKESILPKRNSDKLIAR
	KKDWDPKKYGGFDSPTVAYSVL
	VVAKVEKGKSKKLKSVKELLGI
	TIMERSSFEKNPIDFLEAKGYK
	EVKKDLIIKLPKYSLFELENGR
	KRMLASAGELQKGNELALPSKY
	VNFLYLASHYEKLKGSPEDNEQ
	KQLFVEQHKHYLDEIIEQISEF
	SKRVILADANLDKVLSAYNKHR
	DKPIREQAENIIHLFTLTNLGA
	PAAFKYFDTTIDRKRYTSTKEV
	LDATLIHQSITGLYETRIDLSQ
	LGGD (SEQ ID NO: 7)
Campylobacter	MARILAFDIGISSIGWAFSEND	137	181	45	316	360	45	316	360	45
jejuni	ELKDCGVRIFTKVENPKTGESL
NCTC 11168	ALPRRLARSARKRLARRKARLN
gi|218563121|	HLKHLIANEFKLNYEDYQSFDE
ref|	SLAKAYKGSLISPYELRFRALN
YP_002344900.1	ELLSKQDFARVILHIAKRRGYD
	DIKNSDDKEKGAILKAIKQNEE
	KLANYQSVGEYLYKEYFQKFKE
	NSKEFTNVRNKKESYERCIAQS
	FLKDELKLIFKKQREFGFSFSK
	KFEEEVLSVAFYKRALKDFSHL
	VGNCSFFTDEKRAPKNSPLAFM
	FVALTRIINLLNNLKNTEGILY
	TKDDLNALLNEVLKNGTLTYKQ
	TKKLLGLSDDYEFKGEKGTYFI
	EFKKYKEFIKALGEHNLSQDDL
	NEIAKDITLIKDEIKLKKALAK
	YDLNQNQIDSLSKLEFKDHLNI
	SFKALKLVTPLMLEGKKYDEAC
	NELNLKVAINEDKKDFLPAFNE
	TYYKDEVTNPVVLRAIKEYRKV
	LNALLKKYGKVHKINIELAREV
	GKNHSQRAKIEKEQNENYKAKK
	DAELECEKLGLKINSKNILKLR
	LFKEQKEFCAYSGEKIKISDLQ
	DEKMLEIDHIYPYSRSFDDSYM
	NKVLVFTKQNQEKLNQTPFEAF
	GNDSAKWQKIEVLAKNLPTKKQ
	KRILDKNYKDKEQKNFKDRNLN
	DTRYIARLVLNYTKDYLDFLPL
	SDDENTKLNDTQKGSKVHVEAK
	SGMLTSALRHTWGFSAKDRNNH
	LHHAIDAVIIAYANNSIVKAFS
	DFKKEQESNSAELYAKKISELD
	YKNKRKFFEPFSGFRQKVLDKI
	DEIFVSKPERKKPSGALHEETF
	RKEEEFYQSYGGKEGVLKALEL
	GKIRKVNGKIVKNGDMFRVDIF
	KHKKTNKFYAVPIYTMDFALKV
	LPNKAVARSKKGEIKDWILMDE
	NYEFCFSLYKDSLILIQTKDMQ
	EPEFVYYNAFTSSTVSLIVSKH
	DNKFETLSKNQKILFKNANEKE
	VIAKSIGIQNLKVFEKYIVSAL
	GEVTKAEFRQREDFKK (SEQ
	ID NO: 8)
Bacteroides	MKRILGLDLGTNSIGWALVNEA	148	339	192	524	617	84	524	617	84
fragilis	ENKDERSSIVKLGVRVNPLTVD
NCTC 9343	ELTNFEKGKSITTNADRTLKRG
gi|60683389|	MRRNLQRYKLRRETLTEVLKEH
ref|YP_213533.1|	KLITEDTILSENGNRTTFETYR
	LRAKAVTEEISLEEFARVLLMI
	NKKRGYKSSRKAKGVEEGTLID
	GMDIARELYNNNLTPGELCLQL
	LDAGKKFLPDFYRSDLQNELDR
	IWEKQKEYYPEILTDVLKEELR
	GKKRDAVWAICAKYFVWKENYT
	EWNKEKGKTEQQEREHKLEGIY
	SKRKRDEAKRENLQWRVNGLKE
	KLSLEQLVIVFQEMNTQINNSS
	GYLGAISDRSKELYFNKQTVGQ
	YQMEMLDKNPNASLRNMVFYRQ
	DYLDEFNMLWEKQAVYHKELTE
	ELKKEIRDIIIFYQRRLKSQKG
	LIGFCEFESRQIEVDIDGKKKI
	KTVGNRVISRSSPLFQEFKIWQ
	ILNNIEVTVVGKKRKRRKLKEN
	YSALFEELNDAEQLELNGSRRL
	CQEEKELLAQELFIRDKMTKSE
	VLKLLFDNPQELDLNFKTIDGN
	KTGYALFQAYSKMIEMSGHEPV
	DFKKPVEKVVEYIKAVFDLLNW
	NTDILGFNSNEELDNQPYYKLW
	HLLYSFEGDNTPTGNGRLIQKM
	TELYGFEKEYATILANVSFQDD
	YGSLSAKAIHKILPHLKEGNRY
	DVACVYAGYRHSESSLTREEIA
	NKVLKDRLMLLPKNSLHNPVVE
	KILNQMVNVINVIIDIYGKPDE
	IRVELARELKKNAKEREELTKS
	IAQTTKAHEEYKTLLQTEFGLT
	NVSRTDILRYKLYKELESCGYK
	TLYSNTYISREKLFSKEFDIEH
	IIPQARLFDDSFSNKTLEARSV
	NIEKGNKTAYDFVKEKFGESGA
	DNSLEHYLNNIEDLFKSGKISK
	TKYNKLKMAEQDIPDGFIERDL
	RNTQYIAKKALSMLNEISHRVV
	ATSGSVTDKLREDWQLIDVMKE
	LNWEKYKALGLVEYFEDRDGRQ
	IGRIKDWTKRNDHRHHAMDALT
	VAFTKDVFIQYENNKNASLDPN
	ANEHAIKNKYFQNGRAIAPMPL
	REFRAEAKKHLENTLISIKAKN
	KVITGNINKTRKKGGVNKNMQQ
	TPRGQLHLETIYGSGKQYLTKE
	EKVNASFDMRKIGTVSKSAYRD
	ALLKRLYENDNDPKKAFAGKNS
	LDKQPIWLDKEQMRKVPEKVKI
	VTLEAIYTIRKEISPDLKVDKV
	IDVGVRKILIDRLNEYGNDAKK
	AFSNLDKNPIWLNKEKGISIKR
	VTISGISNAQSLHVKKDKDGKP
	ILDENGRNIPVDFVNTGNNHHV
	AVYYRPVIDKRGQLVVDEAGNP
	KYELEEVVVSFFEAVTRANLGL
	PIIDKDYKTTEGWQFLFSMKQN
	EYFVFPNEKTGFNPKEIDLLDV
	ENYGLISPNLFRVQKFSLKNYV
	FRHHLETTIKDTSSILRGITWI
	DFRSSKGLDTIVKVRVNHIGQI
	VSVGEY (SEQ ID NO: 9)
Bifidobacterium	MSRKNYVDDYAISLDIGNASVG	173	335	163	516	607	87	516	607	87
bifidum	WSAFTPNYRLVRAKGHELIGVR
S17	LFDPADTAESRRMARTTRRRYS
gi|310286728|	RRRWRLRLLDALFDQALSEIDP
ref|	SFLARRKYSWVHPDDENNADCW
YP_003937986.	YGSVLFDSNEQDKRFYEKYPTI
	YHLRKALMEDDSQHDIREIYLA
	IHHMVKYRGNFLVEGTLESSNA
	FKEDELLKLLGRITRYEMSEGE
	QNSDIEQDDENKLVAPANGQLA
	DALCATRGSRSMRVDNALEALS
	AVNDLSREQRAIVKAIFAGLEG
	NKLDLAKIFVSKEFSSENKKIL
	GIYFNKSDYEEKCVQIVDSGLL
	DDEEREFLDRMQGQYNAIALKQ
	LLGRSTSVSDSKCASYDAHRAN
	WNLIKLQLRTKENEKDINENYG
	ILVGWKIDSGQRKSVRGESAYE
	NMRKKANVFFKKMIETSDLSET
	DKNRLIHDIEEDKLFPIQRDSD
	NGVIPHQLHQNELKQIIKKQGK
	YYPFLLDAFEKDGKQINKIEGL
	LTFRVPYFVGPLVVPEDLQKSD
	NSENHWMVRKKKGEITPWNFDE
	MVDKDASGRKFIERLVGTDSYL
	LGEPTLPKNSLLYQEYEVLNEL
	NNVRLSVRTGNHWNDKRRMRLG
	REEKTLLCQRLFMKGQTVTKRT
	AENLLRKEYGRTYELSGLSDES
	KFTSSLSTYGKMCRIFGEKYVN
	EHRDLMEKIVELQTVFEDKETL
	LHQLRQLEGISEADCALLVNTH
	YTGWGRLSRKLLTTKAGECKIS
	DDFAPRKHSIIEIMRAEDRNLM
	EIITDKQLGFSDWIEQENLGAE
	NGSSLMEVVDDLRVSPKVKRGI
	IQSIRLIDDISKAVGKRPSRIF
	LELADDIQPSGRTISRKSRLQD
	LYRNANLGKEFKGIADELNACS
	DKDLQDDRLFLYYTQLGKDMYT
	GEELDLDRLSSAYDIDHIIPQA
	VTQNDSIDNRVLVARAENARKT
	DSFTYMPQIADRMRNFWQILLD
	NGLISRVKFERLTRQNEFSERE
	KERFVQRSLVETRQIMKNVATL
	MRQRYGNSAAVIGLNAELTKEM
	HRYLGFSHKNRDINDYHHAQDA
	LCVGIAGQFAANRGFFADGEVS
	DGAQNSYNQYLRDYLRGYREKL
	SAEDRKQGRAFGFIVGSMRSQD
	EQKRVNPRTGEVVWSEEDKDYL
	RKVMNYRKMLVTQKVGDDFGAL
	YDETRYAATDPKGIKGIPFDGA
	KQDTSLYGGFSSAKPAYAVLIE
	SKGKTRLVNVTMQEYSLLGDRP
	SDDELRKVLAKKKSEYAKANIL
	LRHVPKMQLIRYGGGLMVIKSA
	GELNNAQQLWLPYEEYCYFDDL
	SQGKGSLEKDDLKKLLDSILGS
	VQCLYPWHRFTEEELADLHVAF
	DKLPEDEKKNVITGIVSALHAD
	AKTANLSIVGMTGSWRRMNNKS
	GYTFSDEDEFIFQSPSGLFEKR
	VTVGELKRKAKKEVNSKYRTNE
	KRLPTLSGASQP (SEQ ID
	NO: 10)
Veillonella	METQTSNQLITSHLKDYPKQDY	185	339	155	574	663	79	574	663	79
atypica ACS-	FVGLDIGTNSVGWAVTNTSYEL
134-V-Col7a	LKFHSHKMWGSRLFEEGESAVT
gi|303229466|	RRGFRSMRRRLERRKLRLKLLE
ref|	ELFADAMAQVDSTFFIRLHESK
ZP_07316256.1	YHYEDKTTGHSSKHILFIDEDY
	TDQDYFTEYPTIYHLRKDLMEN
	GTDDIRKLFLAVHHILKYRGNF
	LYEGATFNSNAFTFEDVLKQAL
	VNITFNCFDTNSAISSISNILM
	ESGKTKSDKAKAIERLVDTYTV
	FDEVNTPDKPQKEQVKEDKKTL
	KAFANLVLGLSANLIDLFGSVE
	DIDDDLKKLQIVGDTYDEKRDE
	LAKVWGDEIHIIDDCKSVYDAI
	ILMSIKEPGLTISQSKVKAFDK
	HKEDLVILKSLLKLDRNVYNEM
	FKSDKKGLHNYVHYIKQGRTEE
	TSCSREDFYKYTKKIVEGLADS
	KDKEYILNEIELQTLLPLQRIK
	DNGVIPYQLHLEELKVILDKCG
	PKFPFLHTVSDGFSVTEKLIKM
	LEFRIPYYVGPLNTHHNIDNGG
	FSWAVRKQAGRVTPWNFEEKID
	REKSAAAFIKNLTNKCTYLFGE
	DVLPKSSLLYSEFMLLNELNNV
	RIDGKALAQGVKQHLIDSIFKQ
	DHKKMTKNRIELFLKDNNYITK
	KHKPEITGLDGEIKNDLTSYRD
	MVRILGNNFDVSMAEDIITDIT
	IFGESKKMLRQTLRNKFGSQLN
	DETIKKLSKLRYRDWGRLSKKL
	LKGIDGCDKAGNGAPKTIIELM
	RNDSYNLMEILGDKFSFMECIE
	EENAKLAQGQVVNPHDIIDELA
	LSPAVKRAVWQALRIVDEVAHI
	KKALPSRIFVEVARTNKSEKKK
	KDSRQKRLSDLYSAIKKDDVLQ
	SGLQDKEFGALKSGLANYDDAA
	LRSKKLYLYYTQMGRCAYTGNI
	IDLNQLNTDNYDIDHIYPRSLT
	KDDSFDNLVLCERTANAKKSDI
	YPIDNRIQTKQKPFWAFLKHQG
	LISERKYERLTRIAPLTADDLS
	GFIARQLVETNQSVKATTTLLR
	RLYPDIDVVFVKAENVSDFRHN
	NNFIKVRSLNHHHHAKDAYLNI
	VVGNVYHEKFTRNFRLFFKKNG
	ANRTYNLAKMFNYDVICTNAQD
	GKAWDVKTSMNTVKKMMASNDV
	RVTRRLLEQSGALADATIYKAS
	VAAKAKDGAYIGMKTKYSVFAD
	VTKYGGMTKIKNAYSIIVQYTG
	KKGEEIKEIVPLPIYLINRNAT
	DIELIDYVKSVIPKAKDISIKY
	RKLCINQLVKVNGFYYYLGGKT
	NDKIYIDNAIELVVPHDIATYI
	KLLDKYDLLRKENKTLKASSIT
	TSIYNINTSTVVSLNKVGIDVF
	DYFMSKLRTPLYMKMKGNKVDE
	LSSTGRSKFIKMTLEEQSIYLL
	EVLNLLTNSKTTFDVKPLGITG
	SRSTIGVKIHNLDEFKIINESI
	TGLYSNEVTIV (SEQ ID
	NO: 11)
Lactobacillus	MTKLNQPYGIGLDIGSNSIGFA	169	320	152	559	645	78	559	645	78
rhamnosus	VVDANSHLLRLKGETAIGARLF
GG	REGQSAADRRGSRTTRRRLSRT
gi|258509199|	RWRLSFLRDFFAPHITKIDPDF
ref|	FLRQKYSEISPKDKDRFKYEKR
YP_003171950.1	LENDRTDAEFYEDYPSMYHLRL
	HLMTHTHKADPREIFLAIHHIL
	KSRGHFLTPGAAKDFNTDKVDL
	EDIFPALTEAYAQVYPDLELTF
	DLAKADDFKAKLLDEQATPSDT
	QKALVNLLLSSDGEKEIVKKRK
	QVLTEFAKAITGLKTKFNLALG
	TEVDEADASNWQFSMGQLDDKW
	SNIETSMTDQGTEIFEQIQELY
	RARLLNGIVPAGMSLSQAKVAD
	YGQHKEDLELFKTYLKKLNDHE
	LAKTIRGLYDRYINGDDAKPFL
	REDFVKALTKEVTAHPNEVSEQ
	LLNRMGQANFMLKQRTKANGAI
	PIQLQQRELDQIIANQSKYYDW
	LAAPNPVEAHRWKMPYQLDELL
	NFHIPYYVGPLITPKQQAESGE
	NVFAWMVRKDPSGNITPYNFDE
	KVDREASANTFIQRMKTTDTYL
	IGEDVLPKQSLLYQKYEVLNEL
	NNVRINNECLGTDQKQRLIREV
	FERHSSVTIKQVADNLVAHGDF
	ARRPEIRGLADEKRFLSSLSTY
	HQLKEILHEAIDDPTKLLDIEN
	IITWSTVFEDHTIFETKLAEIE
	WLDPKKINELSGIRYRGWGQFS
	RKLLDGLKLGNGHTVIQELMLS
	NHNLMQILADETLKETMTELNQ
	DKLKTDDIEDVINDAYTSPSNK
	KALRQVLRVVEDIKHAANGQDP
	SWLFIETADGTGTAGKRTQSRQ
	KQIQTVYANAAQELIDSAVRGE
	LEDKIADKASFTDRLVLYFMQG
	GRDIYTGAPLNIDQLSHYDIDH
	ILPQSLIKDDSLDNRVLVNATI
	NREKNNVFASTLFAGKMKATWR
	KWHEAGLISGRKLRNLMLRPDE
	IDKFAKGFVARQLVETRQIIKL
	TEQIAAAQYPNTKIIAVKAGLS
	HQLREELDFPKNRDVNHYHHAF
	DAFLAARIGTYLLKRYPKLAPF
	FTYGEFAKVDVKKFREFNFIGA
	LTHAKKNIIAKDTGEIVWDKER
	DIRELDRIYNFKRMLITHEVYF
	ETADLFKQTIYAAKDSKERGGS
	KQLIPKKQGYPTQVYGGYTQES
	GSYNALVRVAEADTTAYQVIKI
	SAQNASKIASANLKSREKGKQL
	LNEIVVKQLAKRRKNWKPSANS
	FKIVIPRFGMGTLFQNAKYGLF
	MVNSDTYYRNYQELWLSRENQK
	LLKKLFSIKYEKTQMNHDALQV
	YKAIIDQVEKFFKLYDINQFRA
	KLSDAIERFEKLPINTDGNKIG
	KTETLRQILIGLQANGTRSNVK
	NLGIKTDLGLLQVGSGIKLDKD
	TQIVYQSPSGLFKRRIPLADL
	(SEQ ID NO: 12)
Filifactor	MTKEYYLGLDVGTNSVGWAVTD	166	314	149	508	592	76	508	592	76
alocis	SQYNLCKFKKKDMWGIRLFESA
ATCC 35896	NTAKDRRLQRGNRRRLERKKQR
gi|374307738|	IDLLQEIFSPEICKIDPTFFIR
ref|	LNESRLHLEDKSNDFKYPLFIE
YP_005054169.1	KDYSDIEYYKEFPTIFHLRKHL
	IESEEKQDIRLIYLALHNIIKT
	RGHFLIDGDLQSAKQLRPILDT
	FLLSLQEEQNLSVSLSENQKDE
	YEEILKNRSIAKSEKVKKLKNL
	FEISDELEKEEKKAQSAVIENF
	CKFIVGNKGDVCKFLRVSKEEL
	EIDSFSFSEGKYEDDIVKNLEE
	KVPEKVYLFEQMKAMYDWNILV
	DILETEEYISFAKVKQYEKHKT
	NLRLLRDIILKYCTKDEYNRMF
	NDEKEAGSYTAYVGKLKKNNKK
	YWIEKKRNPEEFYKSLGKLLDK
	IEPLKEDLEVLTMMIEECKNHT
	LLPIQKNKDNGVIPHQVHEVEL
	KKILENAKKYYSFLTETDKDGY
	SVVQKIESIFRFRIPYYVGPLS
	TRHQEKGSNVWMVRKPGREDRI
	YPWNMEEIIDFEKSNENFITRM
	TNKCTYLIGEDVLPKHSLLYSK
	YMVLNELNNVKVRGKKLPTSLK
	QKVFEDLFENKSKVTGKNLLEY
	LQIQDKDIQIDDLSGFDKDFKT
	SLKSYLDFKKQIFGEEIEKESI
	QNMIEDIIKWITIYGNDKEMLK
	RVIRANYSNQLTEEQMKKITGF
	QYSGWGNFSKMFLKGISGSDVS
	TGETFDIITAMWETDNNLMQIL
	SKKFTFMDNVEDENSGKVGKID
	KITYDSTVKEMFLSPENKRAVW
	QTIQVAEEIKKVMGCEPKKIFI
	EMARGGEKVKKRTKSRKAQLLE
	LYAACEEDCRELIKEIEDRDER
	DFNSMKLFLYYTQFGKCMYSGD
	DIDINELIRGNSKWDRDHIYPQ
	SKIKDDSIDNLVLVNKTYNAKK
	SNELLSEDIQKKMHSFWLSLLN
	KKLITKSKYDRLTRKGDFTDEE
	LSGFIARQLVETRQSTKAIADI
	FKQIYSSEVVYVKSSLVSDFRK
	KPLNYLKSRRVNDYHHAKDAYL
	NIVVGNVYNKKFTSNPIQWMKK
	NRDTNYSLNKVFEHDVVINGEV
	IWEKCTYHEDTNTYDGGTLDRI
	RKIVERDNILYTEYAYCEKGEL
	FNATIQNKNGNSTVSLKKGLDV
	KKYGGYFSANTSYFSLIEFEDK
	KGDRARHIIGVPIYIANMLEHS
	PSAFLEYCEQKGYQNVRILVEK
	IKKNSLLIINGYPLRIRGENEV
	DTSFKRAIQLKLDQKNYELVRN
	IEKFLEKYVEKKGNYPIDENRD
	HITHEKMNQLYEVLLSKMKKEN
	KKGMADPSDRIEKSKPKFIKLE
	DLIDKINVINKMLNLLRCDNDT
	KADLSLIELPKNAGSFVVKKNT
	IGKSKIILVNQSVTGLYENRRE
	L (SEQ ID NO: 13)
Oenococcus	MARDYSVGLDIGTSSVGWAAID	169	317	149	555	639	80	555	639	80
kitaharae	NKYHLIRAKSKNLIGVRLFDSA
DSM 17330	VTAEKRRGYRTTRRRLSRRHWR
gi|366983953|	LRLLNDIFAGPLTDFGDENFLA
gb|EHN59352.1|	RLKYSWVHPQDQSNQAHFAAGL
	LFDSKEQDKDFYRKYPTIYHLR
	LALMNDDQKHDLREVYLAIHHL
	VKYRGHFLIEGDVKADSAFDVH
	TFADAIQRYAESNNSDENLLGK
	IDEKKLSAALTDKHGSKSQRAE
	TAETAFDILDLQSKKQIQAILK
	SVVGNQANLMAIFGLDSSAISK
	DEQKNYKFSFDDADIDEKIADS
	EALLSDTEFEFLCDLKAAFDGL
	TLKMLLGDDKTVSAAMVRRENE
	HQKDWEYIKSHIRNAKNAGNGL
	YEKSKKFDGINAAYLALQSDNE
	DDRKKAKKIFQDEISSADIPDD
	VKADFLKKIDDDQFLPIQRTKN
	NGTIPHQLHRNELEQIIEKQGI
	YYPFLKDTYQENSHELNKITAL
	INFRVPYYVGPLVEEEQKIADD
	GKNIPDPTNHWMVRKSNDTITP
	WNLSQVVDLDKSGRRFIERLTG
	TDTYLIGEPTLPKNSLLYQKED
	VLQELNNIRVSGRRLDIRAKQD
	AFEHLFKVQKTVSATNLKDFLV
	QAGYISEDTQIEGLADVNGKNF
	NNALTTYNYLVSVLGREFVENP
	SNEELLEEITELQTVFEDKKVL
	RRQLDQLDGLSDHNREKLSRKH
	YTGWGRISKKLLTTKIVQNADK
	IDNQTFDVPRMNQSIIDTLYNT
	KMNLMEIINNAEDDFGVRAWID
	KQNTTDGDEQDVYSLIDELAGP
	KEIKRGIVQSFRILDDITKAVG
	YAPKRVYLEFARKTQESHLTNS
	RKNQLSTLLKNAGLSELVTQVS
	QYDAAALQNDRLYLYFLQQGKD
	MYSGEKLNLDNLSNYDIDHIIP
	QAYTKDNSLDNRVLVSNITNRR
	KSDSSNYLPALIDKMRPFWSVL
	SKQGLLSKHKFANLTRTRDFDD
	MEKERFIARSLVETRQIIKNVA
	SLIDSHFGGETKAVAIRSSLTA
	DMRRYVDIPKNRDINDYHHAFD
	ALLFSTVGQYTENSGLMKKGQL
	SDSAGNQYNRYIKEWIHAARLN
	AQSQRVNPFGFVVGSMRNAAPG
	KLNPETGEITPEENADWSIADL
	DYLHKVMNFRKITVTRRLKDQK
	GQLYDESRYPSVLHDAKSKASI
	NFDKHKPVDLYGGFSSAKPAYA
	ALIKFKNKFRLVNVLRQWTYSD
	KNSEDYILEQIRGKYPKAEMVL
	SHIPYGQLVKKDGALVTISSAT
	ELHNFEQLWLPLADYKLINTLL
	KTKEDNLVDILHNRLDLPEMTI
	ESAFYKAFDSILSFAFNRYALH
	QNALVKLQAHRDDFNALNYEDK
	QQTLERILDALHASPASSDLKK
	INLSSGFGRLFSPSHFTLADTD
	EFIFQSVTGLFSTQKTVAQLYQ
	ETK (SEQ ID NO: 14)
Fructobacillus	MVYDVGLDIGTGSVGWVALDEN	168	314	147	488	571	76	488	571	76
fructosus	GKLARAKGKNLVGVRLFDTAQT
KCTC 3544	AADRRGFRTTRRRLSRRKWRLR
gi|339625081|	LLDELFSAEINEIDSSFFQRLK
ref|	YSYVHPKDEENKAHYYGGYLFP
ZP_08660870.1	TEEETKKFHRSYPTIYHLRQEL
	MAQPNKRFDIREIYLAIHHLVK
	YRGHFLSSQEKITIGSTYNPED
	LANAIEVYADEKGLSWELNNPE
	QLTEIISGEAGYGLNKSMKADE
	ALKLFEFDNNQDKVAIKTLLAG
	LTGNQIDFAKLFGKDISDKDEA
	KLWKLKLDDEALEEKSQTILSQ
	LTDEEIELFHAVVQAYDGFVLI
	GLLNGADSVSAAMVQLYDQHRE
	DRKLLKSLAQKAGLKHKRFSEI
	YEQLALATDEATIKNGISTARE
	LVEESNLSKEVKEDTLRRLDEN
	EFLPKQRTKANSVIPHQLHLAE
	LQKILQNQGQYYPFLLDTFEKE
	DGQDNKIEELLRFRIPYYVGPL
	VTKKDVEHAGGDADNHWVERNE
	GFEKSRVTPWNFDKVFNRDKAA
	RDFIERLTGNDTYLIGEKTLPQ
	NSLRYQLFTVLNELNNVRVNGK
	KFDSKTKADLINDLFKARKTVS
	LSALKDYLKAQGKGDVTITGLA
	DESKFNSSLSSYNDLKKTFDAE
	YLENEDNQETLEKIIEIQTVFE
	DSKIASRELSKLPLDDDQVKKL
	SQTHYTGWGRLSEKLLDSKIID
	ERGQKVSILDKLKSTSQNFMSI
	INNDKYGVQAWITEQNTGSSKL
	TFDEKVNELTTSPANKRGIKQS
	FAVLNDIKKAMKEEPRRVYLEF
	AREDQTSVRSVPRYNQLKEKYQ
	SKSLSEEAKVLKKTLDGNKNKM
	SDDRYFLYFQQQGKDMYTGRPI
	NFERLSQDYDIDHIIPQAFTKD
	DSLDNRVLVSRPENARKSDSFA
	YTDEVQKQDGSLWTSLLKSGFI
	NRKKYERLTKAGKYLDGQKTGF
	IARQLVETRQIIKNVASLIEGE
	YENSKAVAIRSEITADMRLLVG
	IKKHREINSFHHAFDALLITAA
	GQYMQNRYPDRDSTNVYNEFDR
	YTNDYLKNLRQLSSRDEVRRLK
	SFGFVVGTMRKGNEDWSEENTS
	YLRKVMMFKNILTTKKTEKDRG
	PLNKETIFSPKSGKKLIPLNSK
	RSDTALYGGYSNVYSAYMTLVR
	ANGKNLLIKIPISIANQIEVGN
	LKINDYIVNNPAIKKFEKILIS
	KLPLGQLVNEDGNLIYLASNEY
	RHNAKQLWLSTTDADKIASISE
	NSSDEELLEAYDILTSENVKNR
	FPFFKKDIDKLSQVRDEFLDSD
	KRIAVIQTILRGLQIDAAYQAP
	VKIISKKVSDWHKLQQSGGIKL
	SDNSEMIYQSATGIFETRVKIS
	DLL (SEQ ID NO: 15)
Catenibacterium	IVDYCIGLDLGTGSVGWAVVDM	173	318	146	511	594	78	511	594	78
mitsuokai	NHRLMKRNGKHLWGSRLFSNAE
DSM 15897	TAANRRASRSIRRRYNKRRERI
gi|224543312|	RLLRAILQDMVLEKDPTFFIRL
ref|	EHTSFLDEEDKAKYLGTDYKDN
ZP_03683851.1	YNLFIDEDENDYTYYHKYPTIY
	HLRKALCESTEKADPRLIYLAL
	HHIVKYRGNFLYEGQKFNMDAS
	NIEDKLSDIFTQFTSFNNIPYE
	DDEKKNLEILEILKKPLSKKAK
	VDEVMTLIAPEKDYKSAFKELV
	TGIAGNKMNVTKMILCEPIKQG
	DSEIKLKFSDSNYDDQFSEVEK
	DLGEYVEFVDALHNVYSWVELQ
	TIMGATHTDNASISEAMVSRYN
	KHHDDLKLLKDCIKNNVPNKYF
	DMFRNDSEKSKGYYNYINRPSK
	APVDEFYKYVKKCIEKVDTPEA
	KQILNDIELENFLLKQNSRING
	SVPYQMQLDEMIKIIDNQAEYY
	PILKEKREQLLSILTFRIPYYF
	GPLNETSEHAWIKRLEGKENQR
	ILPWNYQDIVDVDATAEGFIKR
	MRSYCTYFPDEEVLPKNSLIVS
	KYEVYNELNKIRVDDKLLEVDV
	KNDIYNELFMKNKTVTEKKLKN
	WLVNNQCCSKDAEIKGFQKENQ
	FSTSLTPWIDFTNIFGKIDQSN
	FDLIENIIYDLTVFEDKKIMKR
	RLKKKYALPDDKVKQILKLKYK
	DWSRLSKKLLDGIVADNRFGSS
	VTVLDVLEMSRLNLMEIINDKD
	LGYAQMIEEATSCPEDGKFTYE
	EVERLAGSPALKRGIWQSLQIV
	EEITKVMKCRPKYIYIEFERSE
	EAKERTESKIKKLENVYKDLDE
	QTKKEYKSVLEELKGFDNTKKI
	SSDSLFLYFTQLGKCMYSGKKL
	DIDSLDKYQIDHIVPQSLVKDD
	SFDNRVLVVPSENQRKLDDLVV
	PFDIRDKMYRFWKLLFDHELIS
	PKKFYSLIKTEYTERDEERFIN
	RQLVETRQITKNVTQIIEDHYS
	TTKVAAIRANLSHEFRVKNHIY
	KNRDINDYHHAHDAYIVALIGG
	FMRDRYPNMHDSKAVYSEYMKM
	FRKNKNDQKRWKDGFVINSMNY
	PYEVDGKLIWNPDLINEIKKCF
	YYKDCYCTTKLDQKSGQLFNLT
	VLSNDAHADKGVTKAVVPVNKN
	RSDVHKYGGFSGLQYTIVAIEG
	QKKKGKKTELVKKISGVPLHLK
	AASINEKINYIEEKEGLSDVRI
	IKDNIPVNQMIEMDGGEYLLTS
	PTEYVNARQLVLNEKQCALIAD
	IYNAIYKQDYDNLDDILMIQLY
	IELTNKMKVLYPAYRGIAEKFE
	SMNENYVVISKEEKANIIKQML
	IVMHRGPQNGNIVYDDFKISDR
	IGRLKTKNHNLNNIVFISQSPT
	GIYTKKYKL (SEQ ID NO:
	16)
Finegoldia	MKSEKKYYIGLDVGTNSVGWAV	168	313	146	452	534	77	452	534	77
magna	TDEFYNILRAKGKDLWGVRLFE
ATCC 29328	KADTAANTRIFRSGRRRNDRKG
gi|169823755|	MRLQILREIFEDEIKKVDKDFY
ref|	DRLDESKFWAEDKKVSGKYSLF
YP_001691366.1	NDKNFSDKQYFEKFPTIFHLRK
	YLMEEHGKVDIRYYFLAINQMM
	KRRGHFLIDGQISHVTDDKPLK
	EQLILLINDLLKIELEEELMDS
	IFEILADVNEKRTDKKNNLKEL
	IKGQDFNKQEGNILNSIFESIV
	TGKAKIKNIISDEDILEKIKED
	NKEDFVLTGDSYEENLQYFEEV
	LQENITLFNTLKSTYDFLILQS
	ILKGKSTLSDAQVERYDEHKKD
	LEILKKVIKKYDEDGKLFKQVF
	KEDNGNGYVSYIGYYLNKNKKI
	TAKKKISNIEFTKYVKGILEKQ
	CDCEDEDVKYLLGKIEQENFLL
	KQISSINSVIPHQIHLFELDKI
	LENLAKNYPSFNNKKEEFTKIE
	KIRKTFTFRIPYYVGPLNDYHK
	NNGGNAWIFRNKGEKIRPWNFE
	KIVDLHKSEEEFIKRMLNQCTY
	LPEETVLPKSSILYSEYMVLNE
	LNNLRINGKPLDTDVKLKLIEE
	LFKKKTKVTLKSIRDYMVRNNF
	ADKEDFDNSEKNLEIASNMKSY
	IDFNNILEDKEDVEMVEDLIEK
	ITIHTGNKKLLKKYIEETYPDL
	SSSQIQKIINLKYKDWGRLSRK
	LLDGIKGTKKETEKTDTVINFL
	RNSSDNLMQIIGSQNYSFNEYI
	DKLRKKYIPQEISYEVVENLYV
	SPSVKKMIWQVIRVTEEITKVM
	GYDPDKIFIEMAKSEEEKKTTI
	SRKNKLLDLYKAIKKDERDSQY
	EKLLTGLNKLDDSDLRSRKLYL
	YYTQMGRDMYTGEKIDLDKLFD
	STHYDKDHIIPQSMKKDDSIIN
	NLVLVNKNANQTTKGNIYPVPS
	SIRNNPKIYNYWKYLMEKEFIS
	KEKYNRLIRNTPLTNEELGGFI
	NRQLVETRQSTKAIKELFEKFY
	QKSKIIPVKASLASDLRKDMNT
	LKSREVNDLHHAHDAFLNIVAG
	DVWNREFTSNPINYVKENREGD
	KVKYSLSKDFTRPRKSKGKVIW
	TPEKGRKLIVDTLNKPSVLISN
	ESHVKKGELFNATIAGKKDYKK
	GKIYLPLKKDDRLQDVSKYGGY
	KAINGAFFFLVEHTKSKKRIRS
	IELFPLHLLSKFYEDKNTVLDY
	AINVLQLQDPKIIIDKINYRTE
	IIIDNFSYLISTKSNDGSITVK
	PNEQMYWRVDEISNLKKIENKY
	KKDAILTEEDRKIMESYIDKIY
	QQFKAGKYKNRRTTDTIIEKYE
	IIDLDTLDNKQLYQLLVAFISL
	SYKTSNNAVDFTVIGLGTECGK
	PRITNLPDNTYLVYKSITGIYE
	KRIRIK (SEQ ID NO: 17)
Coriobacterium	MKLRGIEDDYSIGLDMGTSSVG	175	318	144	511	592	82	511	592	82
glomeran	WAVTDERGTLAHFKRKPTWGSR
sPW2	LFREAQTAAVARMPRGQRRRYV
gi|328956315|	RRRWRLDLLQKLFEQQMEQADP
ref|	DFFIRLRQSRLLRDDRAEEHAD
YP_004373648.1	YRWPLFNDCKFTERDYYQRFPT
	IYHVRSWLMETDEQADIRLIYL
	ALHNIVKHRGNFLREGQSLSAK
	SARPDEALNHLRETLRVWSSER
	GFECSIADNGSILAMLTHPDLS
	PSDRRKKIAPLFDVKSDDAAAD
	KKLGIALAGAVIGLKTEFKNIF
	GDFPCEDSSIYLSNDEAVDAVR
	SACPDDCAELFDRLCEVYSAYV
	LQGLLSYAPGQTISANMVEKYR
	RYGEDLALLKKLVKIYAPDQYR
	MFFSGATYPGTGIYDAAQARGY
	TKYNLGPKKSEYKPSESMQYDD
	FRKAVEKLFAKTDARADERYRM
	MMDRFDKQQFLRRLKTSDNGSI
	YHQLHLEELKAIVENQGRFYPF
	LKRDADKLVSLVSFRIPYYVGP
	LSTRNARTDQHGENRFAWSERK
	PGMQDEPIFPWNWESIIDRSKS
	AEKFILRMTGMCTYLQQEPVLP
	KSSLLYEEFCVLNELNGAHWSI
	DGDDEHRFDAADREGIIEELFR
	RKRTVSYGDVAGWMERERNQIG
	AHVCGGQGEKGFESKLGSYIFF
	CKDVFKVERLEQSDYPMIERII
	LWNTLFEDRKILSQRLKEEYGS
	RLSAEQIKTICKKRFTGWGRLS
	EKFLTGITVQVDEDSVSIMDVL
	REGCPVSGKRGRAMVMMEILRD
	EELGFQKKVDDENRAFFAENAQ
	ALGVNELPGSPAVRRSLNQSIR
	IVDEIASIAGKAPANIFIEVTR
	DEDPKKKGRRTKRRYNDLKDAL
	EAFKKEDPELWRELCETAPNDM
	DERLSLYFMQRGKCLYSGRAID
	IHQLSNAGIYEVDHIIPRTYVK
	DDSLENKALVYREENQRKTDML
	LIDPEIRRRMSGYWRMLHEAKL
	IGDKKFRNLLRSRIDDKALKGF
	IARQLVETGQMVKLVRSLLEAR
	YPETNIISVKASISHDLRTAAE
	LVKCREANDFHHAHDAFLACRV
	GLFIQKRHPCVYENPIGLSQVV
	RNYVRQQADIFKRCRTIPGSSG
	FIVNSFMTSGFDKETGEIFKDD
	WDAEAEVEGIRRSLNFRQCFIS
	RMPFEDHGVFWDATIYSPRAKK
	TAALPLKQGLNPSRYGSFSREQ
	FAYFFIYKARNPRKEQTLFEFA
	QVPVRLSAQIRQDENALERYAR
	ELAKDQGLEFIRIERSKILKNQ
	LIEIDGDRLCITGKEEVRNACE
	LAFAQDEMRVIRMLVSEKPVSR
	ECVISLFNRILLHGDQASRRLS
	KQLKLALLSEAFSEASDNVQRN
	VVLGLIAIFNGSTNMVNLSDIG
	GSKFAGNVRIKYKKELASPKVN
	VHLIDQSVTGMFERRTKIGL
	(SEQ ID NO: 18)
Eubacterium	MENKQYYIGLDVGTNSVGWAVT	169	310	142	552	633	76	552	633	76
yurii	DTSYNLLRAKGKDMWGARLFEK
ATCC 43715	ANTAAERRTKRTSRRRSEREKA
gi|306821691|	RKAMLKELFADEINRVDPSFFI
ref|	RLEESKFFLDDRSENNRQRYTL
ZP_07455288.1	FNDATFTDKDYYEKYKTIFHLR
	SALINSDEKFDVRLVFLAILNL
	FSHRGHFLNASLKGDGDIQGMD
	VFYNDLVESCEYFEIELPRITN
	IDNFEKILSQKGKSRTKILEEL
	SEELSISKKDKSKYNLIKLISG
	LEASVVELYNIEDIQDENKKIK
	IGFRESDYEESSLKVKEIIGDE
	YFDLVERAKSVHDMGLLSNIIG
	NSKYLCEARVEAYENHHKDLLK
	IKELLKKYDKKAYNDMFRKMTD
	KNYSAYVGSVNSNIAKERRSVD
	KRKIEDLYKYIEDTALKNIPDD
	NKDKIEILEKIKLGEFLKKQLT
	ASNGVIPNQLQSRELRAILKKA
	ENYLPFLKEKGEKNLTVSEMII
	QLFEFQIPYYVGPLDKNPKKDN
	KANSWAKIKQGGRILPWNFEDK
	VDVKGSRKEFIEKMVRKCTYIS
	DEHTLPKQSLLYEKFMVLNEIN
	NIKIDGEKISVEAKQKIYNDLF
	VKGKKVSQKDIKKELISLNIMD
	KDSVLSGTDTVCNAYLSSIGKF
	TGVFKEEINKQSIVDMIEDIIF
	LKTVYGDEKRFVKEEIVEKYGD
	EIDKDKIKRILGFKFSNWGNLS
	KSFLELEGADVGTGEVRSIIQS
	LWETNFNLMELLSSRFTYMDEL
	EKRVKKLEKPLSEWTIEDLDDM
	YLSSPVKRMIWQSMKIVDEIQT
	VIGYAPKRIFVEMTRSEGEKVR
	TKSRKDRLKELYNGIKEDSKQW
	VKELDSKDESYFRSKKMYLYYL
	QKGRCMYSGEVIELDKLMDDNL
	YDIDHIYPRSFVKDDSLDNLVL
	VKKEINNRKQNDPITPQIQASC
	QGFWKILHDQGFMSNEKYSRLT
	RKTQEFSDEEKLSFINRQIVET
	GQATKCMAQILQKSMGEDVDVV
	FSKARLVSEFRHKFELFKSRLI
	NDFHHANDAYLNIVVGNSYFVK
	FTRNPANFIKDARKNPDNPVYK
	YHMDRFFERDVKSKSEVAWIGQ
	SEGNSGTIVIVKKTMAKNSPLI
	TKKVEEGHGSITKETIVGVKEI
	KFGRNKVEKADKTPKKPNLQAY
	RPIKTSDERLCNILRYGGRTSI
	SISGYCLVEYVKKRKTIRSLEA
	IPVYLGRKDSLSEEKLLNYFRY
	NLNDGGKDSVSDIRLCLPFIST
	NSLVKIDGYLYYLGGKNDDRIQ
	LYNAYQLKMKKEEVEYIRKIEK
	AVSMSKFDEIDREKNPVLTEEK
	NIELYNKIQDKFENTVFSKRMS
	LVKYNKKDLSFGDFLKNKKSKF
	EEIDLEKQCKVLYNIIFNLSNL
	KEVDLSDIGGSKSTGKCRCKKN
	ITNYKEFKLIQQSITGLYSCEK
	DLMTI (SEQ ID NO: 19)
Peptoniphilus	MKNLKEYYIGLDIGTASVGWAV	171	311	141	535	615	76	535	615	76
duerdenii	TDESYNIPKFNGKKMWGVRLFD
ATCC BAA-	DAKTAEERRTQRGSRRRLNRRK
1640	ERINLLQDLFATEISKVDPNFF
gi|304438954|	LRLDNSDLYREDKDEKLKSKYT
ref|	LFNDKDFKDRDYHKKYPTIHHL
ZP_07398877.1	IMDLIEDEGKKDIRLLYLACHY
	LLKNRGHFIFEGQKFDTKNSFD
	KSINDLKIHLRDEYNIDLEFNN
	EDLIEIITDTTLNKTNKKKELK
	NIVGDTKFLKAISAIMIGSSQK
	LVDLFEDGEFEETTVKSVDFST
	TAFDDKYSEYEEALGDTISLLN
	ILKSIYDSSILENLLKDADKSK
	DGNKYISKAFVKKFNKHGKDLK
	TLKRIIKKYLPSEYANIFRNKS
	INDNYVAYTKSNITSNKRTKAS
	KFTKQEDFYKFIKKHLDTIKET
	KLNSSENEDLKLIDEMLTDIEF
	KTFIPKLKSSDNGVIPYQLKLM
	ELKKILDNQSKYYDFLNESDEY
	GTVKDKVESIMEFRIPYYVGPL
	NPDSKYAWIKRENTKITPWNFK
	DIVDLDSSREEFIDRLIGRCTY
	LKEEKVLPKASLIYNEFMVLNE
	LNNLKLNEFLITEEMKKAIFEE
	LFKTKKKVTLKAVSNLLKKEFN
	LTGDILLSGTDGDFKQGLNSYI
	DFKNIIGDKVDRDDYRIKIEEI
	IKLIVLYEDDKTYLKKKIKSAY
	KNDFTDDEIKKIAALNYKDWGR
	LSKRFLTGIEGVDKTTGEKGSI
	IYFMREYNLNLMELMSGHYTFT
	EEVEKLNPVENRELCYEMVDEL
	YLSPSVKRMLWQSLRVVDEIKR
	IIGKDPKKIFIEMARAKEAKNS
	RKESRKNKLLEFYKFGKKAFIN
	EIGEERYNYLLNEINSEEESKF
	RWDNLYLYYTQLGRCMYSLEPI
	DLADLKSNNIYDQDHIYPKSKI
	YDDSLENRVLVKKNLNHEKGNQ
	YPIPEKVLNKNAYGFWKILFDK
	GLIGQKKYTRLTRRTPFEEREL
	AEFIERQIVETRQATKETANLL
	KNICQDSEIVYSKAENASRFRQ
	EFDIIKCRTVNDLHHMHDAYLN
	IVVGNVYNTKFTKNPLNFIKDK
	DNVRSYNLENMFKYDVVRGSYT
	AWIADDSEGNVKAATIKKVKRE
	LEGKNYRFTRMSYIGTGGLYDQ
	NLMRKGKGQIPQKENTNKSNIE
	KYGGYNKASSAYFALIESDGKA
	GRERTLETIPIMVYNQEKYGNT
	EAVDKYLKDNLELQDPKILKDK
	IKINSLIKLDGFLYNIKGKTGD
	SLSIAGSVQLIVNKEEQKLIKK
	MDKFLVKKKDNKDIKVTSFDNI
	KEEELIKLYKTLSDKLNNGIYS
	NKRNNQAKNISEALDKFKEISI
	EEKIDVLNQIILLFQSYNNGCN
	LKSIGLSAKTGVVFIPKKLNYK
	ECKLINQSITGLFENEVDLLNL
	(SEQ ID NO: 20)
Acidaminococcus	MGKMYYLGLDIGTNSVGYAVTD	167	306	140	511	591	75	511	591	75
sp. D21	PSYHLLKFKGEPMWGAHVFAAG
gi|227824983|	NQSAERRSFRTSRRRLDRRQQR
ref|	VKLVQEIFAPVISPIDPRFFIR
ZP_03989815.1	LHESALWRDDVAETDKHIFFND
	PTYTDKEYYSDYPTIHHLIVDL
	MESSEKHDPRLVYLAVAWLVAH
	RGHFLNEVDKDNIGDVLSFDAF
	YPEFLAFLSDNGVSPWVCESKA
	LQATLLSRNSVNDKYKALKSLI
	FGSQKPEDNFDANISEDGLIQL
	LAGKKVKVNKLFPQESNDASFT
	LNDKEDAIEEILGTLTPDECEW
	IAHIRRLFDWAIMKHALKDGRT
	ISESKVKLYEQHHHDLTQLKYF
	VKTYLAKEYDDIFRNVDSETTK
	NYVAYSYHVKEVKGTLPKNKAT
	QEEFCKYVLGKVKNIECSEADK
	VDFDEMIQRLTDNSFMPKQVSG
	ENRVIPYQLYYYELKTILNKAA
	SYLPFLTQCGKDAISNQDKLLS
	IMTFRIPYFVGPLRKDNSEHAW
	LERKAGKIYPWNFNDKVDLDKS
	EEAFIRRMTNTCTYYPGEDVLP
	LDSLIYEKFMILNEINNIRIDG
	YPISVDVKQQVFGLFEKKRRVT
	VKDIQNLLLSLGALDKHGKLTG
	IDTTIHSNYNTYHHFKSLMERG
	VLTRDDVERIVERMTYSDDTKR
	VRLWLNNNYGTLTADDVKHISR
	LRKHDFGRLSKMFLTGLKGVHK
	ETGERASILDFMWNTNDNLMQL
	LSECYTFSDEITKLQEAYYAKA
	QLSLNDFLDSMYISNAVKRPIY
	RTLAVVNDIRKACGTAPKRIFI
	EMARDGESKKKRSVTRREQIKN
	LYRSIRKDFQQEVDFLEKILEN
	KSDGQLQSDALYLYFAQLGRDM
	YTGDPIKLEHIKDQSFYNIDHI
	YPQSMVKDDSLDNKVLVQSEIN
	GEKSSRYPLDAAIRNKMKPLWD
	AYYNHGLISLKKYQRLTRSTPF
	TDDEKWDFINRQLVETRQSTKA
	LAILLKRKFPDTEIVYSKAGLS
	SDFRHEFGLVKSRNINDLHHAK
	DAFLAIVTGNVYHERFNRRWFM
	VNQPYSVKTKTLFTHSIKNGNF
	VAWNGEEDLGRIVKMLKQNKNT
	IHFTRFSFDRKEGLFDIQPLKA
	STGLVPRKAGLDVVKYGGYDKS
	TAAYYLLVRFTLEDKKTQHKLM
	MIPVEGLYKARIDHDKEFLTDY
	AQTTISEILQKDKQKVINIMFP
	MGTRHIKLNSMISIDGFYLSIG
	GKSSKGKSVLCHAMVPLIVPHK
	IECYIKAMESFARKFKENNKLR
	IVEKFDKITVEDNLNLYELFLQ
	KLQHNPYNKFFSTQFDVLINGR
	STFTKLSPEEQVQTLLNILSIF
	KTCRSSGCDLKSINGSAQAARI
	MISADLTGLSKKYSDIRLVEQS
	ASGLFVSKSQNLLEYL (SEQ
	ID NO: 21)
Lactobacillus	MTKKEQPYNIGLDIGTSSVGWA	171	310	140	542	621	85	542	621	85
farciminis	VTNDNYDLLNIKKKNLWGVRLF
KCTC 3681	EEAQTAKETRLNRSTRRRYRRR
gi|336394882|	KNRINWLNEIFSEELAKTDPSF
ref|	LIRLQNSWVSKKDPDRKRDKYN
ZP_08576281.1	LFIDGPYTDKEYYREFPTIFHL
	RKELILNKDKADIRLIYLALHN
	ILKYRGNFTYEHQKFNISNLNN
	NLSKELIELNQQLIKYDISFPD
	DCDWNHISDILIGRGNATQKSS
	NILKDFTLDKETKKLLKEVINL
	ILGNVAHLNTIFKTSLTKDEEK
	LNFSGKDIESKLDDLDSILDDD
	QFTVLDAANRIYSTITLNEILN
	GESYFSMAKVNQYENHAIDLCK
	LRDMWHTTKNEEAVEQSRQAYD
	DYINKPKYGTKELYTSLKKFLK
	VALPTNLAKEAEEKISKGTYLV
	KPRNSENGVVPYQLNKIEMEKI
	IDNQSQYYPFLKENKEKLLSIL
	SFRIPYYVGPLQSAEKNPFAWM
	ERKSNGHARPWNFDEIVDREKS
	SNKFIRRMTVTDSYLVGEPVLP
	KNSLIYQRYEVLNELNNIRITE
	NLKTNPIGSRLTVETKQRIYNE
	LFKKYKKVTVKKLTKWLIAQGY
	YKNPILIGLSQKDEFNSTLTTY
	LDMKKIFGSSFMEDNKNYDQIE
	ELIEWLTIFEDKQILNEKLHSS
	KYSYTPDQIKKISNMRYKGWGR
	LSKKILMDITTETNTPQLLQLS
	NYSILDLMWATNNNFISIMSND
	KYDFKNYIENHNLNKNEDQNIS
	DLVNDIHVSPALKRGITQSIKI
	VQEIVKFMGHAPKHIFIEVTRE
	TKKSEITTSREKRIKRLQSKLL
	NKANDFKPQLREYLVPNKKIQE
	ELKKHKNDLSSERIMLYFLQNG
	KSLYSEESLNINKLSDYQVDHI
	LPRTYIPDDSLENKALVLAKEN
	QRKADDLLLNSNVIDRNLERWT
	YMLNNNMIGLKKFKNLTRRVIT
	DKDKLGFIHRQLVQTSQMVKGV
	ANILDNMYKNQGTTCIQARANL
	STAFRKALSGQDDTYHFKHPEL
	VKNRNVNDFHHAQDAYLASFLG
	TYRLRRFPTNEMLLMNGEYNKF
	YGQVKELYSKKKKLPDSRKNGF
	IISPLVNGTTQYDRNTGEIIWN
	VGFRDKILKIFNYHQCNVTRKT
	EIKTGQFYDQTIYSPKNPKYKK
	LIAQKKDMDPNIYGGFSGDNKS
	SITIVKIDNNKIKPVAIPIRLI
	NDLKDKKTLQNWLEENVKHKKS
	IQIIKNNVPIGQIIYSKKVGLL
	SLNSDREVANRQQLILPPEHSA
	LLRLLQIPDEDLDQILAFYDKN
	ILVEILQELITKMKKFYPFYKG
	EREFLIANIENFNQATTSEKVN
	SLEELITLLHANSTSAHLIFNN
	IEKKAFGRKTHGLTLNNTDFIY
	QSVTGLYETRIHIE (SEQ ID
	NO: 22)
Streptococcus	MTKFNKNYSIGLDIGVSSVGYA	185	324	140	411	490	85	411	490	85
sanguinis	VVTEDYRVPAFKFKVLGNTEKE
SK49	KIKKNLIGSTTFVSAQPAKGTR
gi|422884106|	VFRVNRRRIDRRNHRITYLRDI
ref|	FQKEIEKVDKNFYRRLDESFRV
ZP_16930555.1	LGDKSEDLQIKQPFFGDKELET
	AYHKKYPTIYHLRKHLADADKN
	SPVADIREVYMAISHILKYRGH
	FLTLDKINPNNINMQNSWIDFI
	ESCQEVEDLEISDESKNIADIF
	KSSENRQEKVKKILPYFQQELL
	KKDKSIFKQLLQLLFGLKTKFK
	DCFELEEEPDLNFSKENYDENL
	ENFLGSLEEDFSDVFAKLKVLR
	DTILLSGMLTYTGATHARFSAT
	MVERYEEHRKDLQRFKFFIKQN
	LSEQDYLDIFGRKTQNGFDVDK
	ETKGYVGYITNKMVLTNPQKQK
	TIQQNFYDYISGKITGIEGAEY
	FLNKISDGTFLRKLRTSDNGAI
	PNQIHAYELEKIIERQGKDYPF
	LLENKDKLLSILTFKIPYYVGP
	LAKGSNSRFAWIKRATSSDILD
	DNDEDTRNGKIRPWNYQKLINM
	DETRDAFITNLIGNDIILLNEK
	VLPKRSLIYEEVMLQNELTRVK
	YKDKYGKAHFFDSELRQNIING
	LFKNNSKRVNAKSLIKYLSDNH
	KDLNAIEIVSGVEKGKSFNSTL
	KTYNDLKTIFSEELLDSEIYQK
	ELEEIIKVITVFDDKKSIKNYL
	TKFFGHLEILDEEKINQLSKLR
	YSGWGRYSAKLLLDIRDEDTGF
	NLLQFLRNDEENRNLTKLISDN
	TLSFEPKIKDIQSKSTIEDDIF
	DEIKKLAGSPAIKRGILNSIKI
	VDELVQIIGYPPHNIVIEMARE
	NMTTEEGQKKAKTRKTKLESAL
	KNIENSLLENGKVPHSDEQLQS
	EKLYLYYLQNGKDMYTLDKTGS
	PAPLYLDQLDQYEVDHIIPYSF
	LPIDSIDNKVLTHRENNQQKLN
	NIPDKETVANMKPFWEKLYNAK
	LISQTKYQRLTTSERTPDGVLT
	ESMKAGFIERQLVETRQIIKHV
	ARILDNRFSDTKIITLKSQLIT
	NFRNTFHIAKIRELNDYHHAHD
	AYLAVVVGQTLLKVYPKLAPEL
	IYGHHAHFNRHEENKATLRKHL
	YSNIMRFFNNPDSKVSKDIWDC
	NRDLPIIKDVIYNSQINFVKRT
	MIKKGAFYNQNPVGKFNKQLAA
	NNRYPLKTKALCLDTSIYGGYG
	PMNSALSIIIIAERFNEKKGKI
	ETVKEFHDIFIIDYEKFNNNPF
	QFLNDTSENGFLKKNNINRVLG
	FYRIPKYSLMQKIDGTRMLFES
	KSNLHKATQFKLTKTQNELFFH
	MKRLLTKSNLMDLKSKSAIKES
	QNFILKHKEEFDNISNQLSAFS
	QKMLGNTTSLKNLIKGYNERKI
	KEIDIRDETIKYFYDNFIKMFS
	FVKSGAPKDINDFFDNKCTVAR
	MRPKPDKKLLNATLIHQSITGL
	YETRIDLSKLGED (SEQ ID
	NO: 23)
Coprococcus	MKQEYFLGLDMGTGSLGWAVTD	172	310	139	556	634	76	556	634	76
catus GD-7	STYQVMRKHGKALWGTRLFESA
gi|291520705|	STAEERRMFRTARRRLDRRNWR
emb|CBK78998.1|	IQVLQEIFSEEISKVDPGFFLR
	MKESKYYPEDKRDAEGNCPELP
	YALFVDDNYTDKNYHKDYPTIY
	HLRKMLMETTEIPDIRLVYLVL
	HHMMKHRGHFLLSGDISQIKEF
	KSTFEQLIQNIQDEELEWHISL
	DDAAIQFVEHVLKDRNLTRSTK
	KSRLIKQLNAKSACEKAILNLL
	SGGTVKLSDIFNNKELDESERP
	KVSFADSGYDDYIGIVEAELAE
	QYYIIASAKAVYDWSVLVEILG
	NSVSISEAKIKVYQKHQADLKT
	LKKIVRQYMTKEDYKRVFVDTE
	EKLNNYSAYIGMTKKNGKKVDL
	KSKQCTQADFYDFLKKNVIKVI
	DHKEITQEIESEIEKENFLPKQ
	VTKDNGVIPYQVHDYELKKILD
	NLGTRMPFIKENAEKIQQLFEF
	RIPYYVGPLNRVDDGKDGKFTW
	SVRKSDARIYPWNFTEVIDVEA
	SAEKFIRRMTNKCTYLVGEDVL
	PKDSLVYSKFMVLNELNNLRLN
	GEKISVELKQRIYEELFCKYRK
	VTRKKLERYLVIEGIAKKGVEI
	TGIDGDFKASLTAYHDFKERLT
	DVQLSQRAKEAIVLNVVLFGDD
	KKLLKQRLSKMYPNLTTGQLKG
	ICSLSYQGWGRLSKTFLEEITV
	PAPGTGEVWNIMTALWQTNDNL
	MQLLSRNYGFTNEVEEFNTLKK
	ETDLSYKTVDELYVSPAVKRQI
	WQTLKVVKEIQKVMGNAPKRVF
	VEMAREKQEGKRSDSRKKQLVE
	LYRACKNEERDWITELNAQSDQ
	QLRSDKLFLYYIQKGRCMYSGE
	TIQLDELWDNTKYDIDHIYPQS
	KTMDDSLNNRVLVKKNYNAIKS
	DTYPLSLDIQKKMMSFWKMLQQ
	QGFITKEKYVRLVRSDELSADE
	LAGFIERQIVETRQSTKAVATI
	LKEALPDTEIVYVKAGNVSNFR
	QTYELLKVREMNDLHHAKDAYL
	NIVVGNAYFVKFTKNAAWFIRN
	NPGRSYNLKRMFEFDIERSGEI
	AWKAGNKGSIVTVKKVMQKNNI
	LVTRKAYEVKGGLFDQQIMKKG
	KGQVPIKGNDERLADIEKYGGY
	NKAAGTYFMLVKSLDKKGKEIR
	TIEFVPLYLKNQIEINHESAIQ
	YLAQERGLNSPEILLSKIKIDT
	LFKVDGFKMWLSGRTGNQLIFK
	GANQLILSHQEAAILKGVVKYV
	NRKNENKDAKLSERDGMTEEKL
	LQLYDTFLDKLSNTVYSIRLSA
	QIKTLTEKRAKFIGLSNEDQCI
	VLNEILHMFQCQSGSANLKLIG
	GPGSAGILVMNNNITACKQISV
	INQSPTGIYEKEIDLIKL
	(SEQ ID NO: 24)
Streptococcus	MKKPYSIGLDIGTNSVGWAVVT	176	314	139	392	470	84	392	470	84
mutans	DDYKVPAKKMKVLGNTDKSHIE
UA159	KNLLGALLFDSGNTAEDRRLKR
gi|24379809|	TARRRYTRRRNRILYLQEIFSE
ref|NP_721764.1|	EMGKVDDSFFHRLEDSFLVTED
	KRGERHPIFGNLEEEVKYHENF
	PTIYHLRQYLADNPEKVDLRLV
	YLALAHIIKFRGHFLIEGKFDT
	RNNDVQRLFQEFLAVYDNTFEN
	SSLQEQNVQVEEILTDKISKSA
	KKDRVLKLFPNEKSNGRFAEFL
	KLIVGNQADFKKHFELEEKAPL
	QFSKDTYEEELEVLLAQIGDNY
	AELFLSAKKLYDSILLSGILTV
	TDVGTKAPLSASMIQRYNEHQM
	DLAQLKQFIRQKLSDKYNEVFS
	DVSKDGYAGYIDGKTNQEAFYK
	YLKGLLNKIEGSGYFLDKIERE
	DFLRKQRTFDNGSIPHQIHLQE
	MRAIIRRQAEFYPFLADNQDRI
	EKLLTFRIPYYVGPLARGKSDF
	AWLSRKSADKITPWNFDEIVDK
	ESSAEAFINRMTNYDLYLPNQK
	VLPKHSLLYEKFTVYNELTKVK
	YKTEQGKTAFFDANMKQEIFDG
	VFKVYRKVTKDKLMDFLEKEFD
	EFRIVDLTGLDKENKVFNASYG
	TYHDLCKILDKDFLDNSKNEKI
	LEDIVLTLTLFEDREMIRKRLE
	NYSDLLTKEQVKKLERRHYTGW
	GRLSAELIHGIRNKESRKTILD
	YLIDDGNSNRNFMQLINDDALS
	FKEEIAKAQVIGETDNLNQVVS
	DIAGSPAIKKGILQSLKIVDEL
	VKIMGHQPENIVVEMARENQFT
	NQGRRNSQQRLKGLTDSIKEFG
	SQILKEHPVENSQLQNDRLFLY
	YLQNGRDMYTGEELDIDYLSQY
	DIDHIIPQAFIKDNSIDNRVLT
	SSKENRGKSDDVPSKDVVRKMK
	SYWSKLLSAKLITQRKFDNLTK
	AERGGLTDDDKAGFIKRQLVET
	RQITKHVARILDERENTETDEN
	NKKIRQVKIVTLKSNLVSNFRK
	EFELYKVREINDYHHAHDAYLN
	AVIGKALLGVYPQLEPEFVYGD
	YPHFHGHKENKATAKKFFYSNI
	MNFFKKDDVRTDKNGEIIWKKD
	EHISNIKKVLSYPQVNIVKKVE
	EQTGGFSKESILPKGNSDKLIP
	RKTKKFYWDTKKYGGFDSPIVA
	YSILVIADIEKGKSKKLKTVKA
	LVGVTIMEKMTFERDPVAFLER
	KGYRNVQEENIIKLPKYSLFKL
	ENGRKRLLASARELQKGNEIVL
	PNHLGTLLYHAKNIHKVDEPKH
	LDYVDKHKDEFKELLDVVSNFS
	KKYTLAEGNLEKIKELYAQNNG
	EDLKELASSFINLLTFTAIGAP
	ATFKFFDKNIDRKRYTSTTEIL
	NATLIHQSITGLYETRIDLNKL
	GGD (SEQ ID NO: 25)
Streptococcus	MDKKYSIGLDIGTNSVGWAVIT	176	314	139	523	600	82	523	600	82
pyogenes	DEYKVPSKKFKVLGNTDRHSIK
M1 GAS	KNLIGALLFDSGETAEATRLKR
gi|13622193|	TARRRYTRRKNRICYLQEIFSN
gb|AAK33936.1|	EMAKVDDSFFHRLEESFLVEED
	KKHERHPIFGNIVDEVAYHEKY
	PTIYHLRKKLVDSTDKADLRLI
	YLALAHMIKFRGHFLIEGDLNP
	DNSDVDKLFIQLVQTYNQLFEE
	NPINASGVDAKAILSARLSKSR
	RLENLIAQLPGEKKNGLFGNLI
	ALSLGLTPNFKSNFDLAEDAKL
	QLSKDTYDDDLDNLLAQIGDQY
	ADLFLAAKNLSDAILLSDILRV
	NTEITKAPLSASMIKRYDEHHQ
	DLTLLKALVRQQLPEKYKEIFF
	DQSKNGYAGYIDGGASQEEFYK
	FIKPILEKMDGTEELLVKLNRE
	DLLRKQRTFDNGSIPHQIHLGE
	LHAILRRQEDFYPFLKDNREKI
	EKILTFRIPYYVGPLARGNSRF
	AWMTRKSEETITPWNFEEVVDK
	GASAQSFIERMTNFDKNLPNEK
	VLPKHSLLYEYFTVYNELTKVK
	YVTEGMRKPAFLSGEQKKAIVD
	LLFKTNRKVTVKQLKEDYFKKI
	ECFDSVEISGVEDRFNASLGTY
	HDLLKIIKDKDFLDNEENEDIL
	EDIVLTLTLFEDREMIEERLKT
	YAHLFDDKVMKQLKRRRYTGWG
	RLSRKLINGIRDKQSGKTILDF
	LKSDGFANRNFMQLIHDDSLTF
	KEDIQKAQVSGQGDSLHEHIAN
	LAGSPAIKKGILQTVKVVDELV
	KVMGRHKPENIVIEMARENQTT
	QKGQKNSRERMKRIEEGIKELG
	SQILKEHPVENTQLQNEKLYLY
	YLQNGRDMYVDQELDINRLSDY
	DVDHIVPQSFLKDDSIDNKVLT
	RSDKNRGKSDNVPSEEVVKKMK
	NYWRQLLNAKLITQRKFDNLTK
	AERGGLSELDKAGFIKRQLVET
	RQITKHVAQILDSRMNTKYDEN
	DKLIREVKVITLKSKLVSDERK
	DFQFYKVREINNYHHAHDAYLN
	AVVGTALIKKYPKLESEFVYGD
	YKVYDVRKMIAKSEQEIGKATA
	KYFFYSNIMNFFKTEITLANGE
	IRKRPLIETNGETGEIVWDKGR
	DFATVRKVLSMPQVNIVKKTEV
	QTGGFSKESILPKRNSDKLIAR
	KKDWDPKKYGGFDSPTVAYSVL
	VVAKVEKGKSKKLKSVKELLGI
	TIMERSSFEKNPIDFLEAKGYK
	EVKKDLIIKLPKYSLFELENGR
	KRMLASAGELQKGNELALPSKY
	VNFLYLASHYEKLKGSPEDNEQ
	KQLFVEQHKHYLDEIIEQISEF
	SKRVILADANLDKVLSAYNKHR
	DKPIREQAENIIHLFTLTNLGA
	PAAFKYFDTTIDRKRYTSTKEV
	LDATLIHQSITGLYETRIDLSQ
	LGGD (SEQ ID NO: 26)
Streptococcus	MTKPYSIGLDIGTNSVGWAVTT	176	314	139	481	558	81	481	558	81
thermophilus	DNYKVPSKKMKVLGNTSKKYIK
LMD-9	KNLLGVLLFDSGITAEGRRLKR
gi|116628213|	TARRRYTRRRNRILYLQEIFST
ref|YP_820832.1|	EMATLDDAFFQRLDDSFLVPDD
	KRDSKYPIFGNLVEEKAYHDEF
	PTIYHLRKYLADSTKKADLRLV
	YLALAHMIKYRGHFLIEGEFNS
	KNNDIQKNFQDFLDTYNAIFES
	DLSLENSKQLEEIVKDKISKLE
	KKDRILKLFPGEKNSGIFSEFL
	KLIVGNQADFRKCFNLDEKASL
	HFSKESYDEDLETLLGYIGDDY
	SDVFLKAKKLYDAILLSGFLTV
	TDNETEAPLSSAMIKRYNEHKE
	DLALLKEYIRNISLKTYNEVFK
	DDTKNGYAGYIDGKTNQEDFYV
	YLKKLLAEFEGADYFLEKIDRE
	DFLRKQRTFDNGSIPYQIHLQE
	MRAILDKQAKFYPFLAKNKERI
	EKILTFRIPYYVGPLARGNSDF
	AWSIRKRNEKITPWNFEDVIDK
	ESSAEAFINRMTSFDLYLPEEK
	VLPKHSLLYETFNVYNELTKVR
	FIAESMRDYQFLDSKQKKDIVR
	LYFKDKRKVTDKDIIEYLHAIY
	GYDGIELKGIEKQFNSSLSTYH
	DLLNIINDKEFLDDSSNEAIIE
	EIIHTLTIFEDREMIKQRLSKF
	ENIFDKSVLKKLSRRHYTGWGK
	LSAKLINGIRDEKSGNTILDYL
	IDDGISNRNFMQLIHDDALSFK
	KKIQKAQIIGDEDKGNIKEVVK
	SLPGSPAIKKGILQSIKIVDEL
	VKVMGGRKPESIVVEMARENQY
	TNQGKSNSQQRLKRLEKSLKEL
	GSKILKENIPAKLSKIDNNALQ
	NDRLYLYYLQNGKDMYTGDDLD
	IDRLSNYDIDHIIPQAFLKDNS
	IDNKVLVSSASNRGKSDDVPSL
	EVVKKRKTFWYQLLKSKLISQR
	KFDNLTKAERGGLSPEDKAGFI
	QRQLVETRQITKHVARLLDEKF
	NNKKDENNRAVRTVKIITLKST
	LVSQFRKDFELYKVREINDFHH
	AHDAYLNAVVASALLKKYPKLE
	PEFVYGDYPKYNSFRERKSATE
	KVYFYSNIMNIFKKSISLADGR
	VIERPLIEVNEETGESVWNKES
	DLATVRRVLSYPQVNVVKKVEE
	QNHGLDRGKPKGLFNANLSSKP
	KPNSNENLVGAKEYLDPKKYGG
	YAGISNSFTVLVKGTIEKGAKK
	KITNVLEFQGISILDRINYRKD
	KLNFLLEKGYKDIELIIELPKY
	SLFELSDGSRRMLASILSTNNK
	RGEIHKGNQIFLSQKFVKLLYH
	AKRISNTINENHRKYVENHKKE
	FEELFYYILEFNENYVGAKKNG
	KLLNSAFQSWQNHSIDELCSSF
	IGPTGSERKGLFELTSRGSAAD
	FEFLGVKIPRYRDYTPSSLLKD
	ATLIHQSVTGLYETRIDLAKLG
	EG (SEQ ID NO: 27)
Fusobacterium	MKKQKFSDYYLGFDIGTNSVGW	171	308	138	537	614	76	537	614	76
nucleatum	CVTDLDYNVLRFNKKDMWGSRL
ATCC49256	FDEAKTAAERRVQRNSRRRLKR
gi|34762592|	RKWRLNLLEEIFSDEIMKIDSN
ref|	FFRRLKESSLWLEDKNSKEKFT
ZP_00143587.1|	LFNDDNYKDYDFYKQYPTIFHL
	RDELIKNPEKKDIRLIYLALHS
	IFKSRGHFLFEGQNLKEIKNFE
	TLYNNLISFLEDNGINKSIDKD
	NIEKLEKIICDSGKGLKDKEKE
	FKGIFNSDKQLVAIFKLSVGSS
	VSLNDLFDTDEYKKEEVEKEKI
	SFREQIYEDDKPIYYSILGEKI
	ELLDIAKSFYDFMVLNNILSDS
	NYISEAKVKLYEEHKKDLKNLK
	YIIRKYNKENYDKLFKDKNENN
	YPAYIGLNKEKDKKEVVEKSRL
	KIDDLIKVIKGYLPKPERIEEK
	DKTIFNEILNKIELKTILPKQR
	ISDNGTLPYQIHEVELEKILEN
	QSKYYDFLNYEENGVSTKDKLL
	KTFKFRIPYYVGPLNSYHKDKG
	GNSWIVRKEEGKILPWNFEQKV
	DIEKSAEEFIKRMTNKCTYLNG
	EDVIPKDSFLYSEYIILNELNK
	VQVNDEFLNEENKRKIIDELFK
	ENKKVSEKKFKEYLLVNQIANR
	TVELKGIKDSFNSNYVSYIKFK
	DIFGEKLNLDIYKEISEKSILW
	KCLYGDDKKIFEKKIKNEYGDI
	LNKDEIKKINSFKENTWGRLSE
	KLLTGIEFINLETGECYSSVME
	ALRRTNYNLMELLSSKFTLQES
	IDNENKEMNEVSYRDLIEESYV
	SPSLKRAILQTLKIYEEIKKIT
	GRVPKKVFIEMARGGDESMKNK
	KIPARQEQLKKLYDSCGNDIAN
	FSIDIKEMKNSLSSYDNNSLRQ
	KKLYLYYLQFGKCMYTGREIDL
	DRLLQNNDTYDIDHIYPRSKVI
	KDDSFDNLVLVLKNENAEKSNE
	YPVKKEIQEKMKSFWRFLKEKN
	FISDEKYKRLTGKDDFELRGFM
	ARQLVNVRQTTKEVGKILQQIE
	PEIKIVYSKAEIASSFREMFDF
	IKVRELNDTHHAKDAYLNIVAG
	NVYNTKFTEKPYRYLQEIKENY
	DVKKIYNYDIKNAWDKENSLEI
	VKKNMEKNTVNITRFIKEEKGE
	LFNLNPIKKGETSNEIISIKPK
	LYDGKDNKLNEKYGYYTSLKAA
	YFIYVEHEKKNKKVKTFERITR
	IDSTLIKNEKNLIKYLVSQKKL
	LNPKIIKKIYKEQTLIIDSYPY
	TFTGVDSNKKVELKNKKQLYLE
	KKYEQILKNALKFVEDNQGETE
	ENYKFIYLKKRNNNEKNETIDA
	VKERYNIEFNEMYDKFLEKLSS
	KDYKNYINNKLYTNFLNSKEKF
	KKLKLWEKSLILREFLKIFNKN
	TYGKYEIKDSQTKEKLFSFPED
	TGRIRLGQSSLGNNKELLEESV
	TGLFVKKIKL (SEQ ID NO:
	28)
Planococcus	MKNYTIGLDIGVASVGWVCIDE	162	299	138	538	614	94	538	614	94
antarcticus	NYKILNYNNRHAFGVHEFESAE
DSM 14505	SAAGRRLKRGMRRRYNRRKKRL
gi|389815359|	QLLQSLFDSYITDSGFFSKTDS
ref|	QHFWKNNNEFENRSLTEVLSSL
ZP_10206685.1	RISSRKYPTIYHLRSDLIESNK
	KMDLRLVYLALHNLVKYRGHFL
	QEGNWSEAASAEGMDDQLLELV
	TRYAELENLSPLDLSESQWKAA
	ETLLLNRNLTKTDQSKELTAMF
	GKEYEPFCKLVAGLGVSLHQLF
	PSSEQALAYKETKTKVQLSNEN
	VEEVMELLLEEESALLEAVQPF
	YQQVVLYELLKGETYVAKAKVS
	AFKQYQKDMASLKNLLDKTFGE
	KVYRSYFISDKNSQREYQKSHK
	VEVLCKLDQFNKEAKFAETFYK
	DLKKLLEDKSKTSIGTTEKDEM
	LRIIKAIDSNQFLQKQKGIQNA
	AIPHQNSLYEAEKILRNQQAHY
	PFITTEWIEKVKQILAFRIPYY
	IGPLVKDTTQSPFSWVERKGDA
	PITPWNFDEQIDKAASAEAFIS
	RMRKTCTYLKGQEVLPKSSLTY
	ERFEVLNELNGIQLRTTGAESD
	FRHRLSYEMKCWIIDNVFKQYK
	TVSTKRLLQELKKSPYADELYD
	EHTGEIKEVFGTQKENAFATSL
	SGYISMKSILGAVVDDNPAMTE
	ELIYWIAVFEDREILHLKIQEK
	YPSITDVQRQKLALVKLPGWGR
	FSRLLIDGLPLDEQGQSVLDHM
	EQYSSVFMEVLKNKGFGLEKKI
	QKMNQHQVDGTKKIRYEDIEEL
	AGSPALKRGIWRSVKIVEELVS
	IFGEPANIVLEVAREDGEKKRT
	KSRKDQWEELTKTTLKNDPDLK
	SFIGEIKSQGDQRFNEQRFWLY
	VTQQGKCLYTGKALDIQNLSMY
	EVDHILPQNFVKDDSLDNLALV
	MPEANQRKNQVGQNKMPLEIIE
	ANQQYAMRTLWERLHELKLISS
	GKLGRLKKPSFDEVDKDKFIAR
	QLVETRQIIKHVRDLLDERFSK
	SDIHLVKAGIVSKFRRFSEIPK
	IRDYNNKHHAMDALFAAALIQS
	ILGKYGKNFLAFDLSKKDRQKQ
	WRSVKGSNKEFFLFKNFGNLRL
	QSPVTGEEVSGVEYMKHVYFEL
	PWQTTKMTQTGDGMFYKESIFS
	PKVKQAKYVSPKTEKFVHDEVK
	NHSICLVEFTFMKKEKEVQETK
	FIDLKVIEHHQFLKEPESQLAK
	FLAEKETNSPIIHARIIRTIPK
	YQKIWIEHFPYYFISTRELHNA
	RQFEISYELMEKVKQLSERSSV
	EELKIVFGLLIDQMNDNYPIYT
	KSSIQDRVQKFVDTQLYDFKSF
	EIGFEELKKAVAANAQRSDTFG
	SRISKKPKPEEVAIGYESITGL
	KYRKPRSVVGTKR (SEQ ID
	NO: 29)
Treponema	MKKEIKDYFLGLDVGTGSVGWA	169	305	137	524	600	81	524	600	81
denticola	VTDTDYKLLKANRKDLWGMRCF
ATCC 35405	ETAETAEVRRLHRGARRRIERR
gi|42525843|	KKRIKLLQELFSQEIAKTDEGF
ref|NP_970941.1|	FQRMKESPFYAEDKTILQENTL
	FNDKDFADKTYHKAYPTINHLI
	KAWIENKVKPDPRLLYLACHNI
	IKKRGHFLFEGDFDSENQFDTS
	IQALFEYLREDMEVDIDADSQK
	VKEILKDSSLKNSEKQSRLNKI
	LGLKPSDKQKKAITNLISGNKI
	NFADLYDNPDLKDAEKNSISFS
	KDDFDALSDDLASILGDSFELL
	LKAKAVYNCSVLSKVIGDEQYL
	SFAKVKIYEKHKTDLTKLKNVI
	KKHFPKDYKKVFGYNKNEKNNN
	NYSGYVGVCKTKSKKLIINNSV
	NQEDFYKFLKTILSAKSEIKEV
	NDILTEIETGTFLPKQISKSNA
	EIPYQLRKMELEKILSNAEKHF
	SFLKQKDEKGLSHSEKIIMLLT
	FKIPYYIGPINDNHKKFFPDRC
	WVVKKEKSPSGKTTPWNFFDHI
	DKEKTAEAFITSRTNFCTYLVG
	ESVLPKSSLLYSEYTVLNEINN
	LQIIIDGKNICDIKLKQKIYED
	LFKKYKKITQKQISTFIKHEGI
	CNKTDEVIILGIDKECTSSLKS
	YIELKNIFGKQVDEISTKNMLE
	EIIRWATIYDEGEGKTILKTKI
	KAEYGKYCSDEQIKKILNLKFS
	GWGRLSRKFLETVTSEMPGFSE
	PVNIITAMRETQNNLMELLSSE
	FTFTENIKKINSGFEDAEKQFS
	YDGLVKPLFLSPSVKKMLWQTL
	KLVKEISHITQAPPKKIFIEMA
	KGAELEPARTKTRLKILQDLYN
	NCKNDADAFSSEIKDLSGKIEN
	EDNLRLRSDKLYLYYTQLGKCM
	YCGKPIEIGHVEDTSNYDIDHI
	YPQSKIKDDSISNRVLVCSSCN
	KNKEDKYPLKSEIQSKQRGFWN
	FLQRNNFISLEKLNRLTRATPI
	SDDETAKFIARQLVETRQATKV
	AAKVLEKMFPETKIVYSKAETV
	SMFRNKFDIVKCREINDFHHAH
	DAYLNIVVGNVYNTKFTNNPWN
	FIKEKRDNPKIADTYNYYKVFD
	YDVKRNNITAWEKGKTIITVKD
	MLKRNTPIYTRQAACKKGELEN
	QTIMKKGLGQHPLKKEGPFSNI
	SKYGGYNKVSAAYYTLIEYEEK
	GNKIRSLETIPLYLVKDIQKDQ
	DVLKSYLTDLLGKKEFKILVPK
	IKINSLLKINGFPCHITGKIND
	SFLLRPAVQFCCSNNEVLYFKK
	IIRFSEIRSQREKIGKTISPYE
	DLSFRSYIKENLWKKTKNDEIG
	EKEFYDLLQKKNLEIYDMLLTK
	HKDTIYKKRPNSATIDILVKGK
	EKFKSLIIENQFEVILEILKLF
	SATRNVSDLQHIGGSKYSGVAK
	IGNKISSLDNCILIYQSITGIF
	EKRIDLLKV (SEQ ID NO:
	30)
Solobacterium	MEGQMKNNGNNLQQGNYYLGLD	179	314	136	544	619	77	544	619	77
moorei	VGTSSVGWAVTDTDYNVLKFRG
F0204	KSMWGARLFDEASTAEERRTHR
gi|320528778|	GNRRRLARRKYRLLLLEQLFEK
ref|	EIRKIDDNFFVRLHESNLWADD
ZP_08029929.1	KSKPSKFLLFNDTNFTDKDYLK
	KYPTIYHLRSDLIHNSTEHDIR
	LVFLALHHLIKYRGHFIYDNSA
	NGDVKTLDEAVSDFEEYLNEND
	IEFNIENKKEFINVLSDKHLTK
	KEKKISLKKLYGDITDSENINI
	SVLIEMLSGSSISLSNLFKDIE
	FDGKQNLSLDSDIEETLNDVVD
	ILGDNIDLLIHAKEVYDIAVLT
	SSLGKHKYLCDAKVELFEKNKK
	DLMILKKYIKKNHPEDYKKIFS
	SPTEKKNYAAYSQTNSKNVCSQ
	EEFCLFIKPYIRDMVKSENEDE
	VRIAKEVEDKSFLTKLKGTNNS
	VVPYQIHERELNQILKNIVAYL
	PFMNDEQEDISVVDKIKLIFKF
	KIPYYVGPLNTKSTRSWVYRSD
	EKIYPWNFSNVIDLDKTAHEFM
	NRLIGRCTYTNDPVLPMDSLLY
	SKYNVLNEINPIKVNGKAIPVE
	VKQAIYTDLFENSKKKVTRKSI
	YIYLLKNGYIEKEDIVSGIDIE
	IKSKLKSHHDFTQIVQENKCTP
	EEIERIIKGILVYSDDKSMLRR
	WLKNNIKGLSENDVKYLAKLNY
	KEWGRLSKTLLTDIYTINPEDG
	EACSILDIMWNTNATLMEILSN
	EKYQFKQNIENYKAENYDEKQN
	LHEELDDMYISPAARRSIWQAL
	RIVDEIVDIKKSAPKKIFIEMA
	REKKSAMKKKRTESRKDTLLEL
	YKSCKSQADGFYDEELFEKLSN
	ESNSRLRRDQLYLYYTQMGRSM
	YTGKRIDFDKLINDKNTYDIDH
	IYPRSKIKDDSITNRVLVEKDI
	NGEKTDIYPISEDIRQKMQPFW
	KILKEKGLINEEKYKRLTRNYE
	LTDEELSSFVARQLVETQQSTK
	ALATLLKKEYPSAKIVYSKAGN
	VSEFRNRKDKELPKFREINDLH
	HAKDAYLNIVVGNVYDTKFTEK
	FFNNIRNENYSLKRVFDFSVPG
	AWDAKGSTFNTIKKYMAKNNPI
	IAFAPYEVKGELFDQQIVPKGK
	GQFPIKQGKDIEKYGGYNKLSS
	AFLFAVEYKGKKARERSLETVY
	IKDVELYLQDPIKYCESVLGLK
	EPQIIKPKILMGSLFSINNKKL
	VVTGRSGKQYVCHHIYQLSIND
	EDSQYLKNIAKYLQEEPDGNIE
	RQNILNITSVNNIKLFDVLCTK
	FNSNTYEIILNSLKNDVNEGRE
	KFSELDILEQCNILLQLLKAFK
	CNRESSNLEKLNNKKQAGVIVI
	PHLFTKCSVFKVIHQSITGLFE
	KEMDLLK (SEQ ID NO:
	31)
Staphylococcus	MGRKPYILSLDIGTGSVGYACM	164	299	136	531	606	92	531	606	92
pseudintermedius	DKGFNVLKYHDKDALGVYLFDG
ED99	ALTAQERRQFRTSRRRKNRRIK
gi|323463801|	RLGLLQELLAPLVQNPNFYQFQ
gb|ADX75954.1|	RQFAWKNDNMDFKNKSLSEVLS
	FLGYESKKYPTIYHLQEALLLK
	DEKFDPELIYMALYHLVKYRGH
	FLFDHLKIENLTNNDNMHDFVE
	LIETYENLNNIKLNLDYEKTKV
	IYEILKDNEMTKNDRAKRVKNM
	EKKLEQFSIMLLGLKFNEGKLF
	NHADNAEELKGANQSHTFADNY
	EENLTPFLTVEQSEFIERANKI
	YLSLTLQDILKGKKSMAMSKVA
	AYDKERNELKQVKDIVYKADST
	RTQFKKIFVSSKKSLKQYDATP
	NDQTFSSLCLFDQYLIRPKKQY
	SLLIKELKKIIPQDSELYFEAE
	NDTLLKVLNTTDNASIPMQINL
	YEAETILRNQQKYHAEITDEMI
	EKVLSLIQFRIPYYVGPLVNDH
	TASKFGWMERKSNESIKPWNED
	EVVDRSKSATQFIRRMTNKCSY
	LINEDVLPKNSLLYQEMEVLNE
	LNATQIRLQTDPKNRKYRMMPQ
	IKLFAVEHIFKKYKTVSHSKFL
	EIMLNSNHRENFMNHGEKLSIF
	GTQDDKKFASKLSSYQDMTKIF
	GDIEGKRAQIEEIIQWITIFED
	KKILVQKLKECYPELTSKQINQ
	LKKLNYSGWGRLSEKLLTHAYQ
	GHSIIELLRHSDENFMEILTND
	VYGFQNFIKEENQVQSNKIQHQ
	DIANLTTSPALKKGIWSTIKLV
	RELTSIFGEPEKIIMEFATEDQ
	QKGKKQKSRKQLWDDNIKKNKL
	KSVDEYKYIIDVANKLNNEQLQ
	QEKLWLYLSQNGKCMYSGQSID
	LDALLSPNATKHYEVDHIFPRS
	FIKDDSIDNKVLVIKKMNQTKG
	DQVPLQFIQQPYERIAYWKSLN
	KAGLISDSKLHKLMKPEFTAMD
	KEGFIQRQLVETRQISVHVRDF
	LKEEYPNTKVIPMKAKMVSEFR
	KKFDIPKIRQMNDAHHAIDAYL
	NGVVYHGAQLAYPNVDLFDFNF
	KWEKVREKWKALGEFNTKQKSR
	ELFFFKKLEKMEVSQGERLISK
	IKLDMNHFKINYSRKLANIPQQ
	FYNQTAVSPKTAELKYESNKSN
	EVVYKGLTPYQTYVVAIKSVNK
	KGKEKMEYQMIDHYVFDFYKFQ
	NGNEKELALYLAQRENKDEVLD
	AQIVYSLNKGDLLYINNHPCYF
	VSRKEVINAKQFELTVEQQLSL
	YNVMNNKETNVEKLLIEYDFIA
	EKVINEYHHYLNSKLKEKRVRT
	FFSESNQTHEDFIKALDELFKV
	VTASATRSDKIGSRKNSMTHRA
	FLGKGKDVKIAYTSISGLKTTK
	PKSLFKLAESRNEL (SEQ ID
	NO: 32)
Flavobacterium	MAKILGLDLGTNSIGWAVVERE	162	286	125	538	613	63	538	613	63
branchiophilum	NIDFSLIDKGVRIFSEGVKSEK
FL-15	GIESSRAAERTGYRSARKIKYR
gi|347536497|	RKLRKYETLKVLSLNRMCPLSI
ref|	EEVEEWKKSGFKDYPLNPEFLK
YP_004843922.1	WLSTDEESNVNPYFFRDRASKH
	KVSLFELGRAFYHIAQRRGFLS
	NRLDQSAEGILEEHCPKIEAIV
	EDLISIDEISTNITDYFFETGI
	LDSNEKNGYAKDLDEGDKKLVS
	LYKSLLAILKKNESDFENCKSE
	IIERLNKKDVLGKVKGKIKDIS
	QAMLDGNYKTLGQYFYSLYSKE
	KIRNQYTSREEHYLSEFITICK
	VQGIDQINEEEKINEKKFDGLA
	KDLYKAIFFQRPLKSQKGLIGK
	CSFEKSKSRCAISHPDFEEYRM
	WTYLNTIKIGTQSDKKLRFLTQ
	DEKLKLVPKFYRKNDFNFDVLA
	KELIEKGSSFGFYKSSKKNDFF
	YWFNYKPTDTVAACQVAASLKN
	AIGEDWKTKSFKYQTINSNKEQ
	VSRTVDYKDLWHLLTVATSDVY
	LYEFAIDKLGLDEKNAKAFSKT
	KLKKDFASLSLSAINKILPYLK
	EGLLYSHAVEVANIENIVDENI
	WKDEKQRDYIKTQISEIIENYT
	LEKSRFEIINGLLKEYKSENED
	GKRVYYSKEAEQSFENDLKKKL
	VLFYKSNEIENKEQQETIFNEL
	LPIFIQQLKDYEFIKIQRLDQK
	VLIFLKGKNETGQIFCTEEKGT
	AEEKEKKIKNRLKKLYHPSDIE
	KFKKKIIKDEFGNEKIVLGSPL
	TPSIKNPMAMRALHQLRKVLNA
	LILEGQIDEKTIIHIEMARELN
	DANKRKGIQDYQNDNKKFREDA
	IKEIKKLYFEDCKKEVEPTEDD
	ILRYQLWMEQNRSEIYEEGKNI
	SICDIIGSNPAYDIEHTIPRSR
	SQDNSQMNKTLCSQRFNREVKK
	QSMPIELNNHLEILPRIAHWKE
	EADNLTREIEIISRSIKAAATK
	EIKDKKIRRRHYLTLKRDYLQG
	KYDRFIWEEPKVGFKNSQIPDT
	GIITKYAQAYLKSYFKKVESVK
	GGMVAEFRKIWGIQESFIDENG
	MKHYKVKDRSKHTHHTIDAITI
	ACMTKEKYDVLAHAWTLEDQQN
	KKEARSIIEASKPWKTFKEDLL
	KIEEEILVSHYTPDNVKKQAKK
	IVRVRGKKQFVAEVERDVNGKA
	VPKKAASGKTIYKLDGEGKKLP
	RLQQGDTIRGSLHQDSIYGAIK
	NPLNTDEIKYVIRKDLESIKGS
	DVESIVDEVVKEKIKEAIANKV
	LLLSSNAQQKNKLVGTVWMNEE
	KRIAINKVRIYANSVKNPLHIK
	EHSLLSKSKHVHKQKVYGQNDE
	NYAMAIYELDGKRDFELINIFN
	LAKLIKQGQGFYPLHKKKEIKG
	KIVFVPIEKRNKRDVVLKRGQQ
	VVFYDKEVENPKDISEIVDFKG
	RIYIIEGLSIQRIVRPSGKVDE
	YGVIMLRYFKEARKADDIKQDN
	FKPDGVFKLGENKPTRKMNHQF
	TAFVEGIDFKVLPSGKFEKI
	(SEQ ID NO: 33)
Ignavibacterium	MEFKKVLGLDIGTNSIGCALLS	223	329	107	357	432	90	357	432	90
album	LPKSIQDYGKGGRLEWLTSRVI
JCM 16511	PLDADYMKAFIDGKNGLPQVIT
gi|385811609|	PAGKRRQKRGSRRLKHRYKLRR
ref|	SRLIRVFKTLNWLPEDFPLDNP
YP_005848005.1	KRIKETISTEGKFSFRISDYVP
	ISDESYREFYREFGYPENEIEQ
	VIEEINFRRKTKGKNKNPMIKL
	LPEDWVVYYLRKKALIKPTTKE
	ELIRIIYLFNQRRGFKSSRKDL
	TETAILDYDEFAKRLAEKEKYS
	AENYETKFVSITKVKEVVELKT
	DGRKGKKRFKVILEDSRIEPYE
	IERKEKPDWEGKEYTFLVTQKL
	EKGKFKQNKPDLPKEEDWALCT
	TALDNRMGSKHPGEFFFDELLK
	AFKEKRGYKIRQYPVNRWRYKK
	ELEFIWTKQCQLNPELNNLNIN
	KEILRKLATVLYPSQSKFFGPK
	IKEFENSDVLHIISEDIIYYQR
	DLKSQKSLISECRYEKRKGIDG
	EIYGLKCIPKSSPLYQEFRIWQ
	DIHNIKVIRKESEVNGKKKINI
	DETQLYINENIKEKLFELFNSK
	DSLSEKDILELISLNIINSGIK
	ISKKEEETTHRINLFANRKELK
	GNETKSRYRKVFKKLGFDGEYI
	LNHPSKLNRLWHSDYSNDYADK
	EKTEKSILSSLGWKNRNGKWEK
	SKNYDVFNLPLEVAKAIANLPP
	LKKEYGSYSALAIRKMLVVMRD
	GKYWQHPDQIAKDQENTSLMLF
	DKNLIQLTNNQRKVLNKYLLTL
	AEVQKRSTLIKQKLNEIEHNPY
	KLELVSDQDLEKQVLKSFLEKK
	NESDYLKGLKTYQAGYLIYGKH
	SEKDVPIVNSPDELGEYIRKKL
	PNNSLRNPIVEQVIRETIFIVR
	DVWKSFGIIDEIHIELGRELKN
	NSEERKKTSESQEKNFQEKERA
	RKLLKELLNSSNFEHYDENGNK
	IFSSFTVNPNPDSPLDIEKFRI
	WKNQSGLTDEELNKKLKDEKIP
	TEIEVKKYILWLTQKCRSPYTG
	KIIPLSKLFDSNVYEIEHIIPR
	SKMKNDSTNNLVICELGVNKAK
	GDRLAANFISESNGKCKFGEVE
	YTLLKYGDYLQYCKDTFKYQKA
	KYKNLLATEPPEDFIERQINDT
	RYIGRKLAELLTPVVKDSKNII
	FTIGSITSELKITWGLNGVWKD
	ILRPRFKRLESIINKKLIFQDE
	DDPNKYHFDLSINPQLDKEGLK
	RLDHRHHALDATIIAATTREHV
	RYLNSLNAADNDEEKREYFLSL
	CNHKIRDFKLPWENFTSEVKSK
	LLSCVVSYKESKPILSDPFNKY
	LKWEYKNGKWQKVFAIQIKNDR
	WKAVRRSMFKEPIGTVWIKKIK
	EVSLKEAIKIQAIWEEVKNDPV
	RKKKEKYIYDDYAQKVIAKIVQ
	ELGLSSSMRKQDDEKLNKFINE
	AKVSAGVNKNLNTTNKTIYNLE
	GRFYEKIKVAEYVLYKAKRMPL
	NKKEYIEKLSLQKMFNDLPNFI
	LEKSILDNYPEILKELESDNKY
	IIEPHKKNNPVNRLLLEHILEY
	HNNPKEAFSTEGLEKLNKKAIN
	KIGKPIKYITRLDGDINEEEIF
	RGAVFETDKGSNVYFVMYENNQ
	TKDREFLKPNPSISVLKAIEHK
	NKIDFFAPNRLGFSRIILSPGD
	LVYVPTNDQYVLIKDNSSNETI
	INWDDNEFISNRIYQVKKFTGN
	SCYFLKNDIASLILSYSASNGV
	GEFGSQNISEYSVDDPPIRIKD
	VCIKIRVDRLGNVRPL (SEQ
	ID NO: 34)
Bergeyella	MKHILGLDLGTNSIGWALIERN	165	261	97	529	604	56	529	604	56
zoohelcum	IEEKYGKIIGMGSRIVPMGAEL
ATCC 43767	SKFEQGQAQTKNADRRTNRGAR
gi|423317190|	RLNKRYKQRRNKLIYILQKLDM
ref|	LPSQIKLKEDFSDPNKIDKITI
ZP_17295095.1	LPISKKQEQLTAFDLVSLRVKA
	LTEKVGLEDLGKIIYKYNQLRG
	YAGGSLEPEKEDIFDEEQSKDK
	KNKSFIAFSKIVFLGEPQEEIF
	KNKKLNRRAIIVETEEGNFEGS
	TFLENIKVGDSLELLINISASK
	SGDTITIKLPNKTNWRKKMENI
	ENQLKEKSKEMGREFYISEFLL
	ELLKENRWAKIRNNTILRARYE
	SEFEAIWNEQVKHYPFLENLDK
	KTLIEIVSFIFPGEKESQKKYR
	ELGLEKGLKYIIKNQVVFYQRE
	LKDQSHLISDCRYEPNEKAIAK
	SHPVFQEYKVWEQINKLIVNTK
	IEAGTNRKGEKKYKYIDRPIPT
	ALKEWIFEELQNKKEITFSAIF
	KKLKAEFDLREGIDFLNGMSPK
	DKLKGNETKLQLQKSLGELWDV
	LGLDSINRQIELWNILYNEKGN
	EYDLTSDRTSKVLEFINKYGNN
	IVDDNAEETAIRISKIKFARAY
	SSLSLKAVERILPLVRAGKYFN
	NDFSQQLQSKILKLLNENVEDP
	FAKAAQTYLDNNQSVLSEGGVG
	NSIATILVYDKHTAKEYSHDEL
	YKSYKEINLLKQGDLRNPLVEQ
	IINEALVLIRDIWKNYGIKPNE
	IRVELARDLKNSAKERATIHKR
	NKDNQTINNKIKETLVKNKKEL
	SLANIEKVKLWEAQRHLSPYTG
	QPIPLSDLFDKEKYDVDHIIPI
	SRYFDDSFTNKVISEKSVNQEK
	ANRTAMEYFEVGSLKYSIFTKE
	QFIAHVNEYFSGVKRKNLLATS
	IPEDPVQRQIKDTQYIAIRVKE
	ELNKIVGNENVKTTTGSITDYL
	RNHWGLTDKFKLLLKERYEALL
	ESEKFLEAEYDNYKKDFDSRKK
	EYEEKEVLFEEQELTREEFIKE
	YKENYIRYKKNKLIIKGWSKRI
	DHRHHAIDALIVACTEPAHIKR
	LNDLNKVLQDWLVEHKSEFMPN
	FEGSNSELLEEILSLPENERTE
	IFTQIEKFRAIEMPWKGFPEQV
	EQKLKEIIISHKPKDKLLLQYN
	KAGDRQIKLRGQLHEGTLYGIS
	QGKEAYRIPLTKFGGSKFATEK
	NIQKIVSPFLSGFIANHLKEYN
	NKKEEAFSAEGIMDLNNKLAQY
	RNEKGELKPHTPISTVKIYYKD
	PSKNKKKKDEEDLSLQKLDREK
	AFNEKLYVKTGDNYLFAVLEGE
	IKTKKTSQIKRLYDIISFFDAT
	NFLKEEFRNAPDKKTFDKDLLF
	RQYFEERNKAKLLFTLKQGDFV
	YLPNENEEVILDKESPLYNQYW
	GDLKERGKNIYVVQKFSKKQIY
	FIKHTIADIIKKDVEFGSQNCY
	ETVEGRSIKENCFKLEIDRLGN
	IVKVIKR (SEQ ID NO:
	35)
Nitrobacter	MHVEIDFPHFSRGDSHLAMNKN	169	253	85	536	611	48	536	611	48
hamburgensis	EILRGSSVLYRLGLDLGSNSLG
X14	WFVTHLEKRGDRHEPVALGPGG
gi|92109262|	VRIFPDGRDPQSGTSNAVDRRM
ref|	ARGARKRRDRFVERRKELIAAL
YP_571550.1|	IKYNLLPDDARERRALEVLDPY
	ALRKTALTDTLPAHHVGRALFH
	LNQRRGFQSNRKTDSKQSEDGA
	IKQAASRLATDKGNETLGVFFA
	DMHLRKSYEDRQTAIRAELVRL
	GKDHLTGNARKKIWAKVRKRLF
	GDEVLPRADAPHGVRARATITG
	TKASYDYYPTRDMLRDEFNAIW
	AGQSAHHATITDEARTEIEHII
	FYQRPLKPAIVGKCTLDPATRP
	FKEDPEGYRAPWSHPLAQRFRI
	LSEARNLEIRDTGKGSRRLTKE
	QSDLVVAALLANREVKFDKLRT
	LLKLPAEARFNLESDRRAALDG
	DQTAARLSDKKGFNKAWRGFPP
	ERQIAIVARLEETEDENELIAW
	LEKECALDGAAAARVANTTLPD
	GHCRLGLRAIKKIVPIMQDGLD
	EDGVAGAGYHIAAKRAGYDHAK
	LPTGEQLGRLPYYGQWLQDAVV
	GSGDARDQKEKQYGQFPNPTVH
	IGLGQLRRVVNDLIDKYGPPTE
	ISIEFTRALKLSEQQKAERQRE
	QRRNQDKNKARAEELAKFGRPA
	NPRNLLKMRLWEELAHDPLDRK
	CVYTGEQISIERLLSDEVDIDH
	ILPVAMTLDDSPANKIICMRYA
	NRHKRKQTPSEAFGSSPTLQGH
	RYNWDDIAARATGLPRNKRWRF
	DANAREEFDKRGGFLARQLNET
	GWLARLAKQYLGAVTDPNQIWV
	VPGRLTSMLRGKWGLNGLLPSD
	NYAGVQDKAEEFLASTDDMEFS
	GVKNRADHRHHAIDGLVTALTD
	RSLLWKMANAYDEEHEKFVIEP
	PWPTMRDDLKAALEKMVVSHKP
	DHGIEGKLHEDSAYGFVKPLDA
	TGLKEEEAGNLVYRKAIESLNE
	NEVDRIRDIQLRTIVRDHVNVE
	KTKGVALADALRQLQAPSDDYP
	QFKHGLRHVRILKKEKGDYLVP
	IANRASGVAYKAYSAGENFCVE
	VFETAGGKWDGEAVRRFDANKK
	NAGPKIAHAPQWRDANEGAKLV
	MRIHKGDLIRLDHEGRARIMVV
	HRLDAAAGRFKLADHNETGNLD
	KRHATNNDIDPFRWLMASYNTL
	KKLAAVPVRVDELGRVWRVMPN
	(SEQ ID NO: 36)
Odoribacter	METTLGIDLGTNSIGLALVDQE	164	242	79	535	610	63	535	610	63
laneus YIT	EHQILYSGVRIFPEGINKDTIG
12061	LGEKEESRNATRRAKRQMRRQY
gi|374384763|	FRKKLRKAKLLELLIAYDMCPL
ref|	KPEDVRRWKNWDKQQKSTVRQF
ZP_09642280.1	PDTPAFREWLKQNPYELRKQAV
	TEDVTRPELGRILYQMIQRRGF
	LSSRKGKEEGKIFTGKDRMVGI
	DETRKNLQKQTLGAYLYDIAPK
	NGEKYRFRTERVRARYTLRDMY
	IREFEIIWQRQAGHLGLAHEQA
	TRKKNIFLEGSATNVRNSKLIT
	HLQAKYGRGHVLIEDTRITVTF
	QLPLKEVLGGKIEIEEEQLKFK
	SNESVLFWQRPLRSQKSLLSKC
	VFEGRNFYDPVHQKWIIAGPTP
	APLSHPEFEEFRAYQFINNIIY
	GKNEHLTAIQREAVFELMCTES
	KDFNFEKIPKHLKLFEKFNFDD
	TTKVPACTTISQLRKLFPHPVW
	EEKREEIWHCFYFYDDNTLLFE
	KLQKDYALQTNDLEKIKKIRLS
	ESYGNVSLKAIRRINPYLKKGY
	AYSTAVLLGGIRNSFGKRFEYF
	KEYEPEIEKAVCRILKEKNAEG
	EVIRKIKDYLVHNRFGFAKNDR
	AFQKLYHHSQAITTQAQKERLP
	ETGNLRNPIVQQGLNELRRTVN
	KLLATCREKYGPSFKFDHIHVE
	MGRELRSSKTEREKQSRQIREN
	EKKNEAAKVKLAEYGLKAYRDN
	IQKYLLYKEIEEKGGTVCCPYT
	GKTLNISHTLGSDNSVQIEHII
	PYSISLDDSLANKTLCDATFNR
	EKGELTPYDFYQKDPSPEKWGA
	SSWEEIEDRAFRLLPYAKAQRF
	IRRKPQESNEFISRQLNDTRYI
	SKKAVEYLSAICSDVKAFPGQL
	TAELRHLWGLNNILQSAPDITF
	PLPVSATENHREYYVITNEQNE
	VIRLFPKQGETPRTEKGELLLT
	GEVERKVFRCKGMQEFQTDVSD
	GKYWRRIKLSSSVTWSPLFAPK
	PISADGQIVLKGRIEKGVFVCN
	QLKQKLKTGLPDGSYWISLPVI
	SQTFKEGESVNNSKLTSQQVQL
	FGRVREGIFRCHNYQCPASGAD
	GNFWCTLDTDTAQPAFTPIKNA
	PPGVGGGQIILTGDVDDKGIFH
	ADDDLHYELPASLPKGKYYGIF
	TVESCDPTLIPIELSAPKTSKG
	ENLIEGNIWVDEHTGEVREDPK
	KNREDQRHHAIDAIVIALSSQS
	LFQRLSTYNARRENKKRGLDST
	EHFPSPWPGFAQDVRQSVVPLL
	VSYKQNPKTLCKISKTLYKDGK
	KIHSCGNAVRGQLHKETVYGQR
	TAPGATEKSYHIRKDIRELKTS
	KHIGKVVDITIRQMLLKHLQEN
	YHIDITQEFNIPSNAFFKEGVY
	RIFLPNKHGEPVPIKKIRMKEE
	LGNAERLKDNINQYVNPRNNHH
	VMIYQDADGNLKEEIVSFWSVI
	ERQNQGQPIYQLPREGRNIVSI
	LQINDTFLIGLKEEEPEVYRND
	LSTLSKHLYRVQKLSGMYYTFR
	HHLASTLNNEREEFRIQSLEAW
	KRANPVKVQIDEIGRITFLNGP
	LC (SEQ ID NO: 37)
Legionella	MESSQILSPIGIDLGGKFTGVC	164	239	76	402	476	67	402	476	67
pneumophila	LSHLEAFAELPNHANTKYSVIL
str. Paris	IDHNNFQLSQAQRRATRHRVRN
gi|54296138|	KKRNQFVKRVALQLFQHILSRD
ref|	LNAKEETALCHYLNNRGYTYVD
YP_122507.1|	TDLDEYIKDETTINLLKELLPS
	ESEHNFIDWFLQKMQSSEFRKI
	LVSKVEEKKDDKELKNAVKNIK
	NFITGFEKNSVEGHRHRKVYFE
	NIKSDITKDNQLDSIKKKIPSV
	CLSNLLGHLSNLQWKNLHRYLA
	KNPKQFDEQTFGNEFLRMLKNF
	RHLKGSQESLAVRNLIQQLEQS
	QDYISILEKTPPEITIPPYEAR
	TNTGMEKDQSLLLNPEKLNNLY
	PNWRNLIPGIIDAHPFLEKDLE
	HTKLRDRKRIISPSKQDEKRDS
	YILQRYLDLNKKIDKFKIKKQL
	SFLGQGKQLPANLIETQKEMET
	HFNSSLVSVLIQIASAYNKERE
	DAAQGIWFDNAFSLCELSNINP
	PRKQKILPLLVGAILSEDFINN
	KDKWAKFKIFWNTHKIGRTSLK
	SKCKEIEEARKNSGNAFKIDYE
	EALNHPEHSNNKALIKIIQTIP
	DIIQAIQSHLGHNDSQALIYHN
	PFSLSQLYTILETKRDGFHKNC
	VAVTCENYWRSQKTEIDPEISY
	ASRLPADSVRPFDGVLARMMQR
	LAYEIAMAKWEQIKHIPDNSSL
	LIPIYLEQNRFEFEESFKKIKG
	SSSDKTLEQAIEKQNIQWEEKF
	QRIINASMNICPYKGASIGGQG
	EIDHIYPRSLSKKHFGVIFNSE
	VNLIYCSSQGNREKKEEHYLLE
	HLSPLYLKHQFGTDNVSDIKNF
	ISQNVANIKKYISFHLLTPEQQ
	KAARHALFLDYDDEAFKTITKF
	LMSQQKARVNGTQKFLGKQIME
	FLSTLADSKQLQLEFSIKQITA
	EEVHDHRELLSKQEPKLVKSRQ
	QSFPSHAIDATLTMSIGLKEFP
	QFSQELDNSWFINHLMPDEVHL
	NPVRSKEKYNKPNISSTPLFKD
	SLYAERFIPVWVKGETFAIGFS
	EKDLFEIKPSNKEKLFTLLKTY
	STKNPGESLQELQAKSKAKWLY
	FPINKTLALEFLHHYFHKEIVT
	PDDTTVCHFINSLRYYTKKESI
	TVKILKEPMPVLSVKFESSKKN
	VLGSFKHTIALPATKDWERLFN
	HPNFLALKANPAPNPKEFNEFI
	RKYFLSDNNPNSDIPNNGHNIK
	PQKHKAVRKVFSLPVIPGNAGT
	MMRIRRKDNKGQPLYQLQTIDD
	TPSMGIQINEDRLVKQEVLMDA
	YKTRNLSTIDGINNSEGQAYAT
	FDNWLTLPVSTFKPEIIKLEMK
	PHSKTRRYIRITQSLADFIKTI
	DEALMIKPSDSIDDPLNMPNEI
	VCKNKLFGNELKPRDGKMKIVS
	TGKIVTYEFESDSTPQWIQTLY
	VTQLKKQP (SEQ ID NO:
	38)
Bacteroides	MKKIVGLDLGTNSIGWALINAY	198	269	72	530	604	83	530	604	83
sp. 203	INKEHLYGIEACGSRIIPMDAA
gi|301311869|	ILGNFDKGNSISQTADRTSYRG
ref|	IRRLRERHLLRRERLHRILDLL
ZP_07217791.1	GFLPKHYSDSLNRYGKFLNDIE
	CKLPWVKDETGSYKFIFQESFK
	EMLANFTEHHPILIANNKKVPY
	DWTIYYLRKKALTQKISKEELA
	WILLNFNQKRGYYQLRGEEEET
	PNKLVEYYSLKVEKVEDSGERK
	GKDTWYNVHLENGMIYRRTSNI
	PLDWEGKTKEFIVTTDLEADGS
	PKKDKEGNIKRSFRAPKDDDWT
	LIKKKTEADIDKIKMTVGAYIY
	DTLLQKPDQKIRGKLVRTIERK
	YYKNELYQILKTQSEFHEELRD
	KQLYIACLNELYPNNEPRRNSI
	STRDFCHLFIEDIIFYQRPLKS
	KKSLIDNCPYEENRYIDKESGE
	IKHASIKCIAKSHPLYQEFRLW
	QFIVNLRIYRKETDVDVTQELL
	PTEADYVTLFEWLNEKKEIDQK
	AFFKYPPFGFKKTTSNYRWNYV
	EDKPYPCNETHAQIIARLGKAH
	IPKAFLSKEKEETLWHILYSIE
	DKQEIEKALHSFANKNNLSEEF
	IEQFKNFPPFKKEYGSYSAKAI
	KKLLPLMRMGKYWSIENIDNGT
	RIRINKIIDGEYDENIRERVRQ
	KAINLTDITHFRALPLWLACYL
	VYDRHSEVKDIVKWKTPKDIDL
	YLKSFKQHSLRNPIVEQVITET
	LRTVRDIWQQVGHIDEIHIELG
	REMKNPADKRARMSQQMIKNEN
	TNLRIKALLTEFLNPEFGIENV
	RPYSPSQQDLLRIYEEGVLNSI
	LELPEDIGIILGKFNQTDTLKR
	PTRSEILRYKLWLEQKYRSPYT
	GEMIPLSKLFTPAYEIEHIIPQ
	SRYFDDSLSNKVICESEINKLK
	DRSLGYEFIKNHHGEKVELAFD
	KPVEVLSVEAYEKLVHESYSHN
	RSKMKKLLMEDIPDQFIERQLN
	DSRYISKVVKSLLSNIVREENE
	QEAISKNVIPCTGGITDRLKKD
	WGINDVWNKIVLPRFIRLNELT
	ESTRFTSINTNNTMIPSMPLEL
	QKGFNKKRIDHRHHAMDAIIIA
	CANRNIVNYLNNVSASKNTKIT
	RRDLQTLLCHKDKTDNNGNYKW
	VIDKPWETFTQDTLTALQKITV
	SFKQNLRVINKTTNHYQHYENG
	KKIVSNQSKGDSWAIRKSMHKE
	TVHGEVNLRMIKTVSFNEALKK
	PQAIVEMDLKKKILAMLELGYD
	TKRIKNYFEENKDTWQDINPSK
	IKVYYFTKETKDRYFAVRKPID
	TSFDKKKIKESITDTGIQQIML
	RHLETKDNDPTLAFSPDGIDEM
	NRNILILNKGKKHQPIYKVRVY
	EKAEKFTVGQKGNKRTKFVEAA
	KGTNLFFAIYETEEIDKDTKKV
	IRKRSYSTIPLNVVIERQKQGL
	SSAPEDENGNLPKYILSPNDLV
	YVPTQEEINKGEVVMPIDRDRI
	YKMVDSSGITANFIPASTANLI
	FALPKATAEIYCNGENCIQNEY
	GIGSPQSKNQKAITGEMVKEIC
	FPIKVDRLGNIIQVGSCILTN
	(SEQ ID NO: 39)
Akkermansia	MSRSLTFSFDIGYASIGWAVIA	136	202	67	348	418	62	348	418	62
muciniphila	SASHDDADPSVCGCGTVLFPKD
ATCC BAA-	DCQAFKRREYRRLRRNIRSRRV
835	RIERIGRLLVQAQIITPEMKET
gi|187736489|	SGHPAPFYLASEALKGHRTLAP
ref|	IELWHVLRWYAHNRGYDNNASW
YP_001878601.	SNSLSEDGGNGEDTERVKHAQD
	LMDKHGTATMAETICRELKLEE
	GKADAPMEVSTPAYKNLNTAFP
	RLIVEKEVRRILELSAPLIPGL
	TAEIIELIAQHHPLTTEQRGVL
	LQHGIKLARRYRGSLLFGQLIP
	RFDNRIISRCPVTWAQVYEAEL
	KKGNSEQSARERAEKLSKVPTA
	NCPEFYEYRMARILCNIRADGE
	PLSAEIRRELMNQARQEGKLTK
	ASLEKAISSRLGKETETNVSNY
	FTLHPDSEEALYLNPAVEVLQR
	SGIGQILSPSVYRIAANRLRRG
	KSVTPNYLLNLLKSRGESGEAL
	EKKIEKESKKKEADYADTPLKP
	KYATGRAPYARTVLKKVVEEIL
	DGEDPTRPARGEAHPDGELKAH
	DGCLYCLLDTDSSVNQHQKERR
	LDTMTNNHLVRHRMLILDRLLK
	DLIQDFADGQKDRISRVCVEVG
	KELTTFSAMDSKKIQRELTLRQ
	KSHTDAVNRLKRKLPGKALSAN
	LIRKCRIAMDMNWTCPFTGATY
	GDHELENLELEHIVPHSFRQSN
	ALSSLVLTWPGVNRMKGQRTGY
	DFVEQEQENPVPDKPNLHICSL
	NNYRELVEKLDDKKGHEDDRRR
	KKKRKALLMVRGLSHKHQSQNH
	EAMKEIGMTEGMMTQSSHLMKL
	ACKSIKTSLPDAHIDMIPGAVT
	AEVRKAWDVFGVFKELCPEAAD
	PDSGKILKENLRSLTHLHHALD
	ACVLGLIPYIIPAHHNGLLRRV
	LAMRRIPEKLIPQVRPVANQRH
	YVLNDDGRMMLRDLSASLKENI
	REQLMEQRVIQHVPADMGGALL
	KETMQRVLSVDGSGEDAMVSLS
	KKKDGKKEKNQVKASKLVGVFP
	EGPSKLKALKAAIEIDGNYGVA
	LDPKPVVIRHIKVFKRIMALKE
	QNGGKPVRILKKGMLIHLTSSK
	DPKHAGVWRIESIQDSKGGVKL
	DLQRAHCAVPKNKTHECNWREV
	DLISLLKKYQMKRYPTSYTGTP
	R (SEQ ID NO: 40)
Prevotella	MTQKVLGLDLGTNSIGSAVRNL	184	250	67	357	425	78	357	425	78
sp. C561	DLSDDLQWQLEFFSSDIFRSSV
gi|345885718|	NKESNGREYSLAAQRSAHRRSR
ref|	GLNEVRRRRLWATLNLLIKHGF
ZP_08837074.1	CPMSSESLMRWCTYDKRKGLFR
	EYPIDDKDFNAWILLDENGDGR
	PDYSSPYQLRRELVTRQFDFEQ
	PIERYKLGRALYHIAQHRGFKS
	SKGETLSQQETNSKPSSTDEIP
	DVAGAMKASEEKLSKGLSTYMK
	EHNLLTVGAAFAQLEDEGVRVR
	NNNDYRAIRSQFQHEIETIFKF
	QQGLSVESELYERLISEKKNVG
	TIFYKRPLRSQRGNVGKCTLER
	SKPRCAIGHPLFEKFRAWTLIN
	NIKVRMSVDTLDEQLPMKLRLD
	LYNECFLAFVRTEFKFEDIRKY
	LEKRLGIHFSYNDKTINYKDST
	SVAGCPITARFRKMLGEEWESF
	RVEGQKERQAHSKNNISFHRVS
	YSIEDIWHFCYDAEEPEAVLAF
	AQETLRLERKKAEELVRIWSAM
	PQGYAMLSQKAIRNINKILMLG
	LKYSDAVILAKVPELVDVSDEE
	LLSIAKDYYLVEAQVNYDKRIN
	SIVIGLIAKYKSVSEEYRFADH
	NYEYLLDESDEKDIIRQIENSL
	GARRWSLMDANEQTDILQKVRD
	RYQDFFRSHERKFVESPKLGES
	FENYLTKKFPMVEREQWKKLYH
	PSQITIYRPVSVGKDRSVLRLG
	NPDIGAIKNPTVLRVLNTLRRR
	VNQLLDDGVISPDETRVVVETA
	RELNDANRKWALDTYNRIRHDE
	NEKIKKILEEFYPKRDGISTDD
	IDKARYVIDQREVDYFTGSKTY
	NKDIKKYKFWLEQGGQCMYTGR
	TINLSNLFDPNAFDIEHTIPES
	LSFDSSDMNLTLCDAHYNRFIK
	KNHIPTDMPNYDKAITIDGKEY
	PAITSQLQRWVERVERLNRNVE
	YWKGQARRAQNKDRKDQCMREM
	HLWKMELEYWKKKLERFTVTEV
	TDGFKNSQLVDTRVITRHAVLY
	LKSIFPHVDVQRGDVTAKFRKI
	LGIQSVDEKKDRSLHSHHAIDA
	TTLTIIPVSAKRDRMLELFAKI
	EEINKMLSFSGSEDRTGLIQEL
	EGLKNKLQMEVKVCRIGHNVSE
	IGTFINDNIIVNHHIKNQALTP
	VRRRLRKKGYIVGGVDNPRWQT
	GDALRGEIHKASYYGAITQFAK
	DDEGKVLMKEGRPQVNPTIKFV
	IRRELKYKKSAADSGFASWDDL
	GKAIVDKELFALMKGQFPAETS
	FKDACEQGIYMIKKGKNGMPDI
	KLHHIRHVRCEAPQSGLKIKEQ
	TYKSEKEYKRYFYAAVGDLYAM
	CCYTNGKIREFRIYSLYDVSCH
	RKSDIEDIPEFITDKKGNRLML
	DYKLRTGDMILLYKDNPAELYD
	LDNVNLSRRLYKINRFESQSNL
	VLMTHHLSTSKERGRSLGKTVD
	YQNLPESIRSSVKSLNFLIMGE
	NRDFVIKNGKIIFNHR
	(SEQ ID NO: 41)
Wolinella	MLVSPISVDLGGKNTGFFSFTD	157	218	36	401	468	60	401	468	60
succinogenes	SLDNSQSGTVIYDESFVLSQVG
DSM 1740	RRSKRHSKRNNLRNKLVKRLFL
gi|34557932|	LILQEHHGLSIDVLPDEIRGLF
ref|	NKRGYTYAGFELDEKKKDALES
NP_907747.1|	DTLKEFLSEKLQSIDRDSDVED
	FLNQIASNAESFKDYKKGFEAV
	FASATHSPNKKLELKDELKSEY
	GENAKELLAGLRVTKEILDEFD
	KQENQGNLPRAKYFEELGEYIA
	TNEKVKSFFDSNSLKLTDMTKL
	IGNISNYQLKELRRYFNDKEME
	KGDIWIPNKLHKITERFVRSWH
	PKNDADRQRRAELMKDLKSKEI
	MELLTTTEPVMTIPPYDDMNNR
	GAVKCQTLRLNEEYLDKHLPNW
	RDIAKRLNHGKENDDLADSTVK
	GYSEDSTLLHRLLDTSKEIDIY
	ELRGKKPNELLVKTLGQSDANR
	LYGFAQNYYELIRQKVRAGIWV
	PVKNKDDSLNLEDNSNMLKRCN
	HNPPHKKNQIHNLVAGILGVKL
	DEAKFAEFEKELWSAKVGNKKL
	SAYCKNIEELRKTHGNTFKIDI
	EELRKKDPAELSKEEKAKLRLT
	DDVILNEWSQKIANFFDIDDKH
	RQRFNNLFSMAQLHTVIDTPRS
	GFSSTCKRCTAENRFRSETAFY
	NDETGEFHKKATATCQRLPADT
	QRPFSGKIERYIDKLGYELAKI
	KAKELEGMEAKEIKVPIILEQN
	AFEYEESLRKSKTGSNDRVINS
	KKDRDGKKLAKAKENAEDRLKD
	KDKRIKAFSSGICPYCGDTIGD
	DGEIDHILPRSHTLKIYGTVEN
	PEGNLIYVHQKCNQAKADSIYK
	LSDIKAGVSAQWIEEQVANIKG
	YKTFSVLSAEQQKAFRYALFLQ
	NDNEAYKKVVDWLRTDQSARVN
	GTQKYLAKKIQEKLTKMLPNKH
	LSFEFILADATEVSELRRQYAR
	QNPLLAKAEKQAPSSHAIDAVM
	AFVARYQKVFKDGTPPNADEVA
	KLAMLDSWNPASNEPLTKGLST
	NQKIEKMIKSGDYGQKNMREVF
	GKSIFGENAIGERYKPIVVQEG
	GYYIGYPATVKKGYELKNCKVV
	TSKNDIAKLEKIIKNQDLISLK
	ENQYIKIFSINKQTISELSNRY
	FNMNYKNLVERDKEIVGLLEFI
	VENCRYYTKKVDVKFAPKYIHE
	TKYPFYDDWRRFDEAWRYLQEN
	QNKTSSKDRFVIDKSSLNEYYQ
	PDKNEYKLDVDTQPIWDDFCRW
	YFLDRYKTANDKKSIRIKARKT
	FSLLAESGVQGKVFRAKRKIPT
	GYAYQALPMDNNVIAGDYANIL
	LEANSKTLSLVPKSGISIEKQL
	DKKLDVIKKTDVRGLAIDNNSF
	FNADFDTHGIRLIVENTSVKVG
	NFPISAIDKSAKRMIFRALFEK
	EKGKRKKKTTISFKESGPVQDY
	LKVFLKKIVKIQLRTDGSISNI
	VVRKNAADFTLSFRSEHIQKLL
	K (SEQ ID NO: 42)
Alicyclobacillus	MAYRLGLDIGITSVGWAVVALE	142	196	55	416	482	61	416	482	61
hesperidum	KDESGLKPVRIQDLGVRIFDKA
URH17-3-68	EDSKTGASLALPRREARSARRR
gi|403744858|	TRRRRHRLWRVKRLLEQHGILS
ref|	MEQIEALYAQRTSSPDVYALRV
ZP_10953934.1	AGLDRCLIAEEIARVLIHIAHR
	RGFQSNRKSEIKDSDAGKLLKA
	VQENENLMQSKGYRTVAEMLVS
	EATKTDAEGKLVHGKKHGYVSN
	VRNKAGEYRHTVSRQAIVDEVR
	KIFAAQRALGNDVMSEELEDSY
	LKILCSQRNFDDGPGGDSPYGH
	GSVSPDGVRQSIYERMVGSCTF
	ETGEKRAPRSSYSFERFQLLTK
	VVNLRIYRQQEDGGRYPCELTQ
	TERARVIDCAYEQTKITYGKLR
	KLLDMKDTESFAGLTYGLNRSR
	NKTEDTVFVEMKFYHEVRKALQ
	RAGVFIQDLSIETLDQIGWILS
	VWKSDDNRRKKLSTLGLSDNVI
	EELLPLNGSKFGHLSLKAIRKI
	LPFLEDGYSYDVACELAGYQFQ
	GKTEYVKQRLLPPLGEGEVTNP
	VVRRALSQAIKVVNAVIRKHGS
	PESIHIELARELSKNLDERRKI
	EKAQKENQKNNEQIKDEIREIL
	GSAHVTGRDIVKYKLFKQQQEF
	CMYSGEKLDVTRLFEPGYAEVD
	HIIPYGISFDDSYDNKVLVKTE
	QNRQKGNRTPLEYLRDKPEQKA
	KFIALVESIPLSQKKKNHLLMD
	KRAIDLEQEGFRERNLSDTRYI
	TRALMNHIQAWLLFDETASTRS
	KRVVCVNGAVTAYMRARWGLTK
	DRDAGDKHHAADAVVVACIGDS
	LIQRVTKYDKFKRNALADRNRY
	VQQVSKSEGITQYVDKETGEVF
	TWESFDERKFLPNEPLEPWPFF
	RDELLARLSDDPSKNIRAIGLL
	TYSETEQIDPIFVSRMPTRKVT
	GAAHKETIRSPRIVKVDDNKGT
	EIQVVVSKVALTELKLTKDGEI
	KDYFRPEDDPRLYNTLRERLVQ
	FGGDAKAAFKEPVYKISKDGSV
	RTPVRKVKIQEKLTLGVPVHGG
	RGIAENGGMVRIDVFAKGGKYY
	FVPIYVADVLKRELPNRLATAH
	KPYSEWRVVDDSYQFKFSLYPN
	DAVMIKPSREVDITYKDRKEPV
	GCRIMYFVSANIASASISLRTH
	DNSGELEGLGIQGLEVFEKYVV
	GPLGDTHPVYKERRMPFRVERK
	MN (SEQ ID NO: 43)
Caenispirillum	MPVLSPLSPNAAQGRRRWSLAL	161	214	54	330	393	68	330	393	68
salinarum	DIGEGSIGWAVAEVDAEGRVLQ
AK4	LTGTGVTLFPSAWSNENGTYVA
gi|427429481|	HGAADRAVRGQQQRHDSRRRRL
ref|	AGLARLCAPVLERSPEDLKDLT
ZP_18919511.1	RTPPKADPRAIFFLRADAARRP
	LDGPELFRVLHHMAAHRGIRLA
	ELQEVDPPPESDADDAAPAATE
	DEDGTRRAAADERAFRRLMAEH
	MHRHGTQPTCGEIMAGRLRETP
	AGAQPVTRARDGLRVGGGVAVP
	TRALIEQEFDAIRAIQAPRHPD
	LPWDSLRRLVLDQAPIAVPPAT
	PCLFLEELRRRGETFQGRTITR
	EAIDRGLTVDPLIQALRIRETV
	GNLRLHERITEPDGRQRYVPRA
	MPELGLSHGELTAPERDTLVRA
	LMHDPDGLAAKDGRIPYTRLRK
	LIGYDNSPVCFAQERDTSGGGI
	TVNPTDPLMARWIDGWVDLPLK
	ARSLYVRDVVARGADSAALARL
	LAEGAHGVPPVAAAAVPAATAA
	ILESDIMQPGRYSVCPWAAEAI
	LDAWANAPTEGFYDVTRGLFGF
	APGEIVLEDLRRARGALLAHLP
	RTMAAARTPNRAAQQRGPLPAY
	ESVIPSQLITSLRRAHKGRAAD
	WSAADPEERNPFLRTWTGNAAT
	DHILNQVRKTANEVITKYGNRR
	GWDPLPSRITVELAREAKHGVI
	RRNEIAKENRENEGRRKKESAA
	LDTFCQDNTVSWQAGGLPKERA
	ALRLRLAQRQEFFCPYCAERPK
	LRATDLFSPAETEIDHVIERRM
	GGDGPDNLVLAHKDCNNAKGKK
	TPHEHAGDLLDSPALAALWQGW
	RKENADRLKGKGHKARTPREDK
	DFMDRVGWRFEEDARAKAEENQ
	ERRGRRMLHDTARATRLARLYL
	AAAVMPEDPAEIGAPPVETPPS
	PEDPTGYTAIYRTISRVQPVNG
	SVTHMLRQRLLQRDKNRDYQTH
	HAEDACLLLLAGPAVVQAFNTE
	AAQHGADAPDDRPVDLMPTSDA
	YHQQRRARALGRVPLATVDAAL
	ADIVMPESDRQDPETGRVHWRL
	TRAGRGLKRRIDDLTRNCVILS
	RPRRPSETGTPGALHNATHYGR
	REITVDGRTDTVVTQRMNARDL
	VALLDNAKIVPAARLDAAAPGD
	TILKEICTEIADRHDRVVDPEG
	THARRWISARLAALVPAHAEAV
	ARDIAELADLDALADADRTPEQ
	EARRSALRQSPYLGRAISAKKA
	DGRARAREQEILTRALLDPHWG
	PRGLRHLIMREARAPSLVRIRA
	NKTDAFGRPVPDAAVWVKTDGN
	AVSQLWRLTSVVTDDGRRIPLP
	KPIEKRIEISNLEYARINGLDE
	GAGVTGNNAPPRPLRQDIDRLT
	PLWRDHGTAPGGYLGTAVGELE
	DKARSALRGKAMRQTLTDAGIT
	AEAGWRLDSEGAVCDLEVAKGD
	TVKKDGKTYKVGVITQGIFGMP
	VDAAGSAPRTPEDCEKFEEQYG
	IKPWKAKGIPLA (SEQ ID
	NO: 44)
Eubacterium	MNYTEKEKLFMKYILALDIGIA	133	185	53	322	384	60	322	384	60
rectale ATCC	SVGWAILDKESETVIEAGSNIF
33656	PEASAADNQLRRDMRGAKRNNR
gi|238924075|	RLKTRINDFIKLWENNNLSIPQ
ref|	FKSTEIVGLKVRAITEEITLDE
YP_002937591.1	LYLILYSYLKHRGISYLEDALD
	DTVSGSSAYANGLKLNAKELET
	HYPCEIQQERLNTIGKYRGQSQ
	IINENGEVLDLSNVFTIGAYRK
	EIQRVFEIQKKYHPELTDEFCD
	GYMLIFNRKRKYYEGPGNEKSR
	TDYGRFTTKLDANGNYITEDNI
	FEKLIGKCSVYPDELRAAAASY
	TAQEYNVLNDLNNLTINGRKLE
	ENEKHEIVERIKSSNTINMRKI
	ISDCMGENIDDFAGARIDKSGK
	EIFHKFEVYNKMRKALLEIGID
	ISNYSREELDEIGYIMTINTDK
	EAMMEAFQKSWIDLSDDVKQCL
	INMRKTNGALENKWQSFSLKIM
	NELIPEMYAQPKEQMTLLTEMG
	VTKGTQEEFAGLKYIPVDVVSE
	DIFNPVVRRSVRISFKILNAVL
	KKYKALDTIVIEMPRDRNSEEQ
	KKRINDSQKLNEKEMEYIEKKL
	AVTYGIKLSPSDFSSQKQLSLK
	LKLWNEQDGICLYSGKTIDPND
	IINNPQLFEIDHIIPRSISFDD
	ARSNKVLVYRSENQKKGNQTPY
	YYLTHSHSEWSFEQYKATVMNL
	SKKKEYAISRKKIQNLLYSEDI
	TKMDVLKGFINRNINDTSYASR
	LVLNTIQNFFMANEADTKVKVI
	KGSYTHQMRCNLKLDKNRDESY
	SHHAVDAMLIGYSELGYEAYHK
	LQGEFIDFETGEILRKDMWDEN
	MSDEVYADYLYGKKWANIRNEV
	VKAEKNVKYWHYVMRKSNRGLC
	NQTIRGTREYDGKQYKINKLDI
	RTKEGIKVFAKLAFSKKDSDRE
	RLLVYLNDRRTFDDLCKIYEDY
	SDAANPFVQYEKETGDIIRKYS
	KKHNGPRIDKLKYKDGEVGACI
	DISHKYGFEKGSKKVILESLVP
	YRMDVYYKEENHSYYLVGVKQS
	DIKFEKGRNVIDEEAYARILVN
	EKMIQPGQSRADLENLGFKFKL
	SFYKNDIIEYEKDGKIYTERLV
	SRTMPKQRNYIETKPIDKAKFE
	KQNLVGLGKTKFIKKYRYDILG
	NKYSCSEEKFTSFC (SEQ ID
	NO: 45)
Mycoplasma	MLRLYCANNLVLNNVQNLWKYL	187	239	53	319	381	80	319	381	80
synoviae53	LLLIFDKKIIFLFKIKVILIRR
gi|71894592|	YMENNNKEKIVIGFDLGVASVG
ref|	WSIVNAETKEVIDLGVRLFSEP
YP_278700.1|	EKADYRRAKRTTRRLLRRKKFK
	REKFHKLILKNAEIFGLQSRNE
	ILNVYKDQSSKYRNILKLKINA
	LKEEIKPSELVWILRDYLQNRG
	YFYKNEKLTDEFVSNSFPSKKL
	HEHYEKYGFFRGSVKLDNKLDN
	KKDKAKEKDEEEESDAKKESEE
	LIFSNKQWINEIVKVFENQSYL
	TESFKEEYLKLFNYVRPFNKGP
	GSKNSRTAYGVFSTDIDPETNK
	FKDYSNIWDKTIGKCSLFEEEI
	RAPKNLPSALIFNLQNEICTIK
	NEFTEFKNWWLNAEQKSEILKF
	VFTELFNWKDKKYSDKKFNKNL
	QDKIKKYLLNFALENFNLNEEI
	LKNRDLENDTVLGLKGVKYYEK
	SNATADAALEFSSLKPLYVFIK
	FLKEKKLDLNYLIGLENTEILY
	FLDSIYLAISYSSDLKERNEWF
	KKLLKELYPKIKNNNLEIIENV
	EDIFEITDQEKFESFSKTHSLS
	REAFNHIIPLLLSNNEGKNYES
	LKHSNEELKKRTEKAELKAQQN
	QKYLKDNFLKEALVPLSVKTSV
	LQAIKIFNQIIKNFGKKYEISQ
	VVIEMARELTKPNLEKLLNNAT
	NSNIKILKEKLDQTEKFDDFTK
	KKFIDKIENSVVFRNKLFLWFE
	QDRKDPYTQLDIKINEIEDETE
	IDHVIPYSKSADDSWFNKLLVK
	KSTNQLKKNKTVWEYYQNESDP
	EAKWNKFVAWAKRIYLVQKSDK
	ESKDNSEKNSIFKNKKPNLKFK
	NITKKLFDPYKDLGFLARNLND
	TRYATKVFRDQLNNYSKHHSKD
	DENKLFKVVCMNGSITSFLRKS
	MWRKNEEQVYRFNFWKKDRDQF
	FHHAVDASIIAIFSLLTKTLYN
	KLRVYESYDVQRREDGVYLINK
	ETGEVKKADKDYWKDQHNFLKI
	RENAIEIKNVLNNVDFQNQVRY
	SRKANTKLNTQLFNETLYGVKE
	FENNFYKLEKVNLFSRKDLRKF
	ILEDLNEESEKNKKNENGSRKR
	ILTEKYIVDEILQILENEEFKD
	SKSDINALNKYMDSLPSKFSEF
	FSQDFINKCKKENSLILTFDAI
	KHNDPKKVIKIKNLKFFREDAT
	LKNKQAVHKDSKNQIKSFYESY
	KCVGFIWLKNKNDLEESIFVPI
	NSRVIHFGDKDKDIFDFDSYNK
	EKLLNEINLKRPENKKENSINE
	IEFVKFVKPGALLLNFENQQIY
	YISTLESSSLRAKIKLLNKMDK
	GKAVSMKKITNPDEYKIIEHVN
	PLGINLNWTKKLENNN
	(SEQ ID NO: 46)
Porphyromonas	MLMSKHVLGLDLGVGSIGWCLI	150	202	53	309	371	60	309	371	60
sp. oral	ALDAQGDPAEILGMGSRVVPLN
taxon 279	NATKAIEAFNAGAAFTASQERT
str. F0450	ARRTMRRGFARYQLRRYRLRRE
gi|402847315|	LEKVGMLPDAALIQLPLLELWE
ref|	LRERAATAGRRLTLPELGRVLC
ZP_10895610.1	HINQKRGYRHVKSDAAAIVGDE
	GEKKKDSNSAYLAGIRANDEKL
	QAEHKTVGQYFAEQLRQNQSES
	PTGGISYRIKDQIFSRQCYIDE
	YDQIMAVQRVHYPDILTDEFIR
	MLRDEVIFMQRPLKSCKHLVSL
	CEFEKQERVMRVQQDDGKGGWQ
	LVERRVKFGPKVAPKSSPLFQL
	CCIYEAVNNIRLTRPNGSPCDI
	TPEERAKIVAHLQSSASLSFAA
	LKKLLKEKALIADQLTSKSGLK
	GNSTRVALASALQPYPQYHHLL
	DMELETRMMTVQLTDEETGEVT
	EREVAVVTDSYVRKPLYRLWHI
	LYSIEEREAMRRALITQLGMKE
	EDLDGGLLDQLYRLDFVKPGYG
	NKSAKFICKLLPQLQQGLGYSE
	ACAAVGYRHSNSPTSEEITERT
	LLEKIPLLQRNELRQPLVEKIL
	NQMINLVNALKAEYGIDEVRVE
	LARELKMSREERERMARNNKDR
	EERNKGVAAKIRECGLYPTKPR
	IQKYMLWKEAGRQCLYCGRSIE
	EEQCLREGGMEVEHIIPKSVLY
	DDSYGNKTCACRRCNKEKGNRT
	ALEYIRAKGREAEYMKRINDLL
	KEKKISYSKHQRLRWLKEDIPS
	DFLERQLRLTQYISRQAMAILQ
	QGIRRVSASEGGVTARLRSLWG
	YGKILHTLNLDRYDSMGETERV
	SREGEATEELHITNWSKRMDHR
	HHAIDALVVACTRQSYIQRLNR
	LSSEFGREDKKKEDQEAQEQQA
	TETGRLSNLERWLTQRPHFSVR
	TVSDKVAEILISYRPGQRVVTR
	GRNIYRKKMADGREVSCVQRGV
	LVPRGELMEASFYGKILSQGRV
	RIVKRYPLHDLKGEVVDPHLRE
	LITTYNQELKSREKGAPIPPLC
	LDKDKKQEVRSVRCYAKTLSLD
	KAIPMCFDEKGEPTAFVKSASN
	HHLALYRTPKGKLVESIVTFWD
	AVDRARYGIPLVITHPREVMEQ
	VLQRGDIPEQVLSLLPPSDWVF
	VDSLQQDEMVVIGLSDEELQRA
	LEAQNYRKISEHLYRVQKMSSS
	YYVFRYHLETSVADDKNTSGRI
	PKFHRVQSLKAYEERNIRKVRV
	DLLGRISLL (SEQ ID NO:
	47)
Streptococcus	MSDLVLGLDIGIGSVGVGILNK	127	178	139	424	486	81	424	486	81
thermophilus	VTGEIIHKNSRIFPAAQAENNL
LMD-9	VRRTNRQGRRLARRKKHRRVRL
gi|116627542|	NRLFEESGLITDFTKISININP
ref|	YQLRVKGLTDELSNEELFIALK
YP_820161.1|	NMVKHRGISYLDDASDDGNSSV
	GDYAQIVKENSKQLETKTPGQI
	QLERYQTYGQLRGDFTVEKDGK
	KHRLINVFPTSAYRSEALRILQ
	TQQEFNPQITDEFINRYLEILT
	GKRKYYHGPGNEKSRTDYGRYR
	TSGETLDNIFGILIGKCTFYPD
	EFRAAKASYTAQEFNLLNDLNN
	LTVPTETKKLSKEQKNQIINYV
	KNEKAMGPAKLFKYIAKLLSCD
	VADIKGYRIDKSGKAEIHTFEA
	YRKMKTLETLDIEQMDRETLDK
	LAYVLTLNTEREGIQEALEHEF
	ADGSFSQKQVDELVQFRKANSS
	IFGKGWHNFSVKLMMELIPELY
	ETSEEQMTILTRLGKQKTTSSS
	NKTKYIDEKLLTEEIYNPVVAK
	SVRQAIKIVNAAIKEYGDFDNI
	VIEMARETNEDDEKKAIQKIQK
	ANKDEKDAAMLKAANQYNGKAE
	LPHSVFHGHKQLATKIRLWHQQ
	GERCLYTGKTISIHDLINNSNQ
	FEVDHILPLSITFDDSLANKVL
	VYATANQEKGQRTPYQALDSMD
	DAWSFRELKAFVRESKTLSNKK
	KEYLLTEEDISKFDVRKKFIER
	NLVDTRYASRVVLNALQEHFRA
	HKIDTKVSVVRGQFTSQLRRHW
	GIEKTRDTYHHHAVDALIIAAS
	SQLNLWKKQKNTLVSYSEDQLL
	DIETGELISDDEYKESVFKAPY
	QHFVDTLKSKEFEDSILFSYQV
	DSKFNRKISDATIYATRQAKVG
	KDKADETYVLGKIKDIYTQDGY
	DAFMKIYKKDKSKFLMYRHDPQ
	TFEKVIEPILENYPNKQINEKG
	KEVPCNPFLKYKEEHGYIRKYS
	KKGNGPEIKSLKYYDSKLGNHI
	DITPKDSNNKVVLQSVSPWRAD
	VYFNKTTGKYEILGLKYADLQF
	EKGTGTYKISQEKYNDIKKKEG
	VDSDSEFKFTLYKNDLLLVKDT
	ETKEQQLFRFLSRTMPKQKHYV
	ELKPYDKQKFEGGEALIKVLGN
	VANSGQCKKGLGKSNISIYKVR
	TDVLGNQHIIKNEGDKPKLDF
	(SEQ ID NO: 48)
Roseburia	MNAEHGKEGLLIMEENFQYRIG	154	204	51	318	380	69	318	380	69
inulinivorans	LDIGITSVGWAVLQNNSQDEPV
DSM 16841	RITDLGVRIFDVAENPKNGDAL
gi|225377804|	AAPRRDARTTRRRLRRRRHRLE
ref|	RIKFLLQENGLIEMDSFMERYY
ZP_03755025.1	KGNLPDVYQLRYEGLDRKLKDE
	ELAQVLIHIAKHRGFRSTRKAE
	TKEKEGGAVLKATTENQKIMQE
	KGYRTVGEMLYLDEAFHTECLW
	NEKGYVLTPRNRPDDYKHTILR
	SMLVEEVHAIFAAQRAHGNQKA
	TEGLEEAYVEIMTSQRSFDMGP
	GLQPDGKPSPYAMEGFGDRVGK
	CTFEKDEYRAPKATYTAELFVA
	LQKINHTKLIDEFGTGRFFSEE
	ERKTIIGLLLSSKELKYGTIRK
	KLNIDPSLKFNSLNYSAKKEGE
	TEEERVLDTEKAKFASMFWTYE
	YSKCLKDRTEEMPVGEKADLFD
	RIGEILTAYKNDDSRSSRLKEL
	GLSGEEIDGLLDLSPAKYQRVS
	LKAMRKMQPYLEDGLIYDKACE
	AAGYDFRALNDGNKKHLLKGEE
	INAIVNDITNPVVKRSVSQTIK
	VINAIIQKYGSPQAVNIELARE
	MSKNFQDRTNLEKEMKKRQQEN
	ERAKQQIIELGKQNPTGQDILK
	YRLWNDQGGYCLYSGKKIPLEE
	LFDGGYDIDHILPYSITFDDSY
	RNKVLVTAQENRQKGNRTPYEY
	FGADEKRWEDYEASVRLLVRDY
	KKQQKLLKKNFTEEERKEFKER
	NLNDTKYITRVVYNMIRQNLEL
	EPFNHPEKKKQVWAVNGAVTSY
	LRKRWGLMQKDRSTDRHHAMDA
	VVIACCTDGMIHKISRYMQGRE
	LAYSRNFKFPDEETGEILNRDN
	FTREQWDEKFGVKVPLPWNSFR
	DELDIRLLNEDPKNFLLTHADV
	QRELDYPGWMYGEEESPIEEGR
	YINYIRPLFVSRMPNHKVTGSA
	HDATIRSARDYETRGVVITKVP
	LTDLKLNKDNEIEGYYDKDSDR
	LLYQALVRQLLLHGNDGKKAFA
	EDFHKPKADGTEGPVVRKVKIE
	KKQTSGVMVRGGTGIAANGEMV
	RIDVFRENGKYYFVPVYTADVV
	RKVLPNRAATHTKPYSEWRVMD
	DANFVFSLYSRDLIHVKSKKDI
	KTNLVNGGLLLQKEIFAYYTGA
	DIATASIAGFANDSNFKFRGLG
	IQSLEIFEKCQVDILGNISVVR
	HENRQEFH (SEQ ID NO:
	49)
Methylosinus	MRVLGLDAGIASLGWALIEIEE	144	193	50	426	488	64	426	488	64
trichosporium	SNRGELSQGTIIGAGTWMFDAP
OB3b	EEKTQAGAKLKSEQRRTFRGQR
gi|296446027|	RVVRRRRQRMNEVRRILHSHGL
ref|	LPSSDRDALKQPGLDPWRIRAE
ZP_06887976.1	ALDRLLGPVELAVALGHIARHR
	GFKSNSKGAKTNDPADDTSKMK
	RAVNETREKLARFGSAAKMLVE
	DESFVLRQTPTKNGASEIVRRF
	RNREGDYSRSLLRDDLAAEMRA
	LFTAQARFQSAIATADLQTAFT
	KAAFFQRPLQDSEKLVGPCPFE
	VDEKRAPKRGYSFELFRFLSRL
	NHVTLRDGKQERTLTRDELALA
	AADFGAAAKVSFTALRKKLKLP
	ETTVFVGVKADEESKLDVVARS
	GKAAEGTARLRSVIVDALGELA
	WGALLCSPEKLDKIAEVISFRS
	DIGRISEGLAQAGCNAPLVDAL
	TAAASDGRFDPFTGAGHISSKA
	ARNILSGLRQGMTYDKACCAAD
	YDHTASRERGAFDVGGHGREAL
	KRILQEERISRELVGSPTARKA
	LIESIKQVKAIVERYGVPDRIH
	VELARDVGKSIEEREEITRGIE
	KRNRQKDKLRGLFEKEVGRPPQ
	DGARGKEELLRFELWSEQMGRC
	LYTDDYISPSQLVATDDAVQVD
	HILPWSRFADDSYANKTLCMAK
	ANQDKKGRTPYEWFKAEKTDTE
	WDAFIVRVEALADMKGFKKRNY
	KLRNAEEAAAKFRNRNLNDTRW
	ACRLLAEALKQLYPKGEKDKDG
	KERRRVFSRPGALTDRLRRAWG
	LQWMKKSTKGDRIPDDRHHALD
	AIVIAATTESLLQRATREVQEI
	EDKGLHYDLVKNVTPPWPGFRE
	QAVEAVEKVFVARAERRRARGK
	AHDATIRHIAVREGEQRVYERR
	KVAELKLADLDRVKDAERNARL
	IEKLRNWIEAGSPKDDPPLSPK
	GDPIFKVRLVTKSKVNIALDTG
	NPKRPGTVDRGEMARVDVFRKA
	SKKGKYEYYLVPIYPHDIATMK
	TPPIRAVQAYKPEDEWPEMDSS
	YEFCWSLVPMTYLQVISSKGEI
	FEGYYRGMNRSVGAIQLSAHSN
	SSDVVQGIGARTLTEFKKENVD
	RFGRKHEVERELRTWRGETWRG
	KAYI (SEQ ID NO: 50)
Ruminococcus	MGNYYLGLDVGIGSIGWAVINI	139	187	49	351	412	55	351	412	55
albus 8	EKKRIEDFNVRIFKSGEIQEKN
gi|325677756|	RNSRASQQCRRSRGLRRLYRRK
ref|	SHRKLRLKNYLSIIGLTTSEKI
ZP_08157403.1	DYYYETADNNVIQLRNKGLSEK
	LTPEEIAACLIHICNNRGYKDF
	YEVNVEDIEDPDERNEYKEEHD
	SIVLISNLMNEGGYCTPAEMIC
	NCREFDEPNSVYRKFHNSAASK
	NHYLITRHMLVKEVDLILENQS
	KYYGILDDKTIAKIKDIIFAQR
	DFEIGPGKNERFRRFTGYLDSI
	GKCQFFKDQERGSRFTVIADIY
	AFVNVLSQYTYTNNRGESVFDT
	SFANDLINSALKNGSMDKRELK
	AIAKSYHIDISDKNSDTSLTKC
	FKYIKVVKPLFEKYGYDWDKLI
	ENYTDTDNNVLNRIGIVLSQAQ
	TPKRRREKLKALNIGLDDGLIN
	ELTKLKLSGTANVSYKYMQGSI
	EAFCEGDLYGKYQAKFNKEIPD
	IDENAKPQKLPPFKNEDDCEFF
	KNPVVFRSINETRKLINAIIDK
	YGYPAAVNIETADELNKTFEDR
	AIDTKRNNDNQKENDRIVKEII
	ECIKCDEVHARHLIEKYKLWEA
	QEGKCLYSGETITKEDMLRDKD
	KLFEVDHIVPYSLILDNTINNK
	ALVYAEENQKKGQRTPLMYMNE
	AQAADYRVRVNTMFKSKKCSKK
	KYQYLMLPDLNDQELLGGWRSR
	NLNDTRYICKYLVNYLRKNLRF
	DRSYESSDEDDLKIRDHYRVFP
	VKSRFTSMFRRWWLNEKTWGRY
	DKAELKKLTYLDHAADAIIIAN
	CRPEYVVLAGEKLKLNKMYHQA
	GKRITPEYEQSKKACIDNLYKL
	FRMDRRTAEKLLSGHGRLTPII
	PNLSEEVDKRLWDKNIYEQFWK
	DDKDKKSCEELYRENVASLYKG
	DPKFASSLSMPVISLKPDHKYR
	GTITGEEAIRVKEIDGKLIKLK
	RKSISEITAESINSIYTDDKIL
	IDSLKTIFEQADYKDVGDYLKK
	TNQHFFTTSSGKRVNKVTVIEK
	VPSRWLRKEIDDNNFSLLNDSS
	YYCIELYKDSKGDNNLQGIAMS
	DIVHDRKTKKLYLKPDFNYPDD
	YYTHVMYIFPGDYLRIKSTSKK
	SGEQLKFEGYFISVKNVNENSF
	RFISDNKPCAKDKRVSITKKDI
	VIKLAVDLMGKVQGENNGKGIS
	CGEPLSLLKEKN (SEQ ID
	NO: 51)
Bifidobacterium	MLSRQLLGASHLARPVSYSYNV	183	230	48	370	431	44	370	431	44
longum	QDNDVHCSYGERCFMRGKRYRI
DJO10A	GIDVGLNSVGLAAVEVSDENSP
gi|189440764|	VRLLNAQSVIHDGGVDPQKNKE
ref|	AITRKNMSGVARRTRRMRRRKR
YP_001955845.	ERLHKLDMLLGKFGYPVIEPES
	LDKPFEEWHVRAELATRYIEDD
	ELRRESISIALRHMARHRGWRN
	PYRQVDSLISDNPYSKQYGELK
	EKAKAYNDDATAAEEESTPAQL
	VVAMLDAGYAEAPRLRWRTGSK
	KPDAEGYLPVRLMQEDNANELK
	QIFRVQRVPADEWKPLFRSVFY
	AVSPKGSAEQRVGQDPLAPEQA
	RALKASLAFQEYRIANVITNLR
	IKDASAELRKLTVDEKQSIYDQ
	LVSPSSEDITWSDLCDFLGFKR
	SQLKGVGSLTEDGEERISSRPP
	RLTSVQRIYESDNKIRKPLVAW
	WKSASDNEHEAMIRLLSNTVDI
	DKVREDVAYASAIEFIDGLDDD
	ALTKLDSVDLPSGRAAYSVETL
	QKLTRQMLTTDDDLHEARKTLF
	NVTDSWRPPADPIGEPLGNPSV
	DRVLKNVNRYLMNCQQRWGNPV
	SVNIEHVRSSFSSVAFARKDKR
	EYEKNNEKRSIFRSSLSEQLRA
	DEQMEKVRESDLRRLEAIQRQN
	GQCLYCGRTITFRTCEMDHIVP
	RKGVGSTNTRTNFAAVCAECNR
	MKSNTPFAIWARSEDAQTRGVS
	LAEAKKRVTMFTFNPKSYAPRE
	VKAFKQAVIARLQQTEDDAAID
	NRSIESVAWMADELHRRIDWYF
	NAKQYVNSASIDDAEAETMKTT
	VSVFQGRVTASARRAAGIEGKI
	HFIGQQSKTRLDRRHHAVDASV
	IAMMNTAAAQTLMERESLRESQ
	RLIGLMPGERSWKEYPYEGTSR
	YESFHLWLDNMDVLLELLNDAL
	DNDRIAVMQSQRYVLGNSIAHD
	ATIHPLEKVPLGSAMSADLIRR
	ASTPALWCALTRLPDYDEKEGL
	PEDSHREIRVHDTRYSADDEMG
	FFASQAAQIAVQEGSADIGSAI
	HHARVYRCWKTNAKGVRKYFYG
	MIRVFQTDLLRACHDDLFTVPL
	PPQSISMRYGEPRVVQALQSGN
	AQYLGSLVVGDEIEMDFSSLDV
	DGQIGEYLQFFSQFSGGNLAWK
	HWVVDGFFNQTQLRIRPRYLAA
	EGLAKAFSDDVVPDGVQKIVTK
	QGWLPPVNTASKTAVRIVRRNA
	FGEPRLSSAHHMPCSWQWRHE
	(SEQ ID NO: 52)
Enterococcus	MYSIGLDLGISSVGWSVIDERT	123	170	48	327	387	60	327	387	60
faecalis	GNVIDLGVRLFSAKNSEKNLER
TX0012	RTNRGGRRLIRRKTNRLKDAKK
gi|315149830|	ILAAVGFYEDKSLKNSCPYQLR
gb|	VKGLTEPLSRGEIYKVTLHILK
EFT93846.1|	KRGISYLDEVDTEAAKESQDYK
	EQVRKNAQLLTKYTPGQIQLQR
	LKENNRVKTGINAQGNYQLNVF
	KVSAYANELATILKTQQAFYPN
	ELTDDWIALFVQPGIAEEAGLI
	YRKRPYYHGPGNEANNSPYGRW
	SDFQKTGEPATNIFDKLIGKDF
	QGELRASGLSLSAQQYNLLNDL
	TNLKIDGEVPLSSEQKEYILTE
	LMTKEFTRFGVNDVVKLLGVKK
	ERLSGWRLDKKGKPEIHTLKGY
	RNWRKIFAEAGIDLATLPTETI
	DCLAKVLTLNTEREGIENTLAF
	ELPELSESVKLLVLDRYKELSQ
	SISTQSWHRFSLKTLHLLIPEL
	MNATSEQNTLLEQFQLKSDVRK
	RYSEYKKLPTKDVLAEIYNPTV
	NKTVSQAFKVIDALLVKYGKEQ
	IRYITIEMPRDDNEEDEKKRIK
	ELHAKNSQRKNDSQSYFMQKSG
	WSQEKFQTTIQKNRRFLAKLLY
	YYEQDGICAYTGLPISPELLVS
	DSTEIDHIIPISISLDDSINNK
	VLVLSKANQVKGQQTPYDAWMD
	GSFKKINGKFSNWDDYQKWVES
	RHFSHKKENNLLETRNIFDSEQ
	VEKFLARNLNDTRYASRLVLNT
	LQSFFTNQETKVRVVNGSFTHT
	LRKKWGADLDKTRETHHHHAVD
	ATLCAVTSFVKVSRYHYAVKEE
	TGEKVMREIDFETGEIVNEMSY
	WEFKKSKKYERKTYQVKWPNFR
	EQLKPVNLHPRIKFSHQVDRKA
	NRKLSDATIYSVREKTEVKTLK
	SGKQKITTDEYTIGKIKDIYTL
	DGWEAFKKKQDKLLMKDLDEKT
	YERLLSIAETTPDFQEVEEKNG
	KVKRVKRSPFAVYCEENDIPAI
	QKYAKKNNGPLIRSLKYYDGKL
	NKHINITKDSQGRPVEKTKNGR
	KVTLQSLKPYRYDIYQDLETKA
	YYTVQLYYSDLRFVEGKYGITE
	KEYMKKVAEQTKGQVVRFCFSL
	QKNDGLEIEWKDSQRYDVRFYN
	FQSANSINFKGLEQEMMPAENQ
	FKQKPYNNGAINLNIAKYGKEG
	KKLRKFNTDILGKKHYLFYEKE
	PKNIIK (SEQ ID NO: 53)
Mycoplasma	MYFYKNKENKLNKKVVLGLDLG	179	226	48	314	374	79	314	374	79
mobile 163K	IASVGWCLTDISQKEDNKFPII
gi|47458868|	LHGVRLFETVDDSDDKLLNETR
ref|	RKKRGQRRRNRRLFTRKRDFIK
YP_015730.1|	YLIDNNIIELEFDKNPKILVRN
	FIEKYINPFSKNLELKYKSVTN
	LPIGFHNLRKAAINEKYKLDKS
	ELIVLLYFYLSLRGAFFDNPED
	TKSKEMNKNEIEIFDKNESIKN
	AEFPIDKIIEFYKISGKIRSTI
	NLKFGHQDYLKEIKQVFEKQNI
	DFMNYEKFAMEEKSFFSRIRNY
	SEGPGNEKSFSKYGLYANENGN
	PELIINEKGQKIYTKIFKTLWE
	SKIGKCSYDKKLYRAPKNSFSA
	KVFDITNKLTDWKHKNEYISER
	LKRKILLSRFLNKDSKSAVEKI
	LKEENIKFENLSEIAYNKDDNK
	INLPIINAYHSLTTIFKKHLIN
	FENYLISNENDLSKLMSFYKQQ
	SEKLFVPNEKGSYEINQNNNVL
	HIFDAISNILNKFSTIQDRIRI
	LEGYFEFSNLKKDVKSSEIYSE
	IAKLREFSGTSSLSFGAYYKFI
	PNLISEGSKNYSTISYEEKALQ
	NQKNNFSHSNLFEKTWVEDLIA
	SPTVKRSLRQTMNLLKEIFKYS
	EKNNLEIEKIVVEVTRSSNNKH
	ERKKIEGINKYRKEKYEELKKV
	YDLPNENTTLLKKLWLLRQQQG
	YDAYSLRKIEANDVINKPWNYD
	IDHIVPRSISFDDSFSNLVIVN
	KLDNAKKSNDLSAKQFIEKIYG
	IEKLKEAKENWGNWYLRNANGK
	AFNDKGKFIKLYTIDNLDEFDN
	SDFINRNLSDTSYITNALVNHL
	TFSNSKYKYSVVSVNGKQTSNL
	RNQIAFVGIKNNKETEREWKRP
	EGFKSINSNDFLIREEGKNDVK
	DDVLIKDRSFNGHHAEDAYFIT
	IISQYFRSFKRIERLNVNYRKE
	TRELDDLEKNNIKFKEKASFDN
	FLLINALDELNEKLNQMRFSRM
	VITKKNTQLFNETLYSGKYDKG
	KNTIKKVEKLNLLDNRTDKIKK
	IEEFFDEDKLKENELTKLHIFN
	HDKNLYETLKIIWNEVKIEIKN
	KNLNEKNYFKYFVNKKLQEGKI
	SFNEWVPILDNDFKIIRKIRYI
	KFSSEEKETDEIIFSQSNFLKI
	DQRQNFSFHNTLYWVQIWVYKN
	QKDQYCFISIDARNSKFEKDEI
	KINYEKLKTQKEKLQIINEEPI
	LKINKGDLFENEEKELFYIVGR
	DEKPQKLEIKYILGKKIKDQKQ
	IQKPVKKYFPNWKKVNLTYMGE
	IFKK (SEQ ID NO: 54)
Actinomyces	MDNKNYRIGIDVGLNSIGFCAV	147	193	47	358	418	40	358	418	40
coleocanis	EVDQHDTPLGFLNLSVYRHDAG
DSM 15436	IDPNGKKTNTTRLAMSGVARRT
gi|227494853|	RRLFRKRKRRLAALDRFIEAQG
ref|	WTLPDHADYKDPYTPWLVRAEL
ZP_03925169.1	AQTPIRDENDLHEKLAIAVRHI
	ARHRGWRSPWVPVRSLHVEQPP
	SDQYLALKERVEAKTLLQMPEG
	ATPAEMVVALDLSVDVNLRPKN
	REKTDTRPENKKPGFLGGKLMQ
	SDNANELRKIAKIQGLDDALLR
	ELIELVFAADSPKGASGELVGY
	DVLPGQHGKRRAEKAHPAFQRY
	RIASIVSNLRIRHLGSGADERL
	DVETQKRVFEYLLNAKPTADIT
	WSDVAEEIGVERNLLMGTATQT
	ADGERASAKPPVDVTNVAFATC
	KIKPLKEWWLNADYEARCVMVS
	ALSHAEKLTEGTAAEVEVAEFL
	QNLSDEDNEKLDSFSLPIGRAA
	YSVDSLERLTKRMIENGEDLFE
	ARVNEFGVSEDWRPPAEPIGAR
	VGNPAVDRVLKAVNRYLMAAEA
	EWGAPLSVNIEHVREGFISKRQ
	AVEIDRENQKRYQRNQAVRSQI
	ADHINATSGVRGSDVTRYLAIQ
	RQNGECLYCGTAITFVNSEMDH
	IVPRAGLGSTNTRDNLVATCER
	CNKSKSNKPFAVWAAECGIPGV
	SVAEALKRVDFWIADGFASSKE
	HRELQKGVKDRLKRKVSDPEID
	NRSMESVAWMARELAHRVQYYF
	DEKHTGTKVRVFRGSLTSAARK
	ASGFESRVNFIGGNGKTRLDRR
	HHAMDAATVAMLRNSVAKTLVL
	RGNIRASERAIGAAETWKSFRG
	ENVADRQIFESWSENMRVLVEK
	FNLALYNDEVSIFSSLRLQLGN
	GKAHDDTITKLQMHKVGDAWSL
	TEIDRASTPALWCALTRQPDFT
	WKDGLPANEDRTIIVNGTHYGP
	LDKVGIFGKAAASLLVRGGSVD
	IGSAIHHARIYRIAGKKPTYGM
	VRVFAPDLLRYRNEDLENVELP
	PQSVSMRYAEPKVREAIREGKA
	EYLGWLVVGDELLLDLSSETSG
	QIAELQQDFPGTTHWTVAGFFS
	PSRLRLRPVYLAQEGLGEDVSE
	GSKSIIAGQGWRPAVNKVFGSA
	MPEVIRRDGLGRKRRFSYSGLP
	VSWQG (SEQ ID NO: 55)
Dinoroseobacter	MRLGLDIGTSSIGWWLYETDGA	138	184	47	338	398	48	338	398	48
shibae	GSDARITGVVDGGVRIFSDGRD
DFL 12	PKSGASLAVDRRAARAMRRRRD
gi|159042956|	RYLRRRATLMKVLAETGLMPAD
ref|	PAEAKALEALDPFALRAAGLDE
YP_001531750.1	PLPLPHLGRALFHLNQRRGFKS
	NRKTDRGDNESGKIKDATARLD
	MEMMANGARTYGEFLHKRRQKA
	TDPRHVPSVRTRLSIANRGGPD
	GKEEAGYDFYPDRRHLEEEFHK
	LWAAQGAHHPELTETLRDLLFE
	KIFFQRPLKEPEVGLCLFSGHH
	GVPPKDPRLPKAHPLTQRRVLY
	ETVNQLRVTADGREARPLTREE
	RDQVIHALDNKKPTKSLSSMVL
	KLPALAKVLKLRDGERFTLETG
	VRDAIACDPLRASPAHPDRFGP
	RWSILDADAQWEVISRIRRVQS
	DAEHAALVDWLTEAHGLDRAHA
	EATAHAPLPDGYGRLGLTATTR
	ILYQLTADVVTYADAVKACGWH
	HSDGRTGECFDRLPYYGEVLER
	HVIPGSYHPDDDDITRFGRITN
	PTVHIGLNQLRRLVNRIIETHG
	KPHQIVVELARDLKKSEEQKRA
	DIKRIRDTTEAAKKRSEKLEEL
	EIEDNGRNRMLLRLWEDLNPDD
	AMRRFCPYTGTRISAAMIFDGS
	CDVDHILPYSRTLDDSFPNRTL
	CLREANRQKRNQTPWQAWGDTP
	HWHAIAANLKNLPENKRWRFAP
	DAMTRFEGENGFLDRALKDTQY
	LARISRSYLDTLFTKGGHVWVV
	PGRFTEMLRRHWGLNSLLSDAG
	RGAVKAKNRTDHRHHAIDAAVI
	AATDPGLLNRISRAAGQGEAAG
	QSAELIARDTPPPWEGFRDDLR
	VRLDRIIVSHRADHGRIDHAAR
	KQGRDSTAGQLHQETAYSIVDD
	IHVASRTDLLSLKPAQLLDEPG
	RSGQVRDPQLRKALRVATGGKT
	GKDFENALRYFASKPGPYQAIR
	RVRIIKPLQAQARVPVPAQDPI
	KAYQGGSNHLFEIWRLPDGEIE
	AQVITSFEAHTLEGEKRPHPAA
	KRLLRVHKGDMVALERDGRRVV
	GHVQKMDIANGLFIVPHNEANA
	DTRNNDKSDPFKWIQIGARPAI
	ASGIRRVSVDEIGRLRDGGTRP
	I (SEQ ID NO: 56)
Actinomyces	MLHCIAVIRVPPSEEPGFFETH	183	228	46	349	409	40	349	409	40
sp. oral	ADSCALCHHGCMTYAANDKAIR
taxon 180	YRVGIDVGLRSIGFCAVEVDDE
str. F0310	DHPIRILNSVVHVHDAGTGGPG
gi|315605738|	ETESLRKRSGVAARARRRGRAE
ref|	KQRLKKLDVLLEELGWGVSSNE
8ZP_0780770.1	LLDSHAPWHIRKRLVSEYIEDE
	TERRQCLSVAMAHIARHRGWRN
	SFSKVDTLLLEQAPSDRMQGLK
	ERVEDRTGLQFSEEVTQGELVA
	TLLEHDGDVTIRGFVRKGGKAT
	KVHGVLEGKYMQSDLVAELRQI
	CRTQRVSETTFEKLVLSIFHSK
	EPAPSAARQRERVGLDELQLAL
	DPAAKQPRAERAHPAFQKFKVV
	ATLANMRIREQSAGERSLTSEE
	LNRVARYLLNHTESESPTWDDV
	ARKLEVPRHRLRGSSRASLETG
	GGLTYPPVDDTTVRVMSAEVDW
	LADWWDCANDESRGHMIDAISN
	GCGSEPDDVEDEEVNELISSAT
	AEDMLKLELLAKKLPSGRVAYS
	LKTLREVTAAILETGDDLSQAI
	TRLYGVDPGWVPTPAPIEAPVG
	NPSVDRVLKQVARWLKFASKRW
	GVPQTVNIEHTREGLKSASLLE
	EERERWERFEARREIRQKEMYK
	RLGISGPFRRSDQVRYEILDLQ
	DCACLYCGNEINFQTFEVDHII
	PRVDASSDSRRTNLAAVCHSCN
	SAKGGLAFGQWVKRGDCPSGVS
	LENAIKRVRSWSKDRLGLTEKA
	MGKRKSEVISRLKTEMPYEEFD
	GRSMESVAWMAIELKKRIEGYF
	NSDRPEGCAAVQVNAYSGRLTA
	CARRAAHVDKRVRLIRLKGDDG
	HHKNRFDRRNHAMDALVIALMT
	PAIARTIAVREDRREAQQLTRA
	FESWKNFLGSEERMQDRWESWI
	GDVEYACDRLNELIDADKIPVT
	ENLRLRNSGKLHADQPESLKKA
	RRGSKRPRPQRYVLGDALPADV
	INRVTDPGLWTALVRAPGFDSQ
	LGLPADLNRGLKLRGKRISADF
	PIDYFPTDSPALAVQGGYVGLE
	FHHARLYRIIGPKEKVKYALLR
	VCAIDLCGIDCDDLFEVELKPS
	SISMRTADAKLKEAMGNGSAKQ
	IGWLVLGDEIQIDPTKFPKQSI
	GKFLKECGPVSSWRVSALDTPS
	KITLKPRLLSNEPLLKTSRVGG
	HESDLVVAECVEKIMKKTGWVV
	EINALCQSGLIRVIRRNALGEV
	RTSPKSGLPISLNLR
	(SEQ ID NO: 57)
Alcanivorax	MRYRVGLDLGTASVGAAVFSMD	139	183	45	344	404	61	344	404	61
sp. W11-5	EQGNPMELIWHYERLFSEPLVP
gi|407803669|	DMGQLKPKKAARRLARQQRRQI
ref|	DRRASRLRRIAIVSRRLGIAPG
ZP_11150502.1	RNDSGVHGNDVPTLRAMAVNER
	IELGQLRAVLLRMGKKRGYGGT
	FKAVRKVGEAGEVASGASRLEE
	EMVALASVQNKDSVTVGEYLAA
	RVEHGLPSKLKVAANNEYYAPE
	YALFRQYLGLPAIKGRPDCLPN
	MYALRHQIEHEFERIWATQSQF
	HDVMKDHGVKEEIRNAIFFQRP
	LKSPADKVGRCSLQTNLPRAPR
	AQIAAQNFRIEKQMADLRWGMG
	RRAEMLNDHQKAVIRELLNQQK
	ELSFRKIYKELERAGCPGPEGK
	GLNMDRAALGGRDDLSGNTTLA
	AWRKLGLEDRWQELDEVTQIQV
	INFLADLGSPEQLDTDDWSCRF
	MGKNGRPRNFSDEFVAFMNELR
	MTDGFDRLSKMGFEGGRSSYSI
	KALKALTEWMIAPHWRETPETH
	RVDEEAAIRECYPESLATPAQG
	GRQSKLEPPPLTGNEVVDVALR
	QVRHTINMMIDDLGSVPAQIVV
	EMAREMKGGVTRRNDIEKQNKR
	FASERKKAAQSIEENGKTPTPA
	RILRYQLWIEQGHQCPYCESNI
	SLEQALSGAYTNFEHILPRTLT
	QIGRKRSELVLAHRECNDEKGN
	RTPYQAFGHDDRRWRIVEQRAN
	ALPKKSSRKTRLLLLKDFEGEA
	LTDESIDEFADRQLHESSWLAK
	VTTQWLSSLGSDVYVSRGSLTA
	ELRRRWGLDTVIPQVRFESGMP
	VVDEEGAEITPEEFEKFRLQWE
	GHRVTREMRTDRRPDKRIDHRH
	HLVDAIVTALTSRSLYQQYAKA
	WKVADEKQRHGRVDVKVELPMP
	ILTIRDIALEAVRSVRISHKPD
	RYPDGRFFEATAYGIAQRLDER
	SGEKVDWLVSRKSLTDLAPEKK
	SIDVDKVRANISRIVGEAIRLH
	ISNIFEKRVSKGMTPQQALREP
	IEFQGNILRKVRCFYSKADDCV
	RIEHSSRRGHHYKMLLNDGFAY
	MEVPCKEGILYGVPNLVRPSEA
	VGIKRAPESGDFIRFYKGDTVK
	NIKTGRVYTIKQILGDGGGKLI
	LTPVTETKPADLLSAKWGRLKV
	GGRNIHLLRLCAE (SEQ ID
	NO: 58)
Aminomonas	MIGEHVRGGCLFDDHWTPNWGA	134	178	45	341	401	63	341	401	63
paucivorans	FRLPNTVRTFTKAENPKDGSSL
DSM 12260	AEPRRQARGLRRRLRRKTQRLE
gi|312879015|	DLRRLLAKEGVLSLSDLETLFR
ref|	ETPAKDPYQLRAEGLDRPLSFP
ZP_07738815.1	EWVRVLYHITKHRGFQSNRRNP
	VEDGQERSRQEEEGKLLSGVGE
	NERLLREGGYRTAGEMLARDPK
	FQDHRRNRAGDYSHTLSRSLLL
	EEARRLFQSQRTLGNPHASSNL
	EEAFLHLVAFQNPFASGEDIRN
	KAGHCSLEPDQIRAPRRSASAE
	TFMLLQKTGNLRLIHRRTGEER
	PLTDKEREQIHLLAWKQEKVTH
	KTLRRHLEIPEEWLFTGLPYHR
	SGDKAEEKLFVHLAGIHEIRKA
	LDKGPDPAVWDTLRSRRDLLDS
	IADTLTFYKNEDEILPRLESLG
	LSPENARALAPLSFSGTAHLSL
	SALGKLLPHLEEGKSYTQARAD
	AGYAAPPPDRHPKLPPLEEADW
	RNPVVFRALTQTRKVVNALVRR
	YGPPWCIHLETARELSQPAKVR
	RRIETEQQANEKKKQQAEREFL
	DIVGTAPGPGDLLKMRLWREQG
	GFCPYCEEYLNPTRLAEPGYAE
	MDHILPYSRSLDNGWHNRVLVH
	GKDNRDKGNRTPFEAFGGDTAR
	WDRLVAWVQASHLSAPKKRNLL
	REDFGEEAERELKDRNLTDTRF
	ITKTAATLLRDRLTFHPEAPKD
	PVMTLNGRLTAFLRKQWGLHKN
	RKNGDLHHALDAAVLAVASRSF
	VYRLSSHNAAWGELPRGREAEN
	GFSLPYPAFRSEVLARLCPTRE
	EILLRLDQGGVGYDEAFRNGLR
	PVFVSRAPSRRLRGKAHMETLR
	SPKWKDHPEGPRTASRIPLKDL
	NLEKLERMVGKDRDRKLYEALR
	ERLAAFGGNGKKAFVAPFRKPC
	RSGEGPLVRSLRIFDSGYSGVE
	LRDGGEVYAVADHESMVRVDVY
	AKKNRFYLVPVYVADVARGIVK
	NRAIVAHKSEEEWDLVDGSFDF
	RFSLFPGDLVEIEKKDGAYLGY
	YKSCHRGDGRLLLDRHDRMPRE
	SDCGTFYVSTRKDVLSMSKYQV
	DPLGEIRLVGSEKPPFVL
	(SEQ ID NO: 59)
Mycoplasma	MEKKRKVTLGFDLGIASVGWAI	139	183	45	319	379	76	319	379	76
canis PG 14	VDSETNQVYKLGSRLFDAPDTN
gi|384393286|	LERRTQRGTRRLLRRRKYRNQK
gb|EIE39736.1|	FYNLVKRTEVFGLSSREAIENR
	FRELSIKYPNIIELKTKALSQE
	VCPDEIAWILHDYLKNRGYFYD
	EKETKEDFDQQTVESMPSYKLN
	EFYKKYGYFKGALSQPTESEMK
	DNKDLKEAFFFDFSNKEWLKEI
	NYFFNVQKNILSETFIEEFKKI
	FSFTRDISKGPGSDNMPSPYGI
	FGEFGDNGQGGRYEHIWDKNIG
	KCSIFTNEQRAPKYLPSALIFN
	FLNELANIRLYSTDKKNIQPLW
	KLSSVDKLNILLNLFNLPISEK
	KKKLTSTNINDIVKKESIKSIM
	ISVEDIDMIKDEWAGKEPNVYG
	VGLSGLNIEESAKENKFKFQDL
	KILNVLINLLDNVGIKFEFKDR
	NDIIKNLELLDNLYLFLIYQKE
	SNNKDSSIDLFIAKNESLNIEN
	LKLKLKEFLLGAGNEFENHNSK
	THSLSKKAIDEILPKLLDNNEG
	WNLEAIKNYDEEIKSQIEDNSS
	LMAKQDKKYLNDNFLKDAILPP
	NVKVTFQQAILIFNKIIQKFSK
	DFEIDKVVIELAREMTQDQEND
	ALKGIAKAQKSKKSLVEERLEA
	NNIDKSVENDKYEKLIYKIFLW
	ISQDFKDPYTGAQISVNEIVNN
	KVEIDHIIPYSLCFDDSSANKV
	LVHKQSNQEKSNSLPYEYIKQG
	HSGWNWDEFTKYVKRVFVNNVD
	SILSKKERLKKSENLLTASYDG
	YDKLGFLARNLNDTRYATILFR
	DQLNNYAEHHLIDNKKMFKVIA
	MNGAVTSFIRKNMSYDNKLRLK
	DRSDFSHHAYDAAIIALFSNKT
	KTLYNLIDPSLNGIISKRSEGY
	WVIEDRYTGEIKELKKEDWTSI
	KNNVQARKIAKEIEEYLIDLDD
	EVFFSRKTKRKTNRQLYNETIY
	GIATKTDEDGITNYYKKEKFSI
	LDDKDIYLRLLREREKFVINQS
	NPEVIDQIIEIIESYGKENNIP
	SRDEAINIKYTKNKINYNLYLK
	QYMRSLTKSLDQFSEEFINQMI
	ANKTFVLYNPTKNTTRKIKFLR
	LVNDVKINDIRKNQVINKENGK
	NNEPKAFYENINSLGAIVFKNS
	ANNFKTLSINTQIAIFGDKNWD
	IEDFKTYNMEKIEKYKEIYGID
	KTYNFHSFIFPGTILLDKQNKE
	FYYISSIQTVRDIIEIKFLNKI
	EFKDENKNQDTSKTPKRLMFGI
	KSIMNNYEQVDISPFGINKKIF
	E (SEQ ID NO: 60)
Lactobacillus	MGYRIGLDVGITSTGYAVLKTD	141	184	44	328	387	61	328	387	61
coryniformis	KNGLPYKILTLDSVIYPRAENP
KCTC 3535	QTGASLAEPRRIKRGLRRRTRR
gi|336393381|	TKFRKQRTQQLFIHSGLLSKPE
ref|	IEQILATPQAKYSVYELRVAGL
ZP_08574780.1	DRRLTNSELFRVLYFFIGHRGF
	KSNRKAELNPENEADKKQMGQL
	LNSIEEIRKAIAEKGYRTVGEL
	YLKDPKYNDHKRNKGYIDGYLS
	TPNRQMLVDEIKQILDKQRELG
	NEKLTDEFYATYLLGDENRAGI
	FQAQRDFDEGPGAGPYAGDQIK
	KMVGKDIFEPTEDRAAKATYTF
	QYFNLLQKMTSLNYQNTTGDTW
	HTLNGLDRQAIIDAVFAKAEKP
	TKTYKPTDFGELRKLLKLPDDA
	RFNLVNYGSLQTQKEIETVEKK
	TRFVDFKAYHDLVKVLPEEMWQ
	SRQLLDHIGTALTLYSSDKRRR
	RYFAEELNLPAELIEKLLPLNF
	SKFGHLSIKSMQNIIPYLEMGQ
	VYSEATTNTGYDFRKKQISKDT
	IREEITNPVVRRAVTKTIKIVE
	QIIRRYGKPDGINIELARELGR
	NFKERGDIQKRQDKNRQTNDKI
	AAELTELGIPVNGQNIIRYKLH
	KEQNGVDPYTGDQIPFERAFSE
	GYEVDHIIPYSISWDDSYTNKV
	LTSAKCNREKGNRIPMVYLANN
	EQRLNALTNIADNIIRNSRKRQ
	KLLKQKLSDEELKDWKQRNIND
	TRFITRVLYNYFRQAIEFNPEL
	EKKQRVLPLNGEVTSKIRSRWG
	FLKVREDGDLHHAIDATVIAAI
	TPKFIQQVTKYSQHQEVKNNQA
	LWHDAEIKDAEYAAEAQRMDAD
	LFNKIFNGFPLPWPEFLDELLA
	RISDNPVEMMKSRSWNTYTPIE
	IAKLKPVFVVRLANHKISGPAH
	LDTIRSAKLFDEKGIVLSRVSI
	TKLKINKKGQVATGDGIYDPEN
	SNNGDKVVYSAIRQALEAHNGS
	GELAFPDGYLEYVDHGTKKLVR
	KVRVAKKVSLPVRLKNKAAADN
	GSMVRIDVFNTGKKFVFVPIYI
	KDTVEQVLPNKAIARGKSLWYQ
	ITESDQFCFSLYPGDMVHIESK
	TGIKPKYSNKENNTSVVPIKNF
	YGYFDGADIATASILVRAHDSS
	YTARSIGIAGLLKFEKYQVDYF
	GRYHKVHEKKRQLFVKRDE
	(SEQ ID NO: 61)
Elusimicrobium	MQKNINTKQNHIYIKQAQKIKE	177	219	43	322	381	47	322	381	47
minutum	KLGDKPYRIGLDLGVGSIGFAI
Pei191	VSMEENDGNVLLPKEIIMVGSR
gi|187250660|	IFKASAGAADRKLSRGQRNNHR
ref|	HTRERMRYLWKVLAEQKLALPV
YP_001875142.1	PADLDRKENSSEGETSAKRFLG
	DVLQKDIYELRVKSLDERLSLQ
	ELGYVLYHIAGHRGSSAIRTFE
	NDSEEAQKENTENKKIAGNIKR
	LMAKKNYRTYGEYLYKEFFENK
	EKHKREKISNAANNHKFSPTRD
	LVIKEAEAILKKQAGKDGFHKE
	LTEEYIEKLTKAIGYESEKLIP
	ESGFCPYLKDEKRLPASHKLNE
	ERRLWETLNNARYSDPIVDIVT
	GEITGYYEKQFTKEQKQKLFDY
	LLTGSELTPAQTKKLLGLKNTN
	FEDIILQGRDKKAQKIKGYKLI
	KLESMPFWARLSEAQQDSFLYD
	WNSCPDEKLLTEKLSNEYHLTE
	EEIDNAFNEIVLSSSYAPLGKS
	AMLIILEKIKNDLSYTEAVEEA
	LKEGKLTKEKQAIKDRLPYYGA
	VLQESTQKIIAKGFSPQFKDKG
	YKTPHTNKYELEYGRIANPVVH
	QTLNELRKLVNEIIDILGKKPC
	EIGLETARELKKSAEDRSKLSR
	EQNDNESNRNRIYEIYIRPQQQ
	VIITRRENPRNYILKFELLEEQ
	KSQCPFCGGQISPNDIINNQAD
	IEHLFPIAESEDNGRNNLVISH
	SACNADKAKRSPWAAFASAAKD
	SKYDYNRILSNVKENIPHKAWR
	FNQGAFEKFIENKPMAARFKTD
	NSYISKVAHKYLACLFEKPNII
	CVKGSLTAQLRMAWGLQGLMIP
	FAKQLITEKESESFNKDVNSNK
	KIRLDNRHHALDAIVIAYASRG
	YGNLLNKMAGKDYKINYSERNW
	LSKILLPPNNIVWENIDADLES
	FESSVKTALKNAFISVKHDHSD
	NGELVKGTMYKIFYSERGYTLT
	TYKKLSALKLTDPQKKKTPKDF
	LETALLKFKGRESEMKNEKIKS
	AIENNKRLFDVIQDNLEKAKKL
	LEEENEKSKAEGKKEKNINDAS
	IYQKAISLSGDKYVQLSKKEPG
	KFFAISKPTPTTTGYGYDTGDS
	LCVDLYYDNKGKLCGEIIRKID
	AQQKNPLKYKEQGFTLFERIYG
	GDILEVDFDIHSDKNSFRNNTG
	SAPENRVFIKVGTFTEITNNNI
	QIWFGNIIKSTGGQDDSFTINS
	MQQYNPRKLILSSCGFIKYRSP
	ILKNKEG (SEQ ID NO:
	62)
Neisseria	MAAFKPNPINYILGLDIGIASV	147	189	43	360	419	61	360	419	61
meningitidis	GWAMVEIDEDENPICLIDLGVR
Z2491	VFERAEVPKTGDSLAMARRLAR
gi|218767588|	SVRRLTRRRAHRLLRARRLLKR
ref|	EGVLQAADFDENGLIKSLPNTP
YP_002342100.1	WQLRAAALDRKLTPLEWSAVLL
	HLIKHRGYLSQRKNEGETADKE
	LGALLKGVADNAHALQTGDFRT
	PAELALNKFEKESGHIRNQRGD
	YSHTFSRKDLQAELILLFEKQK
	EFGNPHVSGGLKEGIETLLMTQ
	RPALSGDAVQKMLGHCTFEPAE
	PKAAKNTYTAERFIWLTKLNNL
	RILEQGSERPLTDTERATLMDE
	PYRKSKLTYAQARKLLGLEDTA
	FFKGLRYGKDNAEASTLMEMKA
	YHAISRALEKEGLKDKKSPLNL
	SPELQDEIGTAFSLFKTDEDIT
	GRLKDRIQPEILEALLKHISFD
	KFVQISLKALRRIVPLMEQGKR
	YDEACAEIYGDHYGKKNTEEKI
	YLPPIPADEIRNPVVLRALSQA
	RKVINGVVRRYGSPARIHIETA
	REVGKSFKDRKEIEKRQEENRK
	DREKAAAKFREYFPNFVGEPKS
	KDILKLRLYEQQHGKCLYSGKE
	INLGRLNEKGYVEIDHALPFSR
	TWDDSFNNKVLVLGSENQNKGN
	QTPYEYFNGKDNSREWQEFKAR
	VETSRFPRSKKQRILLQKFDED
	GFKERNLNDTRYVNRFLCQFVA
	DRMRLTGKGKKRVFASNGQITN
	LLRGFWGLRKVRAENDRHHALD
	AVVVACSTVAMQQKITRFVRYK
	EMNAFDGKTIDKETGEVLHQKT
	HFPQPWEFFAQEVMIRVFGKPD
	GKPEFEEADTPEKLRTLLAEKL
	SSRPEAVHEYVTPLFVSRAPNR
	KMSGQGHMETVKSAKRLDEGVS
	VLRVPLTQLKLKDLEKMVNRER
	EPKLYEALKARLEAHKDDPAKA
	FAEPFYKYDKAGNRTQQVKAVR
	VEQVQKTGVWVRNHNGIADNAT
	MVRVDVFEKGDKYYLVPIYSWQ
	VAKGILPDRAVVQGKDEEDWQL
	IDDSFNFKFSLHPNDLVEVITK
	KARMFGYFASCHRGTGNINIRI
	HDLDHKIGKNGILEGIGVKTAL
	SFQKYQIDELGKEIRPCRLKKR
	PPVR (SEQ ID NO: 63)
Pasteurella	MQTTNLSYILGLDLGIASVGWA	139	181	43	319	378	61	319	378	61
multocida	VVEINENEDPIGLIDVGVRIFE
str. Pm70	RAEVPKTGESLALSRRLARSTR
gi|15602992|	RLIRRRAHRLLLAKRFLKREGI
ref|	LSTIDLEKGLPNQAWELRVAGL
NP_246064.1|	ERRLSAIEWGAVLLHLIKHRGY
	LSKRKNESQTNNKELGALLSGV
	AQNHQLLQSDDYRTPAELALKK
	FAKEEGHIRNQRGAYTHTFNRL
	DLLAELNLLFAQQHQFGNPHCK
	EHIQQYMTELLMWQKPALSGEA
	ILKMLGKCTHEKNEFKAAKHTY
	SAERFVWLTKLNNLRILEDGAE
	RALNEEERQLLINHPYEKSKLT
	YAQVRKLLGLSEQAIFKHLRYS
	KENAESATFMELKAWHAIRKAL
	ENQGLKDTWQDLAKKPDLLDEI
	GTAFSLYKTDEDIQQYLTNKVP
	NSVINALLVSLNFDKFIELSLK
	SLRKILPLMEQGKRYDQACREI
	YGHHYGEANQKTSQLLPAIPAQ
	EIRNPVVLRTLSQARKVINAII
	RQYGSPARVHIETGRELGKSFK
	ERREIQKQQEDNRTKRESAVQK
	FKELFSDFSSEPKSKDILKFRL
	YEQQHGKCLYSGKEINIHRLNE
	KGYVEIDHALPFSRTWDDSFNN
	KVLVLASENQNKGNQTPYEWLQ
	GKINSERWKNFVALVLGSQCSA
	AKKQRLLTQVIDDNKFIDRNLN
	DTRYIARFLSNYIQENLLLVGK
	NKKNVFTPNGQITALLRSRWGL
	IKARENNNRHHALDAIVVACAT
	PSMQQKITRFIRFKEVHPYKIE
	NRYEMVDQESGEIISPHFPEPW
	AYFRQEVNIRVFDNHPDTVLKE
	MLPDRPQANHQFVQPLFVSRAP
	TRKMSGQGHMETIKSAKRLAEG
	ISVLRIPLTQLKPNLLENMVNK
	EREPALYAGLKARLAEFNQDPA
	KAFATPFYKQGGQQVKAIRVEQ
	VQKSGVLVRENNGVADNASIVR
	TDVFIKNNKFFLVPIYTWQVAK
	GILPNKAIVAHKNEDEWEEMDE
	GAKFKFSLFPNDLVELKTKKEY
	FFGYYIGLDRATGNISLKEHDG
	EISKGKDGVYRVGVKLALSFEK
	YQVDELGKNRQICRPQQRQPVR
	(SEQ ID NO: 64)
Rhodovulum	MGIRFAFDLGTNSIGWAVWRTG	141	183	43	319	378	48	319	378	48
sp. PH10	PGVFGEDTAASLDGSGVLIFKD
gi|402849997|	GRNPKDGQSLATMRRVPRQSRK
ref|	RRDRFVLRRRDLLAALRKAGLF
ZP_10898214.1	PVDVEEGRRLAATDPYHLRAKA
	LDESLTPHEMGRVIFHLNQRRG
	FRSNRKADRQDREKGKIAEGSK
	RLAETLAATNCRTLGEFLWSRH
	RGTPRTRSPTRIRMEGEGAKAL
	YAFYPTREMVRAEFERLWTAQS
	RFAPDLLTPERHEEIAGILFRQ
	RDLAPPKIGCCTFEPSERRLPR
	ALPSVEARGIYERLAHLRITTG
	PVSDRGLTRPERDVLASALLAG
	KSLTFKAVRKTLKILPHALVNF
	EEAGEKGLDGALTAKLLSKPDH
	YGAAWHGLSFAEKDTFVGKLLD
	EADEERLIRRLVTENRLSEDAA
	RRCASIPLADGYGRLGRTANTE
	ILAALVEETDETGTVVTYAEAV
	RRAGERTGRNWHHSDERDGVIL
	DRLPYYGEILQRHVVPGSGEPE
	EKNEAARWGRLANPTVHIGLNQ
	LRKVVNRLIAAHGRPDQIVVEL
	ARELKLNREQKERLDRENRKNR
	EENERRTAILAEHGQRDTAENK
	IRLRLFEEQARANAGIALCPYT
	GRAIGIAELFTSEVEIDHILPV
	SLTLDDSLANRVLCRREANREK
	RRQTPFQAFGATPAWNDIVARA
	AKLPPNKRWRFDPAALERFERE
	GGFLGRQLNETKYLSRLAKIYL
	GKICDPDRVYVTPGTLTGLLRA
	RWGLNSILSDSNFKNRSDHRHH
	AVDAVVIGVLTRGMIQRIAHDA
	ARAEDQDLDRVFRDVPVPFEDF
	RDHVRERVSTITVAVKPEHGKG
	GALHEDTSYGLVPDTDPNAALG
	NLVVRKPIRSLTAGEVDRVRDR
	ALRARLGALAAPFRDESGRVRD
	AKGLAQALEAFGAENGIRRVRI
	LKPDASVVTIADRRTGVPYRAV
	APGENHHVDIVQMRDGSWRGFA
	ASVFEVNRPGWRPEWEVKKLGG
	KLVMRLHKGDMVELSDKDGQRR
	VKVVQQIEISANRVRLSPHNDG
	GKLQDRHADADDPFRWDLATIP
	LLKDRGCVAVRVDPIGVVTLRR
	SNV (SEQ ID NO: 65)
Eubacterium	MMEVFMGRLVLGLDIGITSVGF	131	172	42	303	361	59	303	361	59
dolichum	GIIDLDESEIVDYGVRLFKEGT
DSM 3991	AAENETRRTKRGGRRLKRRRVT
gi|160915782|	RREDMLHLLKQAGIISTSFHPL
ref|	NNPYDVRVKGLNERLNGEELAT
ZP_02077990.1	ALLHLCKHRGSSVETIEDDEAK
	AKEAGETKKVLSMNDQLLKSGK
	YVCEIQKERLRTNGHIRGHENN
	FKTRAYVDEAFQILSHQDLSNE
	LKSAIITIISRKRMYYDGPGGP
	LSPTPYGRYTYFGQKEPIDLIE
	KMRGKCSLFPNEPRAPKLAYSA
	ELFNLLNDLNNLSIEGEKLTSE
	QKAMILKIVHEKGKITPKQLAK
	EVGVSLEQIRGFRIDTKGSPLL
	SELTGYKMIREVLEKSNDEHLE
	DHVFYDEIAEILTKTKDIEGRK
	KQISELSSDLNEESVHQLAGLT
	KFTAYHSLSFKALRLINEEMLK
	TELNQMQSITLFGLKQNNELSV
	KGMKNIQADDTAILSPVAKRAQ
	RETFKVVNRLREIYGEFDSIVV
	EMAREKNSEEQRKAIRERQKFF
	EMRNKQVADIIGDDRKINAKLR
	EKLVLYQEQDGKTAYSLEPIDL
	KLLIDDPNAYEVDHIIPISISL
	DDSITNKVLVTHRENQEKGNLT
	PISAFVKGRFTKGSLAQYKAYC
	LKLKEKNIKTNKGYRKKVEQYL
	LNENDIYKYDIQKEFINRNLVD
	TSYASRVVLNTLTTYFKQNEIP
	TKVFTVKGSLTNAFRRKINLKK
	DRDEDYGHHAIDALIIASMPKM
	RLLSTIFSRYKIEDIYDESTGE
	VFSSGDDSMYYDDRYFAFIASL
	KAIKVRKFSHKIDTKPNRSVAD
	ETIYSTRVIDGKEKVVKKYKDI
	YDPKFTALAEDILNNAYQEKYL
	MALHDPQTFDQIVKVVNYYFEE
	MSKSEKYFTKDKKGRIKISGMN
	PLSLYRDEHGMLKKYSKKGDGP
	AITQMKYFDGVLGNHIDISAHY
	QVRDKKVVLQQISPYRTDFYYS
	KENGYKFVTIRYKDVRWSEKKK
	KYVIDQQDYAMKKAEKKIDDTY
	EFQFSMHRDELIGITKAEGEAL
	IYPDETWHNFNFFFHAGETPEI
	LKFTATNNDKSNKIEVKPIHCY
	CKMRLMPTISKKIVRIDKYATD
	VVGNLYKVKKNTLKFEFD
	(SEQ ID NO: 66)
Nitratifractor	MKKILGVDIGITSFGYAILQET	143	184	42	347	404	61	347	404	61
salsuginis	GKDLYRCLDNSVVMRNNPYDEK
DSM 16511	SGESSQSIRSTQKSMRRLIEKR
gi|319957206|	KKRIRCVAQTMERYGILDYSET
ref|	MKINDPKNNPIKNRWQLRAVDA
YP_004168469.1	WKRPLSPQELFAIFAHMAKHRG
	YKSIATEDLIYELELELGLNDP
	EKESEKKADERRQVYNALRHLE
	ELRKKYGGETIAQTIHRAVEAG
	DLRSYRNHDDYEKMIRREDIEE
	EIEKVLLRQAELGALGLPEEQV
	SELIDELKACITDQEMPTIDES
	LFGKCTFYKDELAAPAYSYLYD
	LYRLYKKLADLNIDGYEVTQED
	REKVIEWVEKKIAQGKNLKKIT
	HKDLRKILGLAPEQKIFGVEDE
	RIVKGKKEPRTFVPFFFLADIA
	KFKELFASIQKHPDALQIFREL
	AEILQRSKTPQEALDRLRALMA
	GKGIDTDDRELLELFKNKRSGT
	RELSHRYILEALPLFLEGYDEK
	EVQRILGFDDREDYSRYPKSLR
	HLHLREGNLFEKEENPINNHAV
	KSLASWALGLIADLSWRYGPFD
	EIILETTRDALPEKIRKEIDKA
	MREREKALDKIIGKYKKEFPSI
	DKRLARKIQLWERQKGLDLYSG
	KVINLSQLLDGSADIEHIVPQS
	LGGLSTDYNTIVTLKSVNAAKG
	NRLPGDWLAGNPDYRERIGMLS
	EKGLIDWKKRKNLLAQSLDEIY
	TENTHSKGIRATSYLEALVAQV
	LKRYYPFPDPELRKNGIGVRMI
	PGKVTSKTRSLLGIKSKSRETN
	FHHAEDALILSTLTRGWQNRLH
	RMLRDNYGKSEAELKELWKKYM
	PHIEGLTLADYIDEAFRRFMSK
	GEESLFYRDMFDTIRSISYWVD
	KKPLSASSHKETVYSSRHEVPT
	LRKNILEAFDSLNVIKDRHKLT
	TEEFMKRYDKEIRQKLWLHRIG
	NTNDESYRAVEERATQIAQILT
	RYQLMDAQNDKEIDEKFQQALK
	ELITSPIEVTGKLLRKMRFVYD
	KLNAMQIDRGLVETDKNMLGIH
	ISKGPNEKLIFRRMDVNNAHEL
	QKERSGILCYLNEMLFIFNKKG
	LIHYGCLRSYLEKGQGSKYIAL
	FNPRFPANPKAQPSKFTSDSKI
	KQVGIGSATGIIKAHLDLDGHV
	RSYEVFGTLPEGSIEWFKEESG
	YGRVEDDPHH (SEQ ID NO:
	67)
Rhodospirillum	MRPIEPWILGLDIGTDSLGWAV	139	180	42	314	371	55	314	371	55
rubrum	FSCEEKGPPTAKELLGGGVRLF
ATCC 11170	DSGRDAKDHTSRQAERGAFRRA
gi|83591793|	RRQTRTWPWRRDRLIALFQAAG
ref|	LTPPAAETRQIALALRREAVSR
YP_425545.1|	PLAPDALWAALLHLAHHRGFRS
	NRIDKRERAAAKALAKAKPAKA
	TAKATAPAKEADDEAGFWEGAE
	AALRQRMAASGAPTVGALLADD
	LDRGQPVRMRYNQSDRDGVVAP
	TRALIAEELAEIVARQSSAYPG
	LDWPAVTRLVLDQRPLRSKGAG
	PCAFLPGEDRALRALPTVQDFI
	IRQTLANLRLPSTSADEPRPLT
	DEEHAKALALLSTARFVEWPAL
	RRALGLKRGVKFTAETERNGAK
	QAARGTAGNLTEAILAPLIPGW
	SGWDLDRKDRVFSDLWAARQDR
	SALLALIGDPRGPTRVTEDETA
	EAVADAIQIVLPTGRASLSAKA
	ARAIAQAMAPGIGYDEAVTLAL
	GLHHSHRPRQERLARLPYYAAA
	LPDVGLDGDPVGPPPAEDDGAA
	AEAYYGRIGNISVHIALNETRK
	IVNALLHRHGPILRLVMVETTR
	ELKAGADERKRMIAEQAERERE
	NAEIDVELRKSDRWMANARERR
	QRVRLARRQNNLCPYTSTPIGH
	ADLLGDAYDIDHVIPLARGGRD
	SLDNMVLCQSDANKTKGDKTPW
	EAFHDKPGWIAQRDDFLARLDP
	QTAKALAWRFADDAGERVARKS
	AEDEDQGFLPRQLTDTGYIARV
	ALRYLSLVTNEPNAVVATNGRL
	TGLLRLAWDITPGPAPRDLLPT
	PRDALRDDTAARRFLDGLTPPP
	LAKAVEGAVQARLAALGRSRVA
	DAGLADALGLTLASLGGGGKNR
	ADHRHHFIDAAMIAVTTRGLIN
	QINQASGAGRILDLRKWPRTNF
	EPPYPTFRAEVMKQWDHIHPSI
	RPAHRDGGSLHAATVFGVRNRP
	DARVLVQRKPVEKLFLDANAKP
	LPADKIAEIIDGFASPRMAKRF
	KALLARYQAAHPEVPPALAALA
	VARDPAFGPRGMTANTVIAGRS
	DGDGEDAGLITPFRANPKAAVR
	TMGNAVYEVWEIQVKGRPRWTH
	RVLTRFDRTQPAPPPPPENARL
	VMRLRRGDLVYWPLESGDRLFL
	VKKMAVDGRLALWPARLATGKA
	TALYAQLSCPNINLNGDQGYCV
	QSAEGIRKEKIRTTSCTALGRL
	RLSKKAT (SEQ ID NO:
	68)
Clostridium	MKYTLGLDVGIASVGWAVIDKD	137	176	40	320	376	61	320	376	61
cellulolyticum	NNKIIDLGVRCFDKAEESKTGE
H10	SLATARRIARGMRRRISRRSQR
gi|220930482|	LRLVKKLFVQYEIIKDSSEFNR
ref|	IFDTSRDGWKDPWELRYNALSR
YP_002507391.1	ILKPYELVQVLTHITKRRGFKS
	NRKEDLSTTKEGVVITSIKNNS
	EMLRTKNYRTIGEMIFMETPEN
	SNKRNKVDEYIHTIAREDLLNE
	IKYIFSIQRKLGSPFVTEKLEH
	DELNIWEFQRPFASGDSILSKV
	GKCTLLKEELRAPTSCYTSEYF
	GLLQSINNLVLVEDNNTLTLNN
	DQRAKIIEYAHFKNEIKYSEIR
	KLLDIEPEILFKAHNLTHKNPS
	GNNESKKFYEMKSYHKLKSTLP
	TDIWGKLHSNKESLDNLFYCLT
	VYKNDNEIKDYLQANNLDYLIE
	YIAKLPTFNKFKHLSLVAMKRI
	IPFMEKGYKYSDACNMAELDFT
	GSSKLEKCNKLTVEPIIENVTN
	PVVIRALTQARKVINAIIQKYG
	LPYMVNIELAREAGMTRQDRDN
	LKKEHENNRKAREKISDLIRQN
	GRVASGLDILKWRLWEDQGGRC
	AYSGKPIPVCDLLNDSLTQIDH
	IYPYSRSMDDSYMNKVLVLTDE
	NQNKRSYTPYEVWGSTEKWEDF
	EARIYSMHLPQSKEKRLLNRNF
	ITKDLDSFISRNLNDTRYISRF
	LKNYIESYLQFSNDSPKSCVVC
	VNGQCTAQLRSRWGLNKNREES
	DLHHALDAAVIACADRKIIKEI
	TNYYNERENHNYKVKYPLPWHS
	FRQDLMETLAGVFISRAPRRKI
	TGPAHDETIRSPKHFNKGLTSV
	KIPLTTVTLEKLETMVKNTKGG
	ISDKAVYNVLKNRLIEHNNKPL
	KAFAEKIYKPLKNGTNGAIIRS
	IRVETPSYTGVFRNEGKGISDN
	SLMVRVDVFKKKDKYYLVPIYV
	AHMIKKELPSKAIVPLKPESQW
	ELIDSTHEFLFSLYQNDYLVIK
	TKKGITEGYYRSCHRGTGSLSL
	MPHFANNKNVKIDIGVRTAISI
	EKYNVDILGNKSIVKGEPRRGM
	EKYNSFKSN (SEQ ID NO:
	69)
Helicobacter	MIRTLGIDIGIASIGWAVIEGE	148	187	40	298	354	48	298	354	48
mustelae	YTDKGLENKEIVASGVRVFTKA
12198	ENPKNKESLALPRTLARSARRR
gi|291276265|	NARKKGRIQQVKHYLSKALGLD
ref|	LECFVQGEKLATLFQTSKDFLS
YP_003516037.1	PWELRERALYRVLDKEELARVI
	LHIAKRRGYDDITYGVEDNDSG
	KIKKAIAENSKRIKEEQCKTIG
	EMMYKLYFQKSLNVRNKKESYN
	RCVGRSELREELKTIFQIQQEL
	KSPWVNEELIYKLLGNPDAQSK
	QEREGLIFYQRPLKGFGDKIGK
	CSHIKKGENSPYRACKHAPSAE
	EFVALTKSINFLKNLTNRHGLC
	FSQEDMCVYLGKILQEAQKNEK
	GLTYSKLKLLLDLPSDFEFLGL
	DYSGKNPEKAVFLSLPSTFKLN
	KITQDRKTQDKIANILGANKDW
	EAILKELESLQLSKEQIQTIKD
	AKLNFSKHINLSLEALYHLLPL
	MREGKRYDEGVEILQERGIFSK
	PQPKNRQLLPPLSELAKEESYF
	DIPNPVLRRALSEFRKVVNALL
	EKYGGFHYFHIELTRDVCKAKS
	ARMQLEKINKKNKSENDAASQL
	LEVLGLPNTYNNRLKCKLWKQQ
	EEYCLYSGEKITIDHLKDQRAL
	QIDHAFPLSRSLDDSQSNKVLC
	LTSSNQEKSNKTPYEWLGSDEK
	KWDMYVGRVYSSNFSPSKKRKL
	TQKNFKERNEEDFLARNLVDTG
	YIGRVTKEYIKHSLSFLPLPDG
	KKEHIRIISGSMTSTMRSFWGV
	QEKNRDHHLHHAQDAIIIACIE
	PSMIQKYTTYLKDKETHRLKSH
	QKAQILREGDHKLSLRWPMSNF
	KDKIQESIQNIIPSHHVSHKVT
	GELHQETVRTKEFYYQAFGGEE
	GVKKALKFGKIREINQGIVDNG
	AMVRVDIFKSKDKGKFYAVPIY
	TYDFAIGKLPNKAIVQGKKNGI
	IKDWLEMDENYEFCFSLFKNDC
	IKIQTKEMQEAVLAIYKSTNSA
	KATIELEHLSKYALKNEDEEKM
	FTDTDKEKNKTMTRESCGIQGL
	KVFQKVKLSVLGEVLEHKPRNR
	QNIALKTTPKHV (SEQ ID
	NO: 70)
Ilyobacter	MKYSIGLDIGIASVGWSVINKD	134	173	40	462	517	63	462	517	63
polytropus	KERIEDMGVRIFQKAENPKDGS
DSM 2926	SLASSRREKRGSRRRNRRKKHR
gi|310780384|	LDRIKNILCESGLVKKNEIEKI
ref|	YKNAYLKSPWELRAKSLEAKIS
YP_003968716.1	NKEIAQILLHIAKRRGFKSFRK
	TDRNADDTGKLLSGIQENKKIM
	EEKGYLTIGDMVAKDPKFNTHV
	RNKAGSYLFSFSRKLLEDEVRK
	IQAKQKELGNTHFTDDVLEKYI
	EVFNSQRNFDEGPSKPSPYYSE
	IGQIAKMIGNCTFESSEKRTAK
	NTWSGERFVFLQKLNNFRIVGL
	SGKRPLTEEERDIVEKEVYLKK
	EVRYEKLRKILYLKEEERFGDL
	NYSKDEKQDKKTEKTKFISLIG
	NYTIKKLNLSEKLKSEIEEDKS
	KLDKIIEILTFNKSDKTIESNL
	KKLELSREDIEILLSEEFSGTL
	NLSLKAIKKILPYLEKGLSYNE
	ACEKADYDYKNNGIKFKRGELL
	PVVDKDLIANPVVLRAISQTRK
	VVNAIIRKYGTPHTIHVEVARD
	LAKSYDDRQTIIKENKKRELEN
	EKTKKFISEEFGIKNVKGKLLL
	KYRLYQEQEGRCAYSRKELSLS
	EVILDESMTDIDHIIPYSRSMD
	DSYSNKVLVLSGENRKKSNLLP
	KEYFDRQGRDWDTFVLNVKAMK
	IHPRKKSNLLKEKFTREDNKDW
	KSRALNDTRYISRFVANYLENA
	LEYRDDSPKKRVFMIPGQLTAQ
	LRARWRLNKVRENGDLHHALDA
	AVVAVTDQKAINNISNISRYKE
	LKNCKDVIPSIEYHADEETGEV
	YFEEVKDTRFPMPWSGFDLELQ
	KRLESENPREEFYNLLSDKRYL
	GWFNYEEGFIEKLRPVFVSRMP
	NRGVKGQAHQETIRSSKKISNQ
	IAVSKKPLNSIKLKDLEKMQGR
	DTDRKLYEALKNRLEEYDDKPE
	KAFAEPFYKPTNSGKRGPLVRG
	IKVEEKQNVGVYVNGGQASNGS
	MVRIDVFRKNGKFYTVPIYVHQ
	TLLKELPNRAINGKPYKDWDLI
	DGSFEFLYSFYPNDLIEIEFGK
	SKSIKNDNKLTKTEIPEVNLSE
	VLGYYRGMDTSTGAATIDTQDG
	KIQMRIGIKTVKNIKKYQVDVL
	GNVYKVKREKRQTF (SEQ ID
	NO: 71)
Sphaerochaeta	MSKKVSRRYEEQAQEICQRLGS	163	202	40	335	389	45	335	389	45
globus	RPYSIGLDLGVGSIGVAVAAYD
str. Buddy	PIKKQPSDLVFVSSRIFIPSTG
gi|325972003|	AAERRQKRGQRNSLRHRANRLK
ref|	FLWKLLAERNLMLSYSEQDVPD
YP_004248194.1	PARLRFEDAVVRANPYELRLKG
	LNEQLTLSELGYALYHIANHRG
	SSSVRTFLDEEKSSDDKKLEEQ
	QAMTEQLAKEKGISTFIEVLTA
	FNTNGLIGYRNSESVKSKGVPV
	PTRDIISNEIDVLLQTQKQFYQ
	EILSDEYCDRIVSAILFENEKI
	VPEAGCCPYFPDEKKLPRCHFL
	NEERRLWEAINNARIKMPMQEG
	AAKRYQSASFSDEQRHILFHIA
	RSGTDITPKLVQKEFPALKTSI
	IVLQGKEKAIQKIAGFRFRRLE
	EKSFWKRLSEEQKDDFFSAWTN
	TPDDKRLSKYLMKHLLLTENEV
	VDALKTVSLIGDYGPIGKTATQ
	LLMKHLEDGLTYTEALERGMET
	GEFQELSVWEQQSLLPYYGQIL
	TGSTQALMGKYWHSAFKEKRDS
	EGFFKPNTNSDEEKYGRIANPV
	VHQTLNELRKLMNELITILGAK
	PQEITVELARELKVGAEKREDI
	IKQQTKQEKEAVLAYSKYCEPN
	NLDKRYIERFRLLEDQAFVCPY
	CLEHISVADIAAGRADVDHIFP
	RDDTADNSYGNKVVAHRQCNDI
	KGKRTPYAAFSNTSAWGPIMHY
	LDETPGMWRKRRKFETNEEEYA
	KYLQSKGFVSRFESDNSYIAKA
	AKEYLRCLFNPNNVTAVGSLKG
	METSILRKAWNLQGIDDLLGSR
	HWSKDADTSPTMRKNRDDNRHH
	GLDAIVALYCSRSLVQMINTMS
	EQGKRAVEIEAMIPIPGYASEP
	NLSFEAQRELFRKKILEFMDLH
	AFVSMKTDNDANGALLKDTVYS
	ILGADTQGEDLVFVVKKKIKDI
	GVKIGDYEEVASAIRGRITDKQ
	PKWYPMEMKDKIEQLQSKNEAA
	LQKYKESLVQAAAVLEESNRKL
	IESGKKPIQLSEKTISKKALEL
	VGGYYYLISNNKRTKTFVVKEP
	SNEVKGFAFDTGSNLCLDFYHD
	AQGKLCGEIIRKIQAMNPSYKP
	AYMKQGYSLYVRLYQGDVCELR
	ASDLTEAESNLAKTTHVRLPNA
	KPGRTFVIIITFTEMGSGYQIY
	FSNLAKSKKGQDTSFTLTTIKN
	YDVRKVQLSSAGLVRYVSPLLV
	DKIEKDEVALCGE (SEQ ID
	NO: 72)
Staphylococcus	MNQKFILGLDIGITSVGYGLID	128	167	40	337	391	57	337	391	57
lugdunensis	YETKNIIDAGVRLFPEANVENN
M23590	EGRRSKRGSRRLKRRRIHRLER
gi|315659848|	VKKLLEDYNLLDQSQIPQSTNP
ref|	YAIRVKGLSEALSKDELVIALL
ZP_07912707.1	HIAKRRGIHKIDVIDSNDDVGN
	ELSTKEQLNKNSKLLKDKFVCQ
	IQLERMNEGQVRGEKNRFKTAD
	IIKEIIQLLNVQKNFHQLDENF
	INKYIELVEMRREYFEGPGKGS
	PYGWEGDPKAWYETLMGHCTYF
	PDELRSVKYAYSADLFNALNDL
	NNLVIQRDGLSKLEYHEKYHII
	ENVFKQKKKPTLKQIANEINVN
	PEDIKGYRITKSGKPQFTEFKL
	YHDLKSVLFDQSILENEDVLDQ
	IAEILTIYQDKDSIKSKLTELD
	ILLNEEDKENIAQLTGYTGTHR
	LSLKCIRLVLEEQWYSSRNQME
	IFTHLNIKPKKINLTAANKIPK
	AMIDEFILSPVVKRTFGQAINL
	INKIIEKYGVPEDIIIELAREN
	NSKDKQKFINEMQKKNENTRKR
	INEIIGKYGNQNAKRLVEKIRL
	HDEQEGKCLYSLESIPLEDLLN
	NPNHYEVDHIIPRSVSFDNSYH
	NKVLVKQSENSKKSNLTPYQYF
	NSGKSKLSYNQFKQHILNLSKS
	QDRISKKKKEYLLEERDINKFE
	VQKEFINRNLVDTRYATRELTN
	YLKAYFSANNMNVKVKTINGSF
	TDYLRKVWKFKKERNHGYKHHA
	EDALIIANADFLFKENKKLKAV
	NSVLEKPEIESKQLDIQVDSED
	NYSEMFIIPKQVQDIKDERNFK
	YSHRVDKKPNRQLINDTLYSTR
	KKDNSTYIVQTIKDIYAKDNTT
	LKKQFDKSPEKFLMYQHDPRTF
	EKLEVIMKQYANEKNPLAKYHE
	ETGEYLTKYSKKNNGPIVKSLK
	YIGNKLGSHLDVTHQFKSSTKK
	LVKLSIKPYRFDVYLTDKGYKF
	ITISYLDVLKKDNYYYIPEQKY
	DKLKLGKAIDKNAKFIASFYKN
	DLIKLDGEIYKIIGVNSDTRNM
	IELDLPDIRYKEYCELNNIKGE
	PRIKKTIGKKVNSIEKLTTDVL
	GNVFTNTQYTKPQLLFKRGN
	(SEQ ID NO: 73)
Treponema	MIMKLEKWRLGLDLGTNSIGWS	144	183	40	328	382	63	328	382	63
sp. JC4	VFSLDKDNSVQDLIDMGVRIFS
gi|384109266|	DGRDPKTKEPLAVARRTARSQR
ref|	KLIYRRKLRRKQVFKFLQEQGL
ZP_10010146.1	FPKTKEECMTLKSLNPYELRIK
	ALDEKLEPYELGRALFNLAVRR
	GFKSNRKDGSREEVSEKKSPDE
	IKTQADMQTHLEKAIKENGCRT
	ITEFLYKNQGENGGIRFAPGRM
	TYYPTRKMYEEEFNLIRSKQEK
	YYPQVDWDDIYKAIFYQRPLKP
	QQRGYCIYENDKERTFKAMPCS
	QKLRILQDIGNLAYYEGGSKKR
	VELNDNQDKVLYELLNSKDKVT
	FDQMRKALCLADSNSFNLEENR
	DFLIGNPTAVKMRSKNRFGKLW
	DEIPLEEQDLIIETIITADEDD
	AVYEVIKKYDLTQEQRDFIVKN
	TILQSGTSMLCKEVSEKLVKRL
	EEIADLKYHEAVESLGYKFADQ
	TVEKYDLLPYYGKVLPGSTMEI
	DLSAPETNPEKHYGKISNPTVH
	VALNQTRVVVNALIKEYGKPSQ
	IAIELSRDLKNNVEKKAEIARK
	QNQRAKENIAINDTISALYHTA
	FPGKSFYPNRNDRMKYRLWSEL
	GLGNKCIYCGKGISGAELFTKE
	IEIEHILPFSRTLLDAESNLTV
	AHSSCNAFKAERSPFEAFGTNP
	SGYSWQEIIQRANQLKNTSKKN
	KFSPNAMDSFEKDSSFIARQLS
	DNQYIAKAALRYLKCLVENPSD
	VWTTNGSMTKLLRDKWEMDSIL
	CRKFTEKEVALLGLKPEQIGNY
	KKNRFDHRHHAIDAVVIGLTDR
	SMVQKLATKNSHKGNRIEIPEF
	PILRSDLIEKVKNIVVSFKPDH
	GAEGKLSKETLLGKIKLHGKET
	FVCRENIVSLSEKNLDDIVDEI
	KSKVKDYVAKHKGQKIEAVLSD
	FSKENGIKKVRCVNRVQTPIEI
	TSGKISRYLSPEDYFAAVIWEI
	PGEKKTFKAQYIRRNEVEKNSK
	GLNVVKPAVLENGKPHPAAKQV
	CLLHKDDYLEFSDKGKMYFCRI
	AGYAATNNKLDIRPVYAVSYCA
	DWINSTNETMLTGYWKPTPTQN
	WVSVNVLFDKQKARLVTVSPIG
	RVFRK (SEQ ID NO: 74)
uncultured	MSSKAIDSLEQLDLFKPQEYTL	154	193	40	313	365	55	313	365	55
delta	GLDLGIKSIGWAILSGERIANA
proteobacterium	GVYLFETAEELNSTGNKLISKA
HF007007E19	AERGRKRRIRRMLDRKARRGRH
gi|297182908|	IRYLLEREGLPTDELEEVVVHQ
gb|ADI19058.1|	SNRTLWDVRAEAVERKLTKQEL
	AAVLFHLVRHRGYFPNTKKLPP
	DDESDSADEEQGKINRATSRLR
	EELKASDCKTIGQFLAQNRDRQ
	RNREGDYSNLMARKLVFEEALQ
	ILAFQRKQGHELSKDFEKTYLD
	VLMGQRSGRSPKLGNCSLIPSE
	LRAPSSAPSTEWFKFLQNLGNL
	QISNAYREEWSIDAPRRAQIID
	ACSQRSTSSYWQIRRDFQIPDE
	YRFNLVNYERRDPDVDLQEYLQ
	QQERKTLANFRNWKQLEKIIGT
	GHPIQTLDEAARLITLIKDDEK
	LSDQLADLLPEASDKAITQLCE
	LDFTTAAKISLEAMYRILPHMN
	QGMGFFDACQQESLPEIGVPPA
	GDRVPPFDEMYNPVVNRVLSQS
	RKLINAVIDEYGMPAKIRVELA
	RDLGKGRELRERIKLDQLDKSK
	QNDQRAEDFRAEFQQAPRGDQS
	LRYRLWKEQNCTCPYSGRMIPV
	NSVLSEDTQIDHILPISQSFDN
	SLSNKVLCFTEENAQKSNRTPF
	EYLDAADFQRLEAISGNWPEAK
	RNKLLHKSFGKVAEEWKSRALN
	DTRYLTSALADHLRHHLPDSKI
	QTVNGRITGYLRKQWGLEKDRD
	KHTHHAVDAIVVACTTPAIVQQ
	VTLYHQDIRRYKKLGEKRPTPW
	PETFRQDVLDVEEEIFITRQPK
	KVSGGIQTKDTLRKHRSKPDRQ
	RVALTKVKLADLERLVEKDASN
	RNLYEHLKQCLEESGDQPTKAF
	KAPFYMPSGPEAKQRPILSKVT
	LLREKPEPPKQLTELSGGRRYD
	SMAQGRLDIYRYKPGGKRKDEY
	RVVLQRMIDLMRGEENVHVFQK
	GVPYDQGPEIEQNYTFLFSLYF
	DDLVEFQRSADSEVIRGYYRTF
	NIANGQLKISTYLEGRQDFDFF
	GANRLAHFAKVQVNLLGKVIK
	(SEQ ID NO: 75)
Alicycliphilus	MRSLRYRLALDLGSTSLGWALF	140	178	39	317	366	48	317	366	48
denitrificans	RLDACNRPTAVIKAGVRIFSDG
K601	RNPKDGSSLAVTRRAARAMRRR
gi|330822845|	RDRLLKRKTRMQAKLVEHGFFP
ref|	ADAGKRKALEQLNPYALRAKGL
YP_004386148.1	QEALLPGEFARALFHINQRRGF
	KSNRKTDKKDNDSGVLKKAIGQ
	LRQQMAEQGSRTVGEYLWTRLQ
	QGQGVRARYREKPYTTEEGKKR
	IDKSYDLYIDRAMIEQEFDALW
	AAQAAFNPTLFHEAARADLKDT
	LLHQRPLRPVKPGRCTLLPEEE
	RAPLALPSTQRFRIHQEVNHLR
	LLDENLREVALTLAQRDAVVTA
	LETKAKLSFEQIRKLLKLSGSV
	QFNLEDAKRTELKGNATSAALA
	RKELFGAAWSGFDEALQDEIVW
	QLVTEEGEGALIAWLQTHTGVD
	EARAQAIVDVSLPEGYGNLSRK
	ALARIVPALRAAVITYDKAVQA
	AGFDHHSQLGFEYDASEVEDLV
	HPETGEIRSVFKQLPYYGKALQ
	RHVAFGSGKPEDPDEKRYGKIA
	NPTVHIGLNQVRMVVNALIRRY
	GRPTEVVIELARDLKQSREQKV
	EAQRRQADNQRRNARIRRSIAE
	VLGIGEERVRGSDIQKWICWEE
	LSFDAADRRCPYSGVQISAAML
	LSDEVEVEHILPFSKTLDDSLN
	NRTVAMRQANRIKRNRTPWDAR
	AEFEAQGWSYEDILQRAERMPL
	RKRYRFAPDGYERWLGDDKDFL
	ARALNDTRYLSRVAAEYLRLVC
	PGTRVIPGQLTALLRGKFGLND
	VLGLDGEKNRNDHRHHAVDACV
	IGVTDQGLMQRFATASAQARGD
	GLTRLVDGMPMPWPTYRDHVER
	AVRHIWVSHRPDHGFEGAMMEE
	TSYGIRKDGSIKQRRKADGSAG
	REISNLIRIHEATQPLRHGVSA
	DGQPLAYKGYVGGSNYCIEITV
	NDKGKWEGEVISTFRAYGVVRA
	GGMGRLRNPHEGQNGRKLIMRL
	VIGDSVRLEVDGAERTMRIVKI
	SGSNGQIFMAPIHEANVDARNT
	DKQDAFTYTSKYAGSLQKAKTR
	RVTISPIGEVRDPGFKG
	(SEQ ID NO: 76)
Azospirillum	MARPAFRAPRREHVNGWTPDPH	205	243	39	342	389	46	342	389	46
sp. B510	RISKPFFILVSWHLLSRVVIDS
gi|288957741|	SSGCFPGTSRDHTDKFAEWECA
ref|	VQPYRLSFDLGTNSIGWGLLNL
YP_003448082.1	DRQGKPREIRALGSRIFSDGRD
	PQDKASLAVARRLARQMRRRRD
	RYLTRRTRLMGALVRFGLMPAD
	PAARKRLEVAVDPYLARERATR
	ERLEPFEIGRALFHLNQRRGYK
	PVRTATKPDEEAGKVKEAVERL
	EAAIAAAGAPTLGAWFAWRKTR
	GETLRARLAGKGKEAAYPFYPA
	RRMLEAEFDTLWAEQARHHPDL
	LTAEAREILRHRIFHQRPLKPP
	PVGRCTLYPDDGRAPRALPSAQ
	RLRLFQELASLRVIHLDLSERP
	LTPAERDRIVAFVQGRPPKAGR
	KPGKVQKSVPFEKLRGLLELPP
	GTGFSLESDKRPELLGDETGAR
	IAPAFGPGWTALPLEEQDALVE
	LLLTEAEPERAIAALTARWALD
	EATAAKLAGATLPDFHGRYGRR
	AVAELLPVLERETRGDPDGRVR
	PIRLDEAVKLLRGGKDHSDFSR
	EGALLDALPYYGAVLERHVAFG
	TGNPADPEEKRVGRVANPTVHI
	ALNQLRHLVNAILARHGRPEEI
	VIELARDLKRSAEDRRREDKRQ
	ADNQKRNEERKRLILSLGERPT
	PRNLLKLRLWEEQGPVENRRCP
	YSGETISMRMLLSEQVDIDHIL
	PFSVSLDDSAANKVVCLREANR
	IKRNRSPWEAFGHDSERWAGIL
	ARAEALPKNKRWRFAPDALEKL
	EGEGGLRARHLNDTRHLSRLAV
	EYLRCVCPKVRVSPGRLTALLR
	RRWGIDAILAEADGPPPEVPAE
	TLDPSPAEKNRADHRHHALDAV
	VIGCIDRSMVQRVQLAAASAER
	EAAAREDNIRRVLEGFKEEPWD
	GFRAELERRARTIVVSHRPEHG
	IGGALHKETAYGPVDPPEEGFN
	LVVRKPIDGLSKDEINSVRDPR
	LRRALIDRLAIRRRDANDPATA
	LAKAAEDLAAQPASRGIRRVRV
	LKKESNPIRVEHGGNPSGPRSG
	GPFHKLLLAGEVHHVDVALRAD
	GRRWVGHWVTLFEAHGGRGADG
	AAAPPRLGDGERFLMRLHKGDC
	LKLEHKGRVRVMQVVKLEPSSN
	SVVVVEPHQVKTDRSKHVKISC
	DQLRARGARRVTVDPLGRVRVH
	APGARVGIGGDAGRTAMEPAED
	IS (SEQ ID NO: 77)
Bradyrhizobium	MKRTSLRAYRLGVDLGANSLGW	143	181	39	323	370	48	323	370	48
sp.	FVVWLDDHGQPEGLGPGGVRIF
BTAi 1	PDGRNPQSKQSNAAGRRLARSA
gi|148255343|	RRRRDRYLQRRGKLMGLLVKHG
ref|	LMPADEPARKRLECLDPYGLRA
YP_001239928.1	KALDEVLPLHHVGRALFHLNQR
	RGLFANRAIEQGDKDASAIKAA
	AGRLQTSMQACGARTLGEFLNR
	RHQLRATVRARSPVGGDVQARY
	EFYPTRAMVDAEFEAIWAAQAP
	HHPTMTAEAHDTIREAIFSQRA
	MKRPSIGKCSLDPATSQDDVDG
	FRCAWSHPLAQRFRIWQDVRNL
	AVVETGPTSSRLGKEDQDKVAR
	ALLQTDQLSFDEIRGLLGLPSD
	ARFNLESDRRDHLKGDATGAIL
	SARRHFGPAWHDRSLDRQIDIV
	ALLESALDEAAIIASLGTTHSL
	DEAAAQRALSALLPDGYCRLGL
	RAIKRVLPLMEAGRTYAEAASA
	AGYDHALLPGGKLSPTGYLPYY
	GQWLQNDVVGSDDERDTNERRW
	GRLPNPTVHIGIGQLRRVVNEL
	IRWHGPPAEITVELTRDLKLSP
	RRLAELEREQAENQRKNDKRTS
	LLRKLGLPASTHNLLKLRLWDE
	QGDVASECPYTGEAIGLERLVS
	DDVDIDHLIPFSISWDDSAANK
	VVCMRYANREKGNRTPFEAFGH
	RQGRPYDWADIAERAARLPRGK
	RWRFGPGARAQFEELGDFQARL
	LNETSWLARVAKQYLAAVTHPH
	RIHVLPGRLTALLRATWELNDL
	LPGSDDRAAKSRKDHRHHAIDA
	LVAALTDQALLRRMANAHDDTR
	RKIEVLLPWPTFRIDLETRLKA
	MLVSHKPDHGLQARLHEDTAYG
	TVEHPETEDGANLVYRKTFVDI
	SEKEIDRIRDRRLRDLVRAHVA
	GERQQGKTLKAAVLSFAQRRDI
	AGHPNGIRHVRLTKSIKPDYLV
	PIRDKAGRIYKSYNAGENAFVD
	ILQAESGRWIARATTVFQANQA
	NESHDAPAAQPIMRVFKGDMLR
	IDHAGAEKFVKIVRLSPSNNLL
	YLVEHHQAGVFQTRHDDPEDSF
	RWLFASFDKLREWNAELVRIDT
	LGQPWRRKRGLETGSEDATRIG
	WTRPKKWP (SEQ ID NO:
	78)
Parvibaculum	MERIFGFDIGTTSIGFSVIDYS	138	176	39	327	374	58	327	374	58
lavamentivorans	STQSAGNIQRLGVRIFPEARDP
DS-1	DGTPLNQQRRQKRMMRRQLRRR
gi|154250555|	RIRRKALNETLHEAGFLPAYGS
ref|	ADWPVVMADEPYELRRRGLEEG
YP_001411379.1	LSAYEFGRAIYHLAQHRHFKGR
	ELEESDTPDPDVDDEKEAANER
	AATLKALKNEQTTLGAWLARRP
	PSDRKRGIHAHRNVVAEEFERL
	WEVQSKFHPALKSEEMRARISD
	TIFAQRPVFWRKNTLGECRFMP
	GEPLCPKGSWLSQQRRMLEKLN
	NLAIAGGNARPLDAEERDAILS
	KLQQQASMSWPGVRSALKALYK
	QRGEPGAEKSLKFNLELGGESK
	LLGNALEAKLADMFGPDWPAHP
	RKQEIRHAVHERLWAADYGETP
	DKKRVIILSEKDRKAHREAAAN
	SFVADFGITGEQAAQLQALKLP
	TGWEPYSIPALNLFLAELEKGE
	RFGALVNGPDWEGWRRTNFPHR
	NQPTGEILDKLPSPASKEERER
	ISQLRNPTVVRTQNELRKVVNN
	LIGLYGKPDRIRIEVGRDVGKS
	KREREEIQSGIRRNEKQRKKAT
	EDLIKNGIANPSRDDVEKWILW
	KEGQERCPYTGDQIGFNALFRE
	GRYEVEHIWPRSRSFDNSPRNK
	TLCRKDVNIEKGNRMPFEAFGH
	DEDRWSAIQIRLQGMVSAKGGT
	GMSPGKVKRFLAKTMPEDFAAR
	QLNDTRYAAKQILAQLKRLWPD
	MGPEAPVKVEAVTGQVTAQLRK
	LWTLNNILADDGEKTRADHRHH
	AIDALTVACTHPGMTNKLSRYW
	QLRDDPRAEKPALTPPWDTIRA
	DAEKAVSEIVVSHRVRKKVSGP
	LHKETTYGDTGTDIKTKSGTYR
	QFVTRKKIESLSKGELDEIRDP
	RIKEIVAAHVAGRGGDPKKAFP
	PYPCVSPGGPEIRKVRLTSKQQ
	LNLMAQTGNGYADLGSNHHIAI
	YRLPDGKADFEIVSLFDASRRL
	AQRNPIVQRTRADGASFVMSLA
	AGEAIMIPEGSKKGIWIVQGVW
	ASGQVVLERDTDADHSTTTRPM
	PNPILKDDAKKVSIDPIGRVRP
	SND (SEQ ID NO: 79)
Prevotella	MNKRILGLDTGTNSLGWAVVDW	170	208	39	328	375	61	328	375	61
timonensis	DEHAQSYELIKYGDVIFQEGVK
CRIS 5C-B1	IEKGIESSKAAERSGYKAIRKQ
gi|282880052|	YFRRRLRKIQVLKVLVKYHLCP
ref|	YLSDDDLRQWHLQKQYPKSDEL
ZP_06288774.1	MLWQRTSDEEGKNPYYDRHRCL
	HEKLDLTVEADRYTLGRALYHL
	TQRRGFLSNRLDTSADNKEDGV
	VKSGISQLSTEMEEAGCEYLGD
	YFYKLYDAQGNKVRIRQRYTDR
	NKHYQHEFDAICEKQELSSELI
	EDLQRAIFFQLPLKSQRHGVGR
	CTFERGKPRCADSHPDYEEFRM
	LCFVNNIQVKGPHDLELRPLTY
	EEREKIEPLFFRKSKPNFDFED
	IAKALAGKKNYAWIHDKEERAY
	KFNYRMTQGVPGCPTIAQLKSI
	FGDDWKTGIAETYTLIQKKNGS
	KSLQEMVDDVWNVLYSFSSVEK
	LKEFAHHKLQLDEESAEKFAKI
	KLSHSFAALSLKAIRKFLPFLR
	KGMYYTHASFFANIPTIVGKEI
	WNKEQNRKYIMENVGELVFNYQ
	PKHREVQGTIEMLIKDFLANNF
	ELPAGATDKLYHPSMIETYPNA
	QRNEFGILQLGSPRTNAIRNPM
	AMRSLHILRRVVNQLLKESIID
	ENTEVHVEYARELNDANKRRAI
	ADRQKEQDKQHKKYGDEIRKLY
	KEETGKDIEPTQTDVLKFQLWE
	EQNHHCLYTGEQIGITDFIGSN
	PKFDIEHTIPQSVGGDSTQMNL
	TLCDNRFNREVKKAKLPTELAN
	HEEILTRIEPWKNKYEQLVKER
	DKQRTFAGMDKAVKDIRIQKRH
	KLQMEIDYWRGKYERFTMTEVP
	EGFSRRQGTGIGLISRYAGLYL
	KSLFHQADSRNKSNVYVVKGVA
	TAEFRKMWGLQSEYEKKCRDNH
	SHHCMDAITIACIGKREYDLMA
	EYYRMEETFKQGRGSKPKFSKP
	WATFTEDVLNIYKNLLVVHDTP
	NNMPKHTKKYVQTSIGKVLAQG
	DTARGSLHLDTYYGAIERDGEI
	RYVVRRPLSSFTKPEELENIVD
	ETVKRTIKEAIADKNFKQAIAE
	PIYMNEEKGILIKKVRCFAKSV
	KQPINIRQHRDLSKKEYKQQYH
	VMNENNYLLAIYEGLVKNKVVR
	EFEIVSYIEAAKYYKRSQDRNI
	FSSIVPTHSTKYGLPLKTKLLM
	GQLVLMFEENPDEIQVDNTKDL
	VKRLYKVVGIEKDGRIKFKYHQ
	EARKEGLPIFSTPYKNNDDYAP
	IFRQSINNINILVDGIDFTIDI
	LGKVTLKE (SEQ ID NO:
	80)
Bacillus	MNYKMGLDIGIASVGWAVINLD	134	171	38	401	448	63	401	448	63
smithii 73	LKRIEDLGVRIFDKAEHPQNGE
47FAA	SLALPRRIARSARRRLRRRKHR
gi|365156657|	LERIRRLLVSENVLTKEEMNLL
ref|	FKQKKQIDVWQLRVDALERKLN
ZP_09352959.1	NDELARVLLHLAKRRGFKSNRK
	SERNSKESSEFLKNIEENQSIL
	AQYRSVGEMIVKDSKFAYHKRN
	KLDSYSNMIARDDLEREIKLIF
	EKQREFNNPVCTERLEEKYLNI
	WSSQRPFASKEDIEKKVGFCTF
	EPKEKRAPKATYTFQSFIVWEH
	INKLRLVSPDETRALTEIERNL
	LYKQAFSKNKMTYYDIRKLLNL
	SDDIHFKGLLYDPKSSLKQIEN
	IRFLELDSYHKIRKCIENVYGK
	DGIRMFNETDIDTFGYALTIFK
	DDEDIVAYLQNEYITKNGKRVS
	NLANKVYDKSLIDELLNLSFSK
	FAHLSMKAIRNILPYMEQGEIY
	SKACELAGYNFTGPKKKEKALL
	LPVIPNIANPVVMRALTQSRKV
	VNAIIKKYGSPVSIHIELARDL
	SHSFDERKKIQKDQTENRKKNE
	TAIKQLIEYELTKNPTGLDIVK
	FKLWSEQQGRCMYSLKPIELER
	LLEPGYVEVDHILPYSRSLDDS
	YANKVLVLTKENREKGNHTPVE
	YLGLGSERWKKFEKFVLANKQF
	SKKKKQNLLRLRYEETEEKEFK
	ERNLNDTRYISKFFANFIKEHL
	KFADGDGGQKVYTINGKITAHL
	RSRWDFNKNREESDLHHAVDAV
	IVACATQGMIKKITEFYKAREQ
	NKESAKKKEPIFPQPWPHFADE
	LKARLSKFPQESIEAFALGNYD
	RKKLESLRPVFVSRMPKRSVTG
	AAHQETLRRCVGIDEQSGKIQT
	AVKTKLSDIKLDKDGHFPMYQK
	ESDPRTYEAIRQRLLEHNNDPK
	KAFQEPLYKPKKNGEPGPVIRT
	VKIIDTKNKVVHLDGSKTVAYN
	SNIVRTDVFEKDGKYYCVPVYT
	MDIMKGTLPNKAIEANKPYSEW
	KEMTEEYTFQFSLFPNDLVRIV
	LPREKTIKTSTNEEIIIKDIFA
	YYKTIDSATGGLELISHDRNFS
	LRGVGSKTLKRFEKYQVDVLGN
	IHKVKGEKRVGLAAPTNQKKGK
	TVDSLQSVSD (SEQ ID NO:
	81)
Cand.	MRRLGLDLGTNSIGWCLLDLGD	135	172	38	344	391	53	344	391	53
Puniceispirillum	DGEPVSIFRTGARIFSDGRDPK
marinum	SLGSLKATRREARLTRRRRDRF
IMCC1322	IQRQKNLINALVKYGLMPADEI
gi|294086111|	QRQALAYKDPYPIRKKALDEAI
ref|	DPYEMGRAIFHINQRRGFKSNR
YP_003552871.1	KSADNEAGVVKQSIADLEMKLG
	EAGARTIGEFLADRQATNDTVR
	ARRLSGTNALYEFYPDRYMLEQ
	EFDTLWAKQAAFNPSLYIEAAR
	ERLKEIVFFQRKLKPQEVGRCI
	FLSDEDRISKALPSFQRFRIYQ
	ELSNLAWIDHDGVAHRITASLA
	LRDHLFDELEHKKKLTFKAMRA
	ILRKQGVVDYPVGFNLESDNRD
	HLIGNLTSCIMRDAKKMIGSAW
	DRLDEEEQDSFILMLQDDQKGD
	DEVRSILTQQYGLSDDVAEDCL
	DVRLPDGHGSLSKKAIDRILPV
	LRDQGLIYYDAVKEAGLGEANL
	YDPYAALSDKLDYYGKALAGHV
	MGASGKFEDSDEKRYGTISNPT
	VHIALNQVRAVVNELIRLHGKP
	DEVVIEIGRDLPMGADGKRELE
	RFQKEGRAKNERARDELKKLGH
	IDSRESRQKFQLWEQLAKEPVD
	RCCPFTGKMMSISDLFSDKVEI
	EHLLPFSLTLDDSMANKTVCFR
	QANRDKGNRAPFDAFGNSPAGY
	DWQEILGRSQNLPYAKRWRFLP
	DAMKRFEADGGFLERQLNDTRY
	ISRYTTEYISTIIPKNKIWVVT
	GRLTSLLRGFWGLNSILRGHNT
	DDGTPAKKSRDDHRHHAIDAIV
	VGMTSRGLLQKVSKAARRSEDL
	DLTRLFEGRIDPWDGFRDEVKK
	HIDAIIVSHRPRKKSQGALHND
	TAYGIVEHAENGASTVVHRVPI
	TSLGKQSDIEKVRDPLIKSALL
	NETAGLSGKSFENAVQKWCADN
	SIKSLRIVETVSIIPITDKEGV
	AYKGYKGDGNAYMDIYQDPTSS
	KWKGEIVSRFDANQKGFIPSWQ
	SQFPTARLIMRLRINDLLKLQD
	GEIEEIYRVQRLSGSKILMAPH
	TEANVDARDRDKNDTFKLTSKS
	PGKLQSASARKVHISPTGLIRE
	G (SEQ ID NO: 82)
Barnesiella	MKNILGLDLGLSSIGWSVIREN	140	176	37	371	417	60	371	417	60
intestinihominis	SEEQELVAMGSRVVSLTAAELS
YIT	SFTQGNGVSINSQRTQKRTQRK
11860	GYDRYQLRRTLLRNKLDTLGML
gi|404487228|	PDDSLSYLPKLQLWGLRAKAVT
ref|	QRIELNELGRVLLHLNQKRGYK
ZP_11022414.1	SIKSDFSGDKKITDYVKTVKTR
	YDELKEMRLTIGELFFRRLTEN
	AFFRCKEQVYPRQAYVEEFDCI
	MNCQRKFYPDILTDETIRCIRD
	EIIYYQRPLKSCKYLVSRCEFE
	KRFYLNAAGKKTEAGPKVSPRT
	SPLFQVCRLWESINNIVVKDRR
	NEIVFISAEQRAALFDELNTHE
	KLKGSDLLKLLGLSKTYGYRLG
	EQFKTGIQGNKTRVEIERALGN
	YPDKKRLLQFNLQEESSSMVNT
	ETGEIIPMISLSFEQEPLYRLW
	HVLYSIDDREQLQSVLRQKFGI
	DDDEVLERLSAIDLVKAGFGNK
	SSKAIRRILPFLQLGMNYAEAC
	EAAGYNHSNNYTKAENEARALL
	DRLPAIKKNELRQPVVEKILNQ
	MVNVVNALMEKYGRFDEIRVEL
	ARELKQSKEERSNTYKSINKNQ
	RENEQIAKRIVEYGVPTRSRIQ
	KYKMWEESKHCCIYCGQPVDVG
	DFLRGFDVEVEHIIPKSLYFDD
	SFANKVCSCRSCNKEKNNRTAY
	DYMKSKGEKALSDYVERVNTMY
	TNNQISKTKWQNLLTPVDKISI
	DFIDRQLRESQYIARKAKEILT
	SICYNVTATSGSVTSFLRHVWG
	WDTVLHDLNEDRYKKVGLTEVI
	EVNHRGSVIRREQIKDWSKRED
	HRHHAIDALTIACTKQAYIQRL
	NNLRAEEGPDENKMSLERYIQS
	QPHFSVAQVREAVDRILVSFRA
	GKRAVTPGKRYIRKNRKRISVQ
	SVLIPRGALSEESVYGVIHVWE
	KDEQGHVIQKQRAVMKYPITSI
	NREMLDKEKVVDKRIHRILSGR
	LAQYNDNPKEAFAKPVYIDKEC
	RIPIRTVRCFAKPAINTLVPLK
	KDDKGNPVAWVNPGNNHHVAIY
	RDEDGKYKERTVTFWEAVDRCR
	VGIPAIVTQPDTIWDNILQRND
	ISENVLESLPDVKWQFVLSLQQ
	NEMFILGMNEEDYRYAMDQQDY
	ALLNKYLYRVQKLSKSDYSFRY
	HTETSVEDKYDGKPNLKLSMQM
	GKLKRVSIKSLLGLNPHKVHIS
	VLGEIKEIS (SEQ ID NO:
	83)
Ralstonia	MAEKQHRWGLDIGTNSIGWAVI	140	176	37	395	440	50	395	440	50
syzygii R24	ALIEGRPAGLVATGSRIFSDGR
gi|344171927|	NPKDGSSLAVERRGPRQMRRRR
emb|CCA84553.1|	DRYLRRRDRFMQALINVGLMPG
	DAAARKALVTENPYVLRQRGLD
	QALTLPEFGRALFHLNQRRGFQ
	SNRKTDRATAKESGKVKNAIAA
	FRAGMGNARTVGEALARRLEDG
	RPVRARMVGQGKDEHYELYIAR
	EWIAQEFDALWASQQRFHAEVL
	ADAARDRLRAILLFQRKLLPVP
	VGKCFLEPNQPRVAAALPSAQR
	FRLMQELNHLRVMTLADKRERP
	LSFQERNDLLAQLVARPKCGFD
	MLRKIVFGANKEAYRFTIESER
	RKELKGCDTAAKLAKVNALGTR
	WQALSLDEQDRLVCLLLDGEND
	AVLADALREHYGLTDAQIDTLL
	GLSFEDGHMRLGRSALLRVLDA
	LESGRDEQGLPLSYDKAVVAAG
	YPAHTADLENGERDALPYYGEL
	LWRYTQDAPTAKNDAERKFGKI
	ANPTVHIGLNQLRKLVNALIQR
	YGKPAQIVVELARNLKAGLEEK
	ERIKKQQTANLERNERIRQKLQ
	DAGVPDNRENRLRMRLFEELGQ
	GNGLGTPCIYSGRQISLQRLFS
	NDVQVDHILPFSKTLDDSFANK
	VLAQHDANRYKGNRGPFEAFGA
	NRDGYAWDDIRARAAVLPRNKR
	NRFAETAMQDWLHNETDELARQ
	LTDTAYLSRVARQYLTAICSKD
	DVYVSPGRLTAMLRAKWGLNRV
	LDGVMEEQGRPAVKNRDDHRHH
	AIDAVVIGATDRAMLQQVATLA
	ARAREQDAERLIGDMPTPWPNF
	LEDVRAAVARCVVSHKPDHGPE
	GGLHNDTAYGIVAGPFEDGRYR
	VRHRVSLFDLKPGDLSNVRCDA
	PLQAELEPIFEQDDARAREVAL
	TALAERYRQRKVWLEELMSVLP
	IRPRGEDGKTLPDSAPYKAYKG
	DSNYCYELFINERGRWDGELIS
	TFRANQAAYRRFRNDPARFRRY
	TAGGRPLLMRLCINDYIAVGTA
	AERTIFRVVKMSENKITLAEHF
	EGGTLKQRDADKDDPFKYLTKS
	PGALRDLGARRIFVDLIGRVLD
	PGIKGD (SEQ ID NO: 84)
Wolinella	MIERILGVDLGISSLGWAIVEY	145	180	36	348	392	60	348	392	60
succinogenes	DKDDEAANRIIDCGVRLFTAAE
DSM 1740	TPKKKESPNKARREARGIRRVL
gi|34557790|	NRRRVRMNMIKKLFLRAGLIQD
ref|	VDLDGEGGMFYSKANRADVWEL
NP_907605.1|	RHDGLYRLLKGDELARVLIHIA
	KHRGYKFIGDDEADEESGKVKK
	AGVVLRQNFEAAGCRTVGEWLW
	RERGANGKKRNKHGDYEISIHR
	DLLVEEVEAIFVAQQEMRSTIA
	TDALKAAYREIAFFVRPMQRIE
	KMVGHCTYFPEERRAPKSAPTA
	EKFIAISKFFSTVIIDNEGWEQ
	KIIERKTLEELLDFAVSREKVE
	FRHLRKFLDLSDNEIFKGLHYK
	GKPKTAKKREATLFDPNEPTEL
	EFDKVEAEKKAWISLRGAAKLR
	EALGNEFYGRFVALGKHADEAT
	KILTYYKDEGQKRRELTKLPLE
	AEMVERLVKIGFSDFLKLSLKA
	IRDILPAMESGARYDEAVLMLG
	VPHKEKSAILPPLNKTDIDILN
	PTVIRAFAQFRKVANALVRKYG
	AFDRVHFELAREINTKGEIEDI
	KESQRKNEKERKEAADWIAETS
	FQVPLTRKNILKKRLYIQQDGR
	CAYTGDVIELERLFDEGYCEID
	HILPRSRSADDSFANKVLCLAR
	ANQQKTDRTPYEWFGHDAARWN
	AFETRTSAPSNRVRTGKGKIDR
	LLKKNFDENSEMAFKDRNLNDT
	RYMARAIKTYCEQYWVFKNSHT
	KAPVQVRSGKLTSVLRYQWGLE
	SKDRESHTHHAVDAIIIAFSTQ
	GMVQKLSEYYRFKETHREKERP
	KLAVPLANFRDAVEEATRIENT
	ETVKEGVEVKRLLISRPPRARV
	TGQAHEQTAKPYPRIKQVKNKK
	KWRLAPIDEEKFESFKADRVAS
	ANQKNFYETSTIPRVDVYHKKG
	KFHLVPIYLHEMVLNELPNLSL
	GTNPEAMDENFFKFSIFKDDLI
	SIQTQGTPKKPAKIIMGYFKNM
	HGANMVLSSINNSPCEGFTCTP
	VSMDKKHKDKCKLCPEENRIAG
	RCLQGFLDYWSQEGLRPPRKEF
	ECDQGVKFALDVKKYQIDPLGY
	YYEVKQEKRLGTIPQMRSAKKL
	VKK (SEQ ID NO: 86)
Mycoplasma	MNNSIKSKPEVTIGLDLGVGSV	144	177	34	373	416	71	373	416	71
gallisepticum	GWAIVDNETNIIHHLGSRLFSQ
str. F	AKTAEDRRSFRGVRRLIRRRKY
gi|284931710|	KLKRFVNLIWKYNSYFGFKNKE
gb|ADC31648.1|	DILNNYQEQQKLHNTVLNLKSE
	ALNAKIDPKALSWILHDYLKNR
	GHFYEDNRDFNVYPTKELAKYF
	DKYGYYKGIIDSKEDNDNKLEE
	ELTKYKFSNKHWLEEVKKVLSN
	QTGLPEKFKEEYESLFSYVRNY
	SEGPGSINSVSPYGIYHLDEKE
	GKVVQKYNNIWDKTIGKCNIFP
	DEYRAPKNSPIAMIFNEINELS
	TIRSYSIYLTGWFINQEFKKAY
	LNKLLDLLIKTNGEKPIDARQF
	KKLREETIAESIGKETLKDVEN
	EEKLEKEDHKWKLKGLKLNTNG
	KIQYNDLSSLAKFVHKLKQHLK
	LDFLLEDQYATLDKINFLQSLF
	VYLGKHLRYSNRVDSANLKEFS
	DSNKLFERILQKQKDGLFKLFE
	QTDKDDEKILAQTHSLSTKAML
	LAITRMTNLDNDEDNQKNNDKG
	WNFEAIKNFDQKFIDITKKNNN
	LSLKQNKRYLDDRFINDAILSP
	GVKRILREATKVFNAILKQFSE
	EYDVTKVVIELARELSEEKELE
	NTKNYKKLIKKNGDKISEGLKA
	LGISEDEIKDILKSPTKSYKFL
	LWLQQDHIDPYSLKEIAFDDIF
	TKTEKFEIDHIIPYSISFDDSS
	SNKLLVLAESNQAKSNQTPYEF
	ISSGNAGIKWEDYEAYCRKFKD
	GDSSLLDSTQRSKKFAKMMKTD
	TSSKYDIGFLARNLNDTRYATI
	VFRDALEDYANNHLVEDKPMFK
	VVCINGSVTSFLRKNFDDSSYA
	KKDRDKNIHHAVDASIISIFSN
	ETKTLFNQLTQFADYKLFKNTD
	GSWKKIDPKTGVVTEVTDENWK
	QIRVRNQVSEIAKVIEKYIQDS
	NIERKARYSRKIENKTNISLFN
	DTVYSAKKVGYEDQIKRKNLKT
	LDIHESAKENKNSKVKRQFVYR
	KLVNVSLLNNDKLADLFAEKED
	ILMYRANPWVINLAEQIFNEYT
	ENKKIKSQNVFEKYMLDLTKEF
	PEKFSEFLVKSMLRNKTAIIYD
	DKKNIVHRIKRLKMLSSELKEN
	KLSNVIIRSKNQSGTKLSYQDT
	INSLALMIMRSIDPTAKKQYIR
	VPLNTLNLHLGDHDFDLHNMDA
	YLKKPKFVKYLKANEIGDEYKP
	WRVLTSGTLLIHKKDKKLMYIS
	SFQNLNDVIEIKNLIETEYKEN
	DDSDSKKKKKANRFLMTLSTIL
	NDYILLDAKDNFDILGLSKNRI
	DEILNSKLGLDKIVK (SEQ
	ID NO: 87)
Acidothermus	MGGSEVGTVPVTWRLGVDVGER	150	182	33	341	380	58	341	380	58
cellulolyticus	SIGLAAVSYEEDKPKEILAAVS
11B	WIHDGGVGDERSGASRLALRGM
gi|117929158|	ARRARRLRRFRRARLRDLDMLL
ref|	SELGWTPLPDKNVSPVDAWLAR
YP_873709.1|	KRLAEEYVVDETERRRLLGYAV
	SHMARHRGWRNPWTTIKDLKNL
	PQPSDSWERTRESLEARYSVSL
	EPGTVGQWAGYLLQRAPGIRLN
	PTQQSAGRRAELSNATAFETRL
	RQEDVLWELRCIADVQGLPEDV
	VSNVIDAVFCQKRPSVPAERIG
	RDPLDPSQLRASRACLEFQEYR
	IVAAVANLRIRDGSGSRPLSLE
	ERNAVIEALLAQTERSLTWSDI
	ALEILKLPNESDLTSVPEEDGP
	SSLAYSQFAPFDETSARIAEFI
	AKNRRKIPTFAQWWQEQDRTSR
	SDLVAALADNSIAGEEEQELLV
	HLPDAELEALEGLALPSGRVAY
	SRLTLSGLTRVMRDDGVDVHNA
	RKTCFGVDDNWRPPLPALHEAT
	GHPVVDRNLAILRKFLSSATMR
	WGPPQSIVVELARGASESRERQ
	AEEEAARRAHRKANDRIRAELR
	ASGLSDPSPADLVRARLLELYD
	CHCMYCGAPISWENSELDHIVP
	RTDGGSNRHENLAITCGACNKE
	KGRRPFASWAETSNRVQLRDVI
	DRVQKLKYSGNMYWTRDEFSRY
	KKSVVARLKRRTSDPEVIQSIE
	STGYAAVALRDRLLSYGEKNGV
	AQVAVFRGGVTAEARRWLDISI
	ERLFSRVAIFAQSTSTKRLDRR
	HHAVDAVVLTTLTPGVAKTLAD
	ARSRRVSAEFWRRPSDVNRHST
	EEPQSPAYRQWKESCSGLGDLL
	ISTAARDSIAVAAPLRLRPTGA
	LHEETLRAFSEHTVGAAWKGAE
	LRRIVEPEVYAAFLALTDPGGR
	FLKVSPSEDVLPADENRHIVLS
	DRVLGPRDRVKLFPDDRGSIRV
	RGGAAYIASFHHARVFRWGSSH
	SPSFALLRVSLADLAVAGLLRD
	GVDVFTAELPPWTPAWRYASIA
	LVKAVESGDAKQVGWLVPGDEL
	DFGPEGVTTAAGDLSMFLKYFP
	ERHWVVTGFEDDKRINLKPAFL
	SAEQAEVLRTERSDRPDTLTEA
	GEILAQFFPRCWRATVAKVLCH
	PGLTVIRRTALGQPRWRRGHLP
	YSWRPWSADPWSGGTP (SEQ
	ID NO: 88)
Mycoplasma	MHNKKNITIGFDLGIASIGWAI	156	184	29	381	420	62	381	420	62
ovipneumoniae	IDSTTSKILDWGTRTFEERKTA
SC01	NERRAFRSTRRNIRRKAYRNQR
gi|363542550|	FINLILKYKDLFELKNISDIQR
ref|	ANKKDTENYEKIISFFTEIYKK
ZP_09312133.1	CAAKHSNILEVKVKALDSKIEK
	LDLIWILHDYLENRGFFYDLEE
	ENVADKYEGIEHPSILLYDFFK
	KNGFFKSNSSIPKDLGGYSFSN
	LQWVNEIKKLFEVQEINPEFSE
	KFLNLFTSVRDYAKGPGSEHSA
	SEYGIFQKDEKGKVFKKYDNIW
	DKTIGKCSFFVEENRSPVNYPS
	YEIFNLLNQLINLSTDLKTTNK
	KIWQLSSNDRNELLDELLKVKE
	KAKIISISLKKNEIKKIILKDF
	GFEKSDIDDQDTIEGRKIIKEE
	PTTKLEVTKHLLATIYSHSSDS
	NWININNILEFLPYLDAICIIL
	DREKSRGQDEVLKKLTEKNIFE
	VLKIDREKQLDFVKSIFSNTKF
	NFKKIGNFSLKAIREFLPKMFE
	QNKNSEYLKWKDEEIRRKWEEQ
	KSKLGKTDKKTKYLNPRIFQDE
	IISPGTKNTFEQAVLVLNQIIK
	KYSKENIIDAIIIESPREKNDK
	KTIEEIKKRNKKGKGKTLEKLF
	QILNLENKGYKLSDLETKPAKL
	LDRLRFYHQQDGIDLYTLDKIN
	IDQLINGSQKYEIEHIIPYSMS
	YDNSQANKILTEKAENLKKGKL
	IASEYIKRNGDEFYNKYYEKAK
	ELFINKYKKNKKLDSYVDLDED
	SAKNRFRFLTLQDYDEFQVEFL
	ARNLNDTRYSTKLFYHALVEHF
	ENNEFFTYIDENSSKHKVKIST
	IKGHVTKYFRAKPVQKNNGPNE
	NLNNNKPEKIEKNRENNEHHAV
	DAAIVAIIGNKNPQIANLLTLA
	DNKTDKKFLLHDENYKENIETG
	ELVKIPKFEVDKLAKVEDLKKI
	IQEKYEEAKKHTAIKFSRKTRT
	ILNGGLSDETLYGFKYDEKEDK
	YFKIIKKKLVTSKNEELKKYFE
	NPFGKKADGKSEYTVLMAQSHL
	SEFNKLKEIFEKYNGFSNKTGN
	AFVEYMNDLALKEPTLKAEIES
	AKSVEKLLYYNFKPSDQFTYHD
	NINNKSFKRFYKNIRIIEYKSI
	PIKFKILSKHDGGKSFKDTLFS
	LYSLVYKVYENGKESYKSIPVT
	SQMRNFGIDEFDFLDENLYNKE
	KLDIYKSDFAKPIPVNCKPVFV
	LKKGSILKKKSLDIDDFKETKE
	TEEGNYYFISTISKRENRDTAY
	GLKPLKLSVVKPVAEPSTNPIF
	KEYIPIHLDELGNEYPVKIKEH
	TDDEKLMCTIK (SEQ ID
	NO: 89)

If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C-terminus, it is understood that the stop codon will be removed.

Exemplary PAM sequences and their corresponding RKR motifs are provided in Table 250.

TABLE 250
Identified PAM sequences and corresponding
RKR motifs.
		RKR
	PAM sequence	motif
Strain Name	(NA)	(AA)
Streptococcus pyogenes	NGG	RKR
Streptococcus mutans	NGG	RKR
Streptococcus thermophilus A	NGGNG (SEQ ID NO: 122)	RYR
Treponema denticola	NAAAAN (SEQ ID NO: 134)	VAK
Streptococcus thermophilus B	NNAAAAW (SEQ ID NO: 135)	IYK
Campylobacter jejuni	NNNNACA (SEQ ID NO: 136)	NLK
Pasteurella multocida	GNNNCNNA (SEQ ID NO: 137)	KDG
Neisseria meningitidis	NNNNGATT (SEQ ID NO: 106) or	IGK
	NNGRRT (SEQ ID NO: 104)
	(R = A or G)
Staphylococcus aureus	NNGRR (R = A or G)	NDK
	(SEQ ID NO: 125)

Exemplary Cas9 core domains are provided in Table 200.

TABLE 200
Amino Acid Sequence of Cas9 Core Domains
		Cas9 Start	Cas9 Stop
	Strain Name	(AA pos)	(AA pos)
		Start and Stop numbers refer
		to the sequence in Table 100
	Staphylococcus aureus	1	772
	Streptococcus pyogenes	1	1099
	Campulobacter jejuni	1	741

Exemplary PI domains, e.g., altered PI domains, are provided in Tables 400 and 500.

TABLE 400
Altered PI Domain
	PI Start	PI Stop	Length of	RKR
Strain Name	(AA pos)	(AA pos)	PI ( AA)	motif (AA)
Alicycliphilus denitrificans	837	1029	193	--Y
K601	Start and Stop numbers
	refer to the sequences
	in Table 100
Campylobacter jejuni	741	984	244	-NG
NCTC 11168
Helicobacter mustelae	771	1024	254	-NQ
12198

TABLE 500
Other Altered PI Domains
	PI Start	PI Stop	Length of	RKR
Strain Name	(AA pos)	(AA pos)	PI ( AA)	motif (AA)
	Start and Stop numbers
	refer to the sequences
	in Table 100
Akkermansia muciniphila ATCC BAA-835	871	1101	231	ALK
Ralstonia syzygii R24	821	1062	242	APY
Cand. Puniceispirillum marinum IMCC1322	815	1035	221	AYK
Fructobacillus fructosus KCTC 3544	1074	1323	250	DGN
Eubacterium yurii ATCC 43715	1107	1391	285	DGY
Eubacterium dolichum DSM 3991	779	1096	318	DKK
Dinoroseobacters hibae DFL 12	851	1079	229	DPI
Clostridium cellulolyticum H10	767	1021	255	EGK
Pasteurellamultocida str. Pm 70	815	1056	242	ENN
Mycoplasma canis PG 14	907	1233	327	EPK
Porphyromonas sp. oraltaxon 279 s tr. F0450	935	1197	263	EPT
Filifactor alocis ATCC 35896	1094	1365	272	EVD
Aminomonas paucivorans DSM 12260	801	1052	252	EVY
Wolinella succinogenes DSM 1740	1034	1409	376	EYK
Oenococcus kitaharae DSM 17330	1119	1389	271	GAL
Coriobacterium glomerans PW2	1126	1384	259	GDR
Peptoniphilus duerdenii ATCC BAA-1640	1091	1364	274	GDS
Bifidobacterium bifidum S17	1138	1420	283	GGL
Alicyclobacillus hesperidum URH17-3-68	876	1146	271	GGR
Rose buria inulinivorans DSM 16841	895	1152	258	GGT
Actinomyces coleocanis DSM 15436	843	1105	263	GKK
Odori bacter laneus YIT 12061	1103	1498	396	GKV
Coprococcus catus GD-7	1063	1338	276	GNQ
Enterococcus faecalis TX0012	829	1150	322	GRK
Bacillus smithii 7 3 47FAA	809	1088	280	GSK
Legionella pneumophila str. Paris	1021	1372	352	GTM
Bacteroides fragilis NCTC 9343	1140	1436	297	IPV
Mycoplasma ovipneumoniae SC01	923	1265	343	IRI
Actinomyces sp. oral taxon 180 s tr. F0310	895	1181	287	KEK
Treponema sp. JC4	832	1062	231	KIS
Fusobacterium nucleatum ATCC49256	1073	1374	302	KKV
Lactobacillus farciminis KCTC 3681	1101	1356	256	KKV
Nitratifractors alsuginis DSM 16511	840	1132	293	KMR
Lactobacillus coryniformis KCTC 3535	850	1119	270	KNK
Mycoplasma mobile 163K	916	1236	321	KNY
Flavobacterium branchiophilum FL-15	1182	1473	292	KQK
Prevotellatimonensis CRIS 5C-B1	957	1218	262	KQQ
Methylosinus trichosporium OB3b	830	1082	253	KRP
Prevotella sp. C561	1099	1424	326	KRY
Mycoplasma gallisepticum str. F	911	1269	359	KTA
Lactobacillus rhamnosus GG	1077	1363	287	KYG
Wolinella succinogenes DSM 1740	811	1059	249	LPN
Streptococcus thermophilus LMD-9	1099	1388	290	MLA
Treponema denticola ATCC 35405	1092	1395	304	NDS
Bergeyella zoohelcum ATCC 43767	1098	1415	318	NEK
Veillonella atypica ACS-134-V-Col7a	1107	1398	292	NGF
Neisseria meningitidis Z2491	835	1082	248	NHN
Ignavibacterium album JCM 16511	1296	1688	393	NKK
Ruminococcus albus 8	853	1156	304	NNF
Streptococcus thermophilus LMD-9	811	1121	311	NNK
Barnesiella intestinihominis YIT 11860	871	1153	283	NPV
Azos pirillums p. B510	911	1168	258	PFH
Rhodospirillum rubrum ATCC 11170	863	1173	311	PRG
Planococcus antarcticus DSM 14505	1087	1333	247	PYY
Staphylococcus pseudintermedius ED99	1073	1334	262	QIV
Alca nivorax sp. W11-5	843	1113	271	RIE
Bradyrhizobium sp. BTAi1	811	1064	254	RIY
Streptococcus pyogenes M1 GAS	1099	1368	270	RKR
Streptococcus mutans UA159	1078	1345	268	RKR
Streptococcus pyogenes	1099	1368	270	RKR
Bacteroides sp. 20 3	1147	1517	371	RNI
S. aureus	772	1053	282	RNK
Solobacterium moorei F0204	1062	1327	266	RSG
Finegoldia magna ATCC 29328	1081	1348	268	RTE
uncultured delta proteobacterium HF0070 07E19	770	1011	242	SGG
Acida minococcus sp. D21	1064	1358	295	SIG
Eubacterium rectale ATCC 33656	824	1114	291	SKK
Caenispirillum salinarum AK4	1048	1442	395	SLV
Acidothermus cellulolyticus 11B	830	1138	309	SPS
Catenibacterium mitsuokai DSM 15897	1068	1329	262	SPT
Parvibaculum lavamentivorans DS-1	827	1037	211	TGN
Staphylococcus lugdunensis M23590	772	1054	283	TKK
Streptococcus sanguinis SK49	1123	1421	299	TRM
Elusimicrobium minutum Pei191	910	1195	286	TTG
Nitrobacter hamburgensis X14	914	1166	253	VAY
Mycoplasma synoviae 53	991	1314	324	VGF
Sphaerochaeta globus str. Buddy	877	1179	303	VKG
Ilyobacter polytropus DSM 2926	837	1092	256	VNG
Rhodovulum sp. PH10	821	1059	239	VPY
Bifidobacterium longum DJO10A	904	1187	284	VRK

Nucleic Acids Encoding Cas9 Fusion Molecules

Nucleic acids encoding the Cas9 fusion molecules, the Cas9 molecules or Cas9 polypeptides, e.g., an eaCas9 molecule or eaCas9 polypeptides, are provided herein.

In an embodiment, a nucleic acid encoding a Cas9 fusion molecule, a Cas9 molecule, or Cas9 polypeptide, can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section XI. In an embodiment, the mRNA, e.g., coding for a Cas9 fusion molecule, Cas9 molecule, or Cas9 polypeptide, disclosed herein, has one or more, e.g., all, of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.

In addition, or alternatively, the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a codon that is common in the host cell. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.

In addition, or alternatively, a nucleic acid encoding a Cas9 fusion molecule, a Cas9 molecule, or a Cas9 polypeptide, may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. pyogenes.

(SEQ ID NO: 138)
ATGGATAAAA AGTACAGCAT CGGGCTGGAC ATCGGTACAA
ACTCAGTGGG GTGGGCCGTG ATTACGGACG AGTACAAGGT
ACCCTCCAAA AAATTTAAAG TGCTGGGTAA CACGGACAGA
CACTCTATAA AGAAAAATCT TATTGGAGCC TTGCTGTTCG
ACTCAGGCGA GACAGCCGAA GCCACAAGGT TGAAGCGGAC
CGCCAGGAGG CGGTATACCA GGAGAAAGAA CCGCATATGC
TACCTGCAAG AAATCTTCAG TAACGAGATG GCAAAGGTTG
ACGATAGCTT TTTCCATCGC CTGGAAGAAT CCTTTCTTGT
TGAGGAAGAC AAGAAGCACG AACGGCACCC CATCTTTGGC
AATATTGTCG ACGAAGTGGC ATATCACGAA AAGTACCCGA
CTATCTACCA CCTCAGGAAG AAGCTGGTGG ACTCTACCGA
TAAGGCGGAC CTCAGACTTA TTTATTTGGC ACTCGCCCAC
ATGATTAAAT TTAGAGGACA TTTCTTGATC GAGGGCGACC
TGAACCCGGA CAACAGTGAC GTCGATAAGC TGTTCATCCA
ACTTGTGCAG ACCTACAATC AACTGTTCGA AGAAAACCCT
ATAAATGCTT CAGGAGTCGA CGCTAAAGCA ATCCTGTCCG
CGCGCCTCTC AAAATCTAGA AGACTTGAGA ATCTGATTGC
TCAGTTGCCC GGGGAAAAGA AAAATGGATT GTTTGGCAAC
CTGATCGCCC TCAGTCTCGG ACTGACCCCA AATTTCAAAA
GTAACTTCGA CCTGGCCGAA GACGCTAAGC TCCAGCTGTC
CAAGGACACA TACGATGACG ACCTCGACAA TCTGCTGGCC
CAGATTGGGG ATCAGTACGC CGATCTCTTT TTGGCAGCAA
AGAACCTGTC CGACGCCATC CTGTTGAGCG ATATCTTGAG
AGTGAACACC GAAATTACTA AAGCACCCCT TAGCGCATCT
ATGATCAAGC GGTACGACGA GCATCATCAG GATCTGACCC
TGCTGAAGGC TCTTGTGAGG CAACAGCTCC CCGAAAAATA
CAAGGAAATC TTCTTTGACC AGAGCAAAAA CGGCTACGCT
GGCTATATAG ATGGTGGGGC CAGTCAGGAG GAATTCTATA
AATTCATCAA GCCCATTCTC GAGAAAATGG ACGGCACAGA
GGAGTTGCTG GTCAAACTTA ACAGGGAGGA CCTGCTGCGG
AAGCAGCGGA CCTTTGACAA CGGGTCTATC CCCCACCAGA
TTCATCTGGG CGAACTGCAC GCAATCCTGA GGAGGCAGGA
GGATTTTTAT CCTTTTCTTA AAGATAACCG CGAGAAAATA
GAAAAGATTC TTACATTCAG GATCCCGTAC TACGTGGGAC
CTCTCGCCCG GGGCAATTCA CGGTTTGCCT GGATGACAAG
GAAGTCAGAG GAGACTATTA CACCTTGGAA CTTCGAAGAA
GTGGTGGACA AGGGTGCATC TGCCCAGTCT TTCATCGAGC
GGATGACAAA TTTTGACAAG AACCTCCCTA ATGAGAAGGT
GCTGCCCAAA CATTCTCTGC TCTACGAGTA CTTTACCGTC
TACAATGAAC TGACTAAAGT CAAGTACGTC ACCGAGGGAA
TGAGGAAGCC GGCATTCCTT AGTGGAGAAC AGAAGAAGGC
GATTGTAGAC CTGTTGTTCA AGACCAACAG GAAGGTGACT
GTGAAGCAAC TTAAAGAAGA CTACTTTAAG AAGATCGAAT
GTTTTGACAG TGTGGAAATT TCAGGGGTTG AAGACCGCTT
CAATGCGTCA TTGGGGACTT ACCATGATCT TCTCAAGATC
ATAAAGGACA AAGACTTCCT GGACAACGAA GAAAATGAGG
ATATTCTCGA AGACATCGTC CTCACCCTGA CCCTGTTCGA
AGACAGGGAA ATGATAGAAG AGCGCTTGAA AACCTATGCC
CACCTCTTCG ACGATAAAGT TATGAAGCAG CTGAAGCGCA
GGAGATACAC AGGATGGGGA AGATTGTCAA GGAAGCTGAT
CAATGGAATT AGGGATAAAC AGAGTGGCAA GACCATACTG
GATTTCCTCA AATCTGATGG CTTCGCCAAT AGGAACTTCA
TGCAACTGAT TCACGATGAC TCTCTTACCT TCAAGGAGGA
CATTCAAAAG GCTCAGGTGA GCGGGCAGGG AGACTCCCTT
CATGAACACA TCGCGAATTT GGCAGGTTCC CCCGCTATTA
AAAAGGGCAT CCTTCAAACT GTCAAGGTGG TGGATGAATT
GGTCAAGGTA ATGGGCAGAC ATAAGCCAGA AAATATTGTG
ATCGAGATGG CCCGCGAAAA CCAGACCACA CAGAAGGGCC
AGAAAAATAG TAGAGAGCGG ATGAAGAGGA TCGAGGAGGG
CATCAAAGAG CTGGGATCTC AGATTCTCAA AGAACACCCC
GTAGAAAACA CACAGCTGCA GAACGAAAAA TTGTACTTGT
ACTATCTGCA GAACGGCAGA GACATGTACG TCGACCAAGA
ACTTGATATT AATAGACTGT CCGACTATGA CGTAGACCAT
ATCGTGCCCC AGTCCTTCCT GAAGGACGAC TCCATTGATA
ACAAAGTCTT GACAAGAAGC GACAAGAACA GGGGTAAAAG
TGATAATGTG CCTAGCGAGG AGGTGGTGAA AAAAATGAAG
AACTACTGGC GACAGCTGCT TAATGCAAAG CTCATTACAC
AACGGAAGTT CGATAATCTG ACGAAAGCAG AGAGAGGTGG
CTTGTCTGAG TTGGACAAGG CAGGGTTTAT TAAGCGGCAG
CTGGTGGAAA CTAGGCAGAT CACAAAGCAC GTGGCGCAGA
TTTTGGACAG CCGGATGAAC ACAAAATACG ACGAAAATGA
TAAACTGATA CGAGAGGTCA AAGTTATCAC GCTGAAAAGC
AAGCTGGTGT CCGATTTTCG GAAAGACTTC CAGTTCTACA
AAGTTCGCGA GATTAATAAC TACCATCATG CTCACGATGC
GTACCTGAAC GCTGTTGTCG GGACCGCCTT GATAAAGAAG
TACCCAAAGC TGGAATCCGA GTTCGTATAC GGGGATTACA
AAGTGTACGA TGTGAGGAAA ATGATAGCCA AGTCCGAGCA
GGAGATTGGA AAGGCCACAG CTAAGTACTT CTTTTATTCT
AACATCATGA ATTTTTTTAA GACGGAAATT ACCCTGGCCA
ACGGAGAGAT CAGAAAGCGG CCCCTTATAG AGACAAATGG
TGAAACAGGT GAAATCGTCT GGGATAAGGG CAGGGATTTC
GCTACTGTGA GGAAGGTGCT GAGTATGCCA CAGGTAAATA
TCGTGAAAAA AACCGAAGTA CAGACCGGAG GATTTTCCAA
GGAAAGCATT TTGCCTAAAA GAAACTCAGA CAAGCTCATC
GCCCGCAAGA AAGATTGGGA CCCTAAGAAA TACGGGGGAT
TTGACTCACC CACCGTAGCC TATTCTGTGC TGGTGGTAGC
TAAGGTGGAA AAAGGAAAGT CTAAGAAGCT GAAGTCCGTG
AAGGAACTCT TGGGAATCAC TATCATGGAA AGATCATCCT
TTGAAAAGAA CCCTATCGAT TTCCTGGAGG CTAAGGGTTA
CAAGGAGGTC AAGAAAGACC TCATCATTAA ACTGCCAAAA
TACTCTCTCT TCGAGCTGGA AAATGGCAGG AAGAGAATGT
TGGCCAGCGC CGGAGAGCTG CAAAAGGGAA ACGAGCTTGC
TCTGCCCTCC AAATATGTTA ATTTTCTCTA TCTCGCTTCC
CACTATGAAA AGCTGAAAGG GTCTCCCGAA GATAACGAGC
AGAAGCAGCT GTTCGTCGAA CAGCACAAGC ACTATCTGGA
TGAAATAATC GAACAAATAA GCGAGTTCAG CAAAAGGGTT
ATCCTGGCGG ATGCTAATTT GGACAAAGTA CTGTCTGCTT
ATAACAAGCA CCGGGATAAG CCTATTAGGG AACAAGCCGA
GAATATAATT CACCTCTTTA CACTCACGAA TCTCGGAGCC
CCCGCCGCCT TCAAATACTT TGATACGACT ATCGACCGGA
AACGGTATAC CAGTACCAAA GAGGTCCTCG ATGCCACCCT
CATCCACCAG TCAATTACTG GCCTGTACGA AACACGGATC
GACCTCTCTC AACTGGGCGG CGACTAG

Provided below is the corresponding amino acid sequence of a S. pyogenes Cas9 molecule.

(SEQ ID NO: 139)
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR
LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD
LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP
INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP
NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI
FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD
LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ
LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD
SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV
MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD
SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI
REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK
YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI
TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE
KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE
DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLSQLGGD*

Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of N. meningitidis.

(SEQ ID NO: 140)
ATGGCCGCCTTCAAGCCCAACCCCATCAACTACATCCTGGGCCTGGACAT
CGGCATCGCCAGCGTGGGCTGGGCCATGGTGGAGATCGACGAGGACGAGA
ACCCCATCTGCCTGATCGACCTGGGTGTGCGCGTGTTCGAGCGCGCTGAG
GTGCCCAAGACTGGTGACAGTCTGGCTATGGCTCGCCGGCTTGCTCGCTC
TGTTCGGCGCCTTACTCGCCGGCGCGCTCACCGCCTTCTGCGCGCTCGCC
GCCTGCTGAAGCGCGAGGGTGTGCTGCAGGCTGCCGACTTCGACGAGAAC
GGCCTGATCAAGAGCCTGCCCAACACTCCTTGGCAGCTGCGCGCTGCCGC
TCTGGACCGCAAGCTGACTCCTCTGGAGTGGAGCGCCGTGCTGCTGCACC
TGATCAAGCACCGCGGCTACCTGAGCCAGCGCAAGAACGAGGGCGAGACC
GCCGACAAGGAGCTGGGTGCTCTGCTGAAGGGCGTGGCCGACAACGCCCA
CGCCCTGCAGACTGGTGACTTCCGCACTCCTGCTGAGCTGGCCCTGAACA
AGTTCGAGAAGGAGAGCGGCCACATCCGCAACCAGCGCGGCGACTACAGC
CACACCTTCAGCCGCAAGGACCTGCAGGCCGAGCTGATCCTGCTGTTCGA
GAAGCAGAAGGAGTTCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGG
GCATCGAGACCCTGCTGATGACCCAGCGCCCCGCCCTGAGCGGCGACGCC
GTGCAGAAGATGCTGGGCCACTGCACCTTCGAGCCAGCCGAGCCCAAGGC
CGCCAAGAACACCTACACCGCCGAGCGCTTCATCTGGCTGACCAAGCTGA
ACAACCTGCGCATCCTGGAGCAGGGCAGCGAGCGCCCCCTGACCGACACC
GAGCGCGCCACCCTGATGGACGAGCCCTACCGCAAGAGCAAGCTGACCTA
CGCCCAGGCCCGCAAGCTGCTGGGTCTGGAGGACACCGCCTTCTTCAAGG
GCCTGCGCTACGGCAAGGACAACGCCGAGGCCAGCACCCTGATGGAGATG
AAGGCCTACCACGCCATCAGCCGCGCCCTGGAGAAGGAGGGCCTGAAGGA
CAAGAAGAGTCCTCTGAACCTGAGCCCCGAGCTGCAGGACGAGATCGGCA
CCGCCTTCAGCCTGTTCAAGACCGACGAGGACATCACCGGCCGCCTGAAG
GACCGCATCCAGCCCGAGATCCTGGAGGCCCTGCTGAAGCACATCAGCTT
CGACAAGTTCGTGCAGATCAGCCTGAAGGCCCTGCGCCGCATCGTGCCCC
TGATGGAGCAGGGCAAGCGCTACGACGAGGCCTGCGCCGAGATCTACGGC
GACCACTACGGCAAGAAGAACACCGAGGAGAAGATCTACCTGCCTCCTAT
CCCCGCCGACGAGATCCGCAACCCCGTGGTGCTGCGCGCCCTGAGCCAGG
CCCGCAAGGTGATCAACGGCGTGGTGCGCCGCTACGGCAGCCCCGCCCGC
ATCCACATCGAGACCGCCCGCGAGGTGGGCAAGAGCTTCAAGGACCGCAA
GGAGATCGAGAAGCGCCAGGAGGAGAACCGCAAGGACCGCGAGAAGGCCG
CCGCCAAGTTCCGCGAGTACTTCCCCAACTTCGTGGGCGAGCCCAAGAGC
AAGGACATCCTGAAGCTGCGCCTGTACGAGCAGCAGCACGGCAAGTGCCT
GTACAGCGGCAAGGAGATCAACCTGGGCCGCCTGAACGAGAAGGGCTACG
TGGAGATCGACCACGCCCTGCCCTTCAGCCGCACCTGGGACGACAGCTTC
AACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACAAGGGCAACCA
GACCCCCTACGAGTACTTCAACGGCAAGGACAACAGCCGCGAGTGGCAGG
AGTTCAAGGCCCGCGTGGAGACCAGCCGCTTCCCCCGCAGCAAGAAGCAG
CGCATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAAGGAGCGCAACCT
GAACGACACCCGCTACGTGAACCGCTTCCTGTGCCAGTTCGTGGCCGACC
GCATGCGCCTGACCGGCAAGGGCAAGAAGCGCGTGTTCGCCAGCAACGGC
CAGATCACCAACCTGCTGCGCGGCTTCTGGGGCCTGCGCAAGGTGCGCGC
CGAGAACGACCGCCACCACGCCCTGGACGCCGTGGTGGTGGCCTGCAGCA
CCGTGGCCATGCAGCAGAAGATCACCCGCTTCGTGCGCTACAAGGAGATG
AACGCCTTCGACGGTAAAACCATCGACAAGGAGACCGGCGAGGTGCTGCA
CCAGAAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGA
TGATCCGCGTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGGCC
GACACCCCCGAGAAGCTGCGCACCCTGCTGGCCGAGAAGCTGAGCAGCCG
CCCTGAGGCCGTGCACGAGTACGTGACTCCTCTGTTCGTGAGCCGCGCCC
CCAACCGCAAGATGAGCGGTCAGGGTCACATGGAGACCGTGAAGAGCGCC
AAGCGCCTGGACGAGGGCGTGAGCGTGCTGCGCGTGCCCCTGACCCAGCT
GAAGCTGAAGGACCTGGAGAAGATGGTGAACCGCGAGCGCGAGCCCAAGC
TGTACGAGGCCCTGAAGGCCCGCCTGGAGGCCCACAAGGACGACCCCGCC
AAGGCCTTCGCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGCAC
CCAGCAGGTGAAGGCCGTGCGCGTGGAGCAGGTGCAGAAGACCGGCGTGT
GGGTGCGCAACCACAACGGCATCGCCGACAACGCCACCATGGTGCGCGTG
GACGTGTTCGAGAAGGGCGACAAGTACTACCTGGTGCCCATCTACAGCTG
GCAGGTGGCCAAGGGCATCCTGCCCGACCGCGCCGTGGTGCAGGGCAAGG
ACGAGGAGGACTGGCAGCTGATCGACGACAGCTTCAACTTCAAGTTCAGC
CTGCACCCCAACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGCATGTT
CGGCTACTTCGCCAGCTGCCACCGCGGCACCGGCAACATCAACATCCGCA
TCCACGACCTGGACCACAAGATCGGCAAGAACGGCATCCTGGAGGGCATC
GGCGTGAAGACCGCCCTGAGCTTCCAGAAGTACCAGATCGACGAGCTGGG
CAAGGAGATCCGCCCCTGCCGCCTGAAGAAGCGCCCTCCTGTGCGCTAA

Provided below is the corresponding amino acid sequence of a N. meningitidis Cas9 molecule.

(SEQ ID NO: 141)
MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAE
VPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDEN
GLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGET
ADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS
HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDA
VQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT
ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEM
KAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG
DHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPAR
IHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKS
KDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF
NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ
RILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG
QITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEM
NAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA
DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSA
KRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA
KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV
DVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS
LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGI
GVKTALSFQKYQIDELGKEIRPCRLKKRPPVR*

Provided below is an amino acid sequence of a S. aureus Cas9 molecule.

(SEQ ID NO: 142)
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK
RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL
SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYV
AELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT
YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA
YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA
KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQ
IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV
KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQ
TNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNP
FNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS
YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTR
YATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKH
HAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEY
KEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL
IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE
KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS
RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEA
KKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT
YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII
KKG*

Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. aureus Cas9.

(SEQ ID NO: 143)
ATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGG
GTATGGGATTATTGACTATGAAACAAGGGACGTGATCGACGCAGGCGTCA
GACTGTTCAAGGAGGCCAACGTGGAAAACAATGAGGGACGGAGAAGCAAG
AGGGGAGCCAGGCGCCTGAAACGACGGAGAAGGCACAGAATCCAGAGGGT
GAAGAAACTGCTGTTCGATTACAACCTGCTGACCGACCATTCTGAGCTGA
GTGGAATTAATCCTTATGAAGCCAGGGTGAAAGGCCTGAGTCAGAAGCTG
TCAGAGGAAGAGTTTTCCGCAGCTCTGCTGCACCTGGCTAAGCGCCGAGG
AGTGCATAACGTCAATGAGGTGGAAGAGGACACCGGCAACGAGCTGTCTA
CAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAGTATGTC
GCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTC
AATTAATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGC
TGAAAGTGCAGAAGGCTTACCACCAGCTGGATCAGAGCTTCATCGATACT
TATATCGACCTGCTGGAGACTCGGAGAACCTACTATGAGGGACCAGGAGA
AGGGAGCCCCTTCGGATGGAAAGACATCAAGGAATGGTACGAGATGCTGA
TGGGACATTGCACCTATTTTCCAGAAGAGCTGAGAAGCGTCAAGTACGCT
TATAACGCAGATCTGTACAACGCCCTGAATGACCTGAACAACCTGGTCAT
CACCAGGGATGAAAACGAGAAACTGGAATACTATGAGAAGTTCCAGATCA
TCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTACACTGAAACAGATTGCT
AAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACAAG
CACTGGAAAACCAGAGTTCACCAATCTGAAAGTGTATCACGATATTAAGG
ACATCACAGCACGGAAAGAAATCATTGAGAACGCCGAACTGCTGGATCAG
ATTGCTAAGATCCTGACTATCTACCAGAGCTCCGAGGACATCCAGGAAGA
GCTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATCGAACAGATTA
GTAATCTGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATC
AATCTGATTCTGGATGAGCTGTGGCATACAAACGACAATCAGATTGCAAT
CTTTAACCGGCTGAAGCTGGTCCCAAAAAAGGTGGACCTGAGTCAGCAGA
AAGAGATCCCAACCACACTGGTGGACGATTTCATTCTGTCACCCGTGGTC
AAGCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAA
GTACGGCCTGCCCAATGATATCATTATCGAGCTGGCTAGGGAGAAGAACA
GCAAGGACGCACAGAAGATGATCAATGAGATGCAGAAACGAAACCGGCAG
ACCAATGAACGCATTGAAGAGATTATCCGAACTACCGGGAAAGAGAACGC
AAAGTACCTGATTGAAAAAATCAAGCTGCACGATATGCAGGAGGGAAAGT
GTCTGTATTCTCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAATCCA
TTCAACTACGAGGTCGATCATATTATCCCCAGAAGCGTGTCCTTCGACAA
TTCCTTTAACAACAAGGTGCTGGTCAAGCAGGAAGAGAACTCTAAAAAGG
GCAATAGGACTCCTTTCCAGTACCTGTCTAGTTCAGATTCCAAGATCTCT
TACGAAACCTTTAAAAAGCACATTCTGAATCTGGCCAAAGGAAAGGGCCG
CATCAGCAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACA
GATTCTCCGTCCAGAAGGATTTTATTAACCGGAATCTGGTGGACACAAGA
TACGCTACTCGCGGCCTGATGAATCTGCTGCGATCCTATTTCCGGGTGAA
CAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGGGTTCACATCTTTTC
TGAGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCAC
CATGCCGAAGATGCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGA
GTGGAAAAAGCTGGACAAAGCCAAGAAAGTGATGGAGAACCAGATGTTCG
AAGAGAAGCAGGCCGAATCTATGCCCGAAATCGAGACAGAACAGGAGTAC
AAGGAGATTTTCATCACTCCTCACCAGATCAAGCATATCAAGGATTTCAA
GGACTACAAGTACTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGA
TCAATGACACCCTGTATAGTACAAGAAAAGACGATAAGGGGAATACCCTG
ATTGTGAACAATCTGAACGGACTGTACGACAAAGATAATGACAAGCTGAA
AAAGCTGATCAACAAAAGTCCCGAGAAGCTGCTGATGTACCACCATGATC
CTCAGACATATCAGAAACTGAAGCTGATTATGGAGCAGTACGGCGACGAG
AAGAACCCACTGTATAAGTACTATGAAGAGACTGGGAACTACCTGACCAA
GTATAGCAAAAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATG
GGAACAAGCTGAATGCCCATCTGGACATCACAGACGATTACCCTAACAGT
CGCAACAAGGTGGTCAAGCTGTCACTGAAGCCATACAGATTCGATGTCTA
TCTGGACAACGGCGTGTATAAATTTGTGACTGTCAAGAATCTGGATGTCA
TCAAAAAGGAGAACTACTATGAAGTGAATAGCAAGTGCTACGAAGAGGCT
AAAAAGCTGAAAAAGATTAGCAACCAGGCAGAGTTCATCGCCTCCTTTTA
CAACAACGACCTGATTAAGATCAATGGCGAACTGTATAGGGTCATCGGGG
TGAACAATGATCTGCTGAACCGCATTGAAGTGAATATGATTGACATCACT
TACCGAGAGTATCTGGAAAACATGAATGATAAGCGCCCCCCTCGAATTAT
CAAAACAATTGCCTCTAAGACTCAGAGTATCAAAAAGTACTCAACCGACA
TTCTGGGAAACCTGTATGAGGTGAAGAGCAAAAAGCACCCTCAGATTATC
AAAAAGGGC

If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C-terminus, it is understood that the stop codon will be removed.

Other Cas Molecules and Cas Polypeptides

Various types of Cas molecules or Cas polypeptides can be used to practice the inventions disclosed herein. In some embodiments, Cas molecules of Type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, Type I or Type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, e.g., in Haft et al. (2005) PLOS COMPUTATIONAL BIOLOGY 1(6): e60, and in Makarova et al. (2011) NATURE REVIEW MICROBIOLOGY 9:467-477, the contents of which are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table 600.

TABLE 600
Cas Systems
			Structure of	Families (and
			encoded	superfamily) of
Gene	System type	Name from	protein (PDB	encoded
name‡	or subtype	Haft et al.§	accessions)¶	protein#**	Representatives
cas1	Type I	cas1	3GOD, 3LFX	COG1518	SERP2463,
	Type II		and 2YZS		SPy 1047 and ygbT
	Type III
cas2	Type I	cas2	2IVY, 218E	COG1343 and	SERP2462,
	Type II		and 3EXC	COG3512	SPy 1048, SPy 1723
	Type III				(N-terminal domain)
					and ygbF
cas3′	Type I‡‡	cas3	NA	COG1203	APE1232 and ygcB
cas3″	Subtype I-A	NA	NA	COG2254	APE1231 and
	Subtype I-B				BH0336
cas4	Subtype I-A	cas4 and csa1	NA	COG1468	APE1239 and
	Subtype I-B				BH0340
	Subtype I-C
	Subtype I-D
	Subtype II-B
cas5	Subtype I-A	cas5a, cas5d,	3KG4	COG1688	APE1234, BH0337,
	Subtype I-B	cas5e, cas5h,		(RAMP)	devS and ygcI
	Subtype I-C	cas5p, cas5t
	Subtype I-E	and cmx5
cas6	Subtype I-A	cas6 and cmx6	3I4H	COG1583 and	PF1131 and slr7014
	Subtype I-B			COG5551
	Subtype I-D			(RAMP)
	Subtype III-A
	Subtype III-B
cas6e	Subtype I-E	cse 3	1WJ9	(RAMP)	ygcH
cas6f	Subtype I-F	csy4	2XLJ	(RAMP)	y 1727
cas7	Subtype I-A	csa2, csd2,	NA	COG1857 and	devR and ygcJ
	Subtype I-B	cse4, csh2,		COG3649
	Subtype I-C	csp1 and cst 2		(RAMP)
	Subtype I-E
cas8a1	Subtype I-A‡‡	cmx1, cst1,	NA	BH0338-like	LA3191§§ and
		csx8, csx13			PG2018§§
		and CXXC-
		CXXC
cas8a2	Subtype I-A‡‡	csa4 and csx9	NA	PH0918	AF0070, AF1873,
					MJ0385, PF0637,
					PH0918 and
					SSO1401
cas8b	Subtype I-B‡‡	csh1 and	NA	BH0338-like	MTH1090 and
		TM1802			TM1802
cas8c	Subtype I-C‡‡	csd1 and csp2	NA	BH0338-like	BH0338
cas9	Type II‡‡	csn1 and csx12	NA	COG3513	FTN 0757 and
					SPy 1046
cas10	Type III‡‡	cmr2, csm1	NA	COG1353	MTH326,
		and csx11			Rv2823c§§ and
					TM1794§§
cas10d	Subtype I-D‡‡	csc3	NA	COG1353	slr7011
csy1	Subtype I-F‡‡	csy1	NA	y 1724-like	y 1724
csy2	Subtype I-F	csy2	NA	(RAMP)	y 1725
csy3	Subtype I-F	csy3	NA	(RAMP)	y 1726
cse1	Subtype I-E‡‡	cse I	NA	YgcL-like	ygcL
cse2	Subtype I-E	cse 2	2ZCA	Ygck-like	ygck
csc1	Subtype I-D	csc 1	NA	alr1563-like	alr1563
				(RAMP)
csc2	Subtype I-D	csc1 and csc2	NA	COG1337	slr7012
				(RAMP)
csa5	Subtype I-A	csa5	NA	AF1870	AF1870, MJ0380,
					PF0643 and
					SSO1398
csn2	Subtype II-A	csn 2	NA	SPy 1049-like	SPy 1049
csm2	Subtype III-	csm2	NA	COG1421	MTH1081 and
	A‡‡				SERP2460
csm3	Subtype III-	csc2 and csm3	NA	COG1337	MTH1080 and
	A			(RAMP)	SERP2459
csm4	Subtype III-	csm4	NA	COG1567	MTH1079 and
	A			(RAMP)	SERP2458
csm5	Subtype III-	csm5	NA	COG1332	MTH1078 and
	A			(RAMP)	SERP2457
csm6	Subtype III-	APE2256 and	2WTE	COG1517	APE2256 and
	A	csm6			SSO1445
cmr1	Subtype III-	cmr1	NA	COG1367	PF1130
	B			(RAMP)
cmr3	Subtype III-	cmr3	NA	COG1769	PF1128
	B			(RAMP)
cmr4	Subtype III-	cmr4	NA	COG1336	PF1126
	B			(RAMP)
cmr5	Subtype III-	cmr5	2ZOP and	COG3337	MTH324 and
	B‡‡		2OEB		PF1125
cmr6	Subtype III-	cmr6	NA	COG1604	PF1124
	B			(RAMP)
csb1	Subtype I-U	GSU0053	NA	(RAMP)	Balac_1306 and
					GSU0053
csb2	Subtype I-	NA	NA	(RAMP)	Balac_1305 and
	USS				GSU0054
csb3	Subtype I-U	NA	NA	(RAMP)	Balac_1303§§
csx17	Subtype I-U	NA	NA	NA	Btus_2683
csx14	Subtype I-U	NA	NA	NA	GSU0052
csx10	Subtype I-U	csx10	NA	(RAMP)	Caur_2274
csx16	Subtype III-	VVA 1548	NA	NA	VVA1548
	U
csaX	Subtype III-	csaX	NA	NA	SSO1438
	U
csx3	Subtype III-	csx3	NA	NA	AF1864
	U
csx1	Subtype III-	csa3, csx1,	1XMX and	COG1517 and	MJ1666, NE0113,
	U	csx2, DXTHG,	2I71	COG4006	PF1127 and
		NE0113 and			TM1812
		TIGR02710
csx15	Unknown	NA	NA	TTE2665	TTE2665
csf1	Type U	csf1	NA	NA	AFE_1038
csf2	Type U	csf2	NA	(RAMP)	AFE_1039
csf3	Type U	csf3	NA	(RAMP)	AFE_1040
csf4	Type U	csf4	NA	NA	AFE_1037

IV. Linkers to Connect Cas9 Molecules or Cas9 Polypeptides to a Template Binding Domain

In an embodiment, a linker covalently connects the Cas9 molecule to the template binding domain (e.g., a DNA binding domain).

In an embodiment, a linker is a short peptide sequence that connects protein domains. In another embodiment, a linker is a short peptide sequence that connects a protein domain and a nucleic acid (e.g., DNA or RNA). In an embodiment, a linker is a short peptide sequence that connects a protein domain and a small molecule. In an embodiment, a linker is a short peptide sequence that connects two small molecules. Linkers are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. In certain embodiments, the linker has sufficient length and flexibility to allow the Cas9 molecule to bind to a target nucleic acid and simultaneously allow the target binding domain to associate with a nucleic acid template system, e.g., so that one binding event does not sterically block the other, and/or so that two adjacent domains do not sterically interfere with one another.

The linker can attach to the C-terminus or N-terminus of the Cas9 molecule.

The linker can attach to the C-terminus or N-terminus of the template binding domain.

The linker can attach to the C-terminus or N-terminus of the template binding domain partner.

The linker can attach to the C-terminus or N-terminus of the template nucleic acid.

In some embodiments, the linker length is from about 6 to 60 amino acids. The linker may be, e.g., 6-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-60 amino acids in length. The linker may be, e.g., at least 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 amino acids in length. In other embodiments, the linker is, e.g., at most 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 amino acids in length. Ranges comprising any combination of these endpoints are also envisioned.

In some embodiments, the linker is encoded by a nucleic acid sequence of 6 to 60 nucleotides or base pairs. The nucleic acid may be, e.g., 6-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length. The linker may be, e.g., at least 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 nucleotides in length. In some embodiments, the linker is, e.g., at most 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 nucleotides in length. Ranges comprising any combination of these endpoints are also envisioned.

In some embodiments, the linker comprises glycine and serine residues. In some embodiments the linker consists of glycine and serine residues. For instance, the linker may comprise one of more modules such as GGS, GSGS, GGGS, GGGGS or GGSG. In some embodiments, the linker comprises a plurality of modules comprising glycine and serine, e.g., at least 2, 3, 4, 5, 10, or 15 of these modules, and/or at most 3, 4, 5, 10, 15, or 20 of these modules, or any combination of these endpoints. In some embodiments, each module in the linker has the same sequence, and in other embodiments, at least two modules in a linker have different sequences from each other.

In some embodiments, the linker is an XTEN linker or a variation of an XTEN linker such as SGSETPGTSESA (SEQ ID NO: 144), SGSETPGTSESATPES (SEQ ID NO: 2), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 145). Additional information on the XTEN linker may be found in Schellenberger et al. (2009), NATURE BIOTECHNOLOGY 27: 1186-1190, the entire contents of which are incorporated herein by reference.

Exemplary linker modules are given in Table 2:

GGS
(SEQ ID NO: 146)
GSGS
(SEQ ID NO: 147)
GGGS
(SEQ ID NO: 148)
GGGGS
(SEQ ID NO: 149)
GGSG
(SEQ ID NO: 150)
SGSETPGTSESA
(SEQ ID NO: 2)
IPGTSESATPES
(SEQ ID NO: 151)
SGSETPGTSESATPEGGSGGS

Additional exemplary linker modules are given in Table 3:

		Length
Name	Description	(nt)
BBa_J176131	PLrigid	60
BBa_J18920	2aa GS linker	6
BBa_J18921	6aa [GS]x linker (SEQ ID NO: 152)	18
BBa_J18922	10aa [GS]x linker (SEQ ID NO: 153)	30
BBa_K105012	10 aa flexible protein domain linker	30
BBa_K133132	8 aa protein domain linker	24
BBa_K1486003	flexible linker 2x (GGGS) (SEQ ID NO: 154)	24
BBa_K1486004	flexible linker 2x (GGGGS) (SEQ ID NO: 155)	30
BBa_K1486037	linker	39
BBa_K157009	Split fluorophore linker; Freiburg standard	51
BBa_K157013	15 aa flexible glycine-serine protein domain linker;	45
	Freiburg standard
BBa_K243004	Short Linker (Gly-Gly-Ser-Gly) (SEQ ID NO: 156)	12
BBa_K243005	Middle Linker ( Gly-Gly-Ser-Gly)x2	24
	(SEQ ID NO: 157)
BBa_K243006	Long Linker (Gly-Gly-Ser-Gly)x3	36
	(SEQ ID NO: 158)
BBa_K243029	GSAT Linker	108
BBa_K243030	SEG	108
BBa_K404300	SEG-Linker	108
BBa_K404301	GSAT-Linker	108
BBa_K404303	Z-EGFR-1907_Short-Linker	192
BBa_K404304	Z-EGFR-1907_Middle-Linker	204
BBa_K404305	Z-EGFR-1907_Long-Linker	216
BBa_K404306	Z-EGFR-1907_SEG-Linker	288
BBa_K416001	(Gly4Ser)3 Flexible Peptide Linker	45
	(SEQ ID NO: 159)
BBa_K648005	Short Fusion Protein Linker: GGSG with standard	12
	25 prefix/suffix (SEQ ID NO: 160)
BBa_K648006	Long 10AA Fusion Protein Linker with Standard	30
	25 Prefix/Suffix
BBa_K648007	Medium 6AA Fusion Protein Linker: GGSGGS	18
	(SEQ ID NO: 161) with Standard 25 Prefix/Suffix

Linkers can comprise a direct bond or an atom such as, e.g., an oxygen (O) or sulfur (S), a unit such as —NR— wherein R is hydrogen or alkyl, —C(O)—, —C(O)O—, —C(O)NH—, SO, SO₂, —SO₂NH— or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, heteroarylalkyl. In some embodiments, one or more methylenes in the chain of atoms can be replaced with one or more of O, S, S(O), SO₂, —SO₂NH—, —NR—, —NR₂, —C(O)—, —C(O)O—, —C(O)NH—, a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic.

In some embodiments, the template binding domain is attached to the Cas9 molecule through a linker that is itself stable under physiological conditions, such as an alkylene chain, and does not result in release of the template binding domain from the Cas9 molecule for at least 2, 3, 4, 5, 10, 15, 24 or 48 hours or for at least 1, 2, 3, 4, 5 or 10 days when administered to a subject. In some embodiments, the template binding domain and the Cas9 molecule comprise residues of a functional groups through which reaction and linkage of the template binding domain to the Cas9 molecule was achieved. In some embodiments, the functional groups, which may be the same or different, terminal or internal, of the template binding domain or Cas9 molecule comprise an amino, acid, imidazole, hydroxyl, thio, acyl halide, —HC═CH—, —C≡C— group, or derivative thereof. In some embodiments, the linker comprises a hydrocarbylene group wherein one or more methylene groups is optionally replaced by a group Y (provided that none of the Y groups are adjacent to each other), wherein each Y, independently for each occurrence, is selected from, substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or —O—, C(═X) (wherein X is NR₁, O or S), —NR₁—, —NR₁C(O)—, —C(O)NR₁—, S(O)_n—, —NR₁S(O)_n—, S(O)_n—NR₁—, —NR₁C(O)—NR₁—; and R₁, independently for each occurrence, represents H or a lower alkyl and wherein n is 0, 1, or 2.

In some embodiments, the linker comprises an alkylene moiety or a heteroalkylene moiety (e.g., an alkylene glycol moiety such as ethylene glycol). In some embodiments, a linker comprises a poly-L-glutamic acid, polylactic acid, poly(ethyleneimine), an oligosaccharide, an amino acid (e.g., glycine), an amino acid chain, or any other suitable linkage. The linker groups can be biologically inactive, such as a PEG, polyglycolic acid, or polylactic acid chain. In certain embodiments, the linker group represents a derivatized or non-derivatized amino acid (e.g., glycine).

The Cas9 molecule attached to the linker may be any Cas9 molecule described herein, e.g., a Cas9 molecule of Section III, e.g., a nickase or a Cas9 molecule capable of making a double stranded break. The template binding domain attached to the linker may be any template binding domain described herein, e.g., a template binding domain of Section V, e.g., a DNA-binding domain.

V. Template Binding Domains

In some embodiments, the template binding domain comprises a plurality of domains that bind a template binding domain partner, e.g., DNA-binding molecules. For instance, a first DNA-binding molecule and a second DNA-binding molecule may, on their own, be poor choices because each has a low affinity and optionally also a short recognition sequence that is present in multiple copies in the genome of the cell to be altered. However, when combined, the first and second molecules could give a higher affinity and optionally also a longer recognition sequence that is rarer in the genome of the cell to be altered. Accordingly, in some embodiments, the template binding domain comprises a plurality of domains that bind a template binding domain partner, e.g., DNA-binding molecules. For instance, the template binding domain may comprise two or more, three or more, four or more, five or more, ten or more, 2-4, 4-6, 6-8, or 8-10 domains that bind a template binding domain partner, e.g., DNA-binding molecules.

The template binding domain can be covalently bound to the Cas9 molecule, e.g., using a linker. The template binding domain and the Cas9 can be part of the same fusion protein. In some embodiments, the Cas9 molecule and the template binding domain are part of the same gene and are transcribed to form a single mRNA which is then translated into a single protein. In other embodiments, the Cas9 molecule and the template binding domain are produced separately and then joined covalently or non-covalently. An example of a template binding domain which binds non-covalently to the Cas9 molecule is a template-binding domain that comprises a region with specific affinity for Cas9, e.g., an antibody that recognizes Cas9, e.g., an scFv antibody or another type of antibody that contains sufficient CDR sequences for binding to Cas9. In some embodiments, the template binding domain is derived from a wild-type protein. For example, the template binding domain may be a fragment of a wild type protein, a mutagenized wild type protein, a mutagenized wild-type protein fragment, a synthetic protein that has been modeled after the three dimensional structure of a naturally-occurring protein. In some embodiments, the template binding domain is mutagenized to increase its affinity for a template binding domain partner. In some embodiments, the template binding domain is mutagenized to decrease its affinity for a template binding domain partner.

V.1 Template Binding Domains that are DNA-Binding Polypeptides

In some embodiments, the template binding domain is a polypeptide, e.g., a protein or protein domain. This polypeptide can bind to the major groove of a target DNA sequence and/or a minor groove of a target DNA sequence. It can comprise one or more of the following domains: zinc finger, helix-turn-helix, leucine zipper, winged helix, winged helix turn helix, helix-loop-helix, HMG-box, and Wor3 domain. It can bind single stranded DNA or double stranded DNA. In some embodiments, the DNA-binding polypeptide is identical in sequence to a wild-type protein, and in other embodiments it comprises one or more mutations, e.g., deletions, relative to a wild-type protein.

In some embodiments, the DNA-binding polypeptide comprises a mutation relative to a wild-type DNA-binding protein. For example, if the wild-type DNA-binding protein must bind a ligand or co-activator before it can bind DNA, the DNA-binding polypeptide is optionally mutated to a constitutively active form. Similarly, if the wild-type DNA-binding protein is incapable of binding to DNA in the presence of a ligand or co-activator before it can bind DNA, the DNA-binding polypeptide can also be mutated to a constitutively active form. In some embodiments, the DNA-binding polypeptide carries a deletion relative to a wild-type protein, e.g., a transcriptional activation or repression domain or a catalytic domain is removed. In some embodiments, the DNA-binding polypeptide consists only of the DNA-binding region of the corresponding wild-type DNA-binding protein.

In some embodiments, the DNA-binding polypeptide recognizes chemically modified DNA, e.g., methylated DNA. In some embodiments, the DNA-binding polypeptide recognizes a chemical modification that is rare in or absent from the genome of the cell to be altered. This can help avoid the DNA-binding polypeptide non-specifically binding to the cell's genome.

Several exemplary DNA binding proteins are given below.

Operon

In some embodiments, the DNA-binding polypeptide is, or is derived from, a DNA-binding protein from an operon, e.g., a bacterial operon. The DNA-binding polypeptide may be, e.g., a repressor or an activator in the context of the operon. Generally, the DNA-binding polypeptide will not activate or repress transcription in the methods described herein. This can be achieved by, e.g., mutating transcriptional regulation domains, or choosing a DNA-binding polypeptide that does not engage the transcriptional machinery of the cell to be altered. For example, when altering the genome of a human cell, one could choose a DNA-binding peptide from a prokaryote, Archaea, single celled eukaryote, plant, or fungus.

DNA-binding proteins from operons, and the nucleotide sequences to which they bind, are known in the art (see, e.g., Postle et al. (1984) NUCLEIC ACIDS RES. 12: 4849-63; Buvinger and Riley (1985) J. BACTERIOL. 163: 850-7; Laughon and Gesteland (1984) MOL. CELL BIOL. 4:260-7; Bram et al. (1986) EMBO J. 5: 603-8; Von Wilcken-Bergmann & Muller-Hill (1982) PROC. NAT'L. ACAD. SCI. 79: 2427-31; Heinrich et al. (1989) NUCLEIC ACIDS RES. 17: 7681-92; Osborne et al. (1989) NUCLEIC ACIDS RES. 17: 7671-80; Singleton et al. (1980) NUCLEIC ACIDS RES. 8: 1551-60; Widdowson et al. (1996) ANTIMICROB. AGENTS CHEMOTHER. 40: 2891-93; Oehler et al. (1994) EMBO J. 13: 3348-55; Bailone and Galibert (1980) NUCLEIC ACIDS RES. 8: 2147-64; and, Staacke et al. (1990) EMBO J. 9: 1963-7).

Exemplary DNA-binding proteins from operons are given in the table below. The template-binding domain may comprise one or more of these proteins or polypeptides derived therefrom.

TABLE V.1
DNA-binding proteins from operons
Template binding     DNA sequence recognized by
domain               the template binding domain
TetR repressor       Tet-O
LacI repressor       Lac operon 01
Gal4 repressor       UAS
Repressor protein C1 Operator L and R
Trp repressor        Trp operator

Transcription Factors

In some embodiments, the DNA-binding polypeptide is, or is derived from, a transcription factor. The DNA-binding polypeptide may be or be derived from, e.g., a repressor or an activator in its wild-type context. Generally, the DNA-binding polypeptide will not activate or repress transcription in the methods described herein. This can be achieved by, e.g., mutating transcriptional regulation domains, such as the trans-activating domain (TAD) or any other domain that binds a transcription co-regulator. This can also be achieved by choosing a DNA-binding polypeptide that does not engage the transcriptional machinery of the cell to be altered. For example, when altering the genome of a human cell, one could choose a DNA-binding peptide from a prokaryote, Archaea, single celled eukaryote, plant, or fungus.

The transcription factor, in some embodiments, falls into one or more of several categories as set out here. The transcription factor may be a specific transcription factor and/or an upstream transcription factor. It may be constitutively active or conditionally active. If conditionally active, it may be developmental or signal-dependent. In some embodiments, the transcription factor is a resident nuclear factor and/or comprises a nuclear localization signal (NLS).

Exemplary transcription factors are given in the table below. The template-binding domain may comprise one or more of these transcription factors or polypeptides derived therefrom.

TABLE V.2
Transcription factors
Template binding domain
Yeast transcription factors
FHL1
ROX1
CMR3
SUT2
GALA
USV1
AFT2
CUP9
TBF1
GCR1
MET31
ECM23
RDR1
HAP5
TYE7
YRM1
YRR1
AZF1
CIN5
MSN1
MSN1
INO4
HAL9
HAL9
YAP7
YAP7
DAL82
RAP1
SKO1
FKH2
CRZ1
RGM1
CEP3
MCM1
MSN2
MAC1
STB4
SOK2
ARG81
ORC1
YOX1
YAP1
LEU3
LEU3
SFP1
HAP1
ECM22
ECM22
ACE2
CHA4
GAT3
BAS1
ABF1
HAP4
MSN4
PHD1
PHD1
RGT1
RSF2
CBF1
GZF3
ZAP1
YAP5
GAT4
FKH1
XBP1
CST6
SKN7
STB5
NDT80
STE12
STP2
RIM101
YAP3
YAP3
HAP2
MIG2
TOS8
AFT1
MIG1
PDR1
PHO4
HAC1
GAT1
RPH1
SPT15
COM2
SWI4
DOT6
GLN3
MIG3
GCN4
URC2
STP1
YHP 1
CAD1
CAD1
ARO80
SUM1
RSC3
YAP6
MET32
ADR1
UPC2
UME6
STB3
SWI5
INO2
GIS1
NRG1
LYS14
LYS14
UGA3
PHO2
MBP1
RPN4
RDS1
HCM1
MATALPHA2
REI1
THI2
TBS1
TBS1
TEC1
NRG2
REB1
EDS1
TOD6
HAP3
Transcription factor families found, e.g., in plants
ABI3VP1 family
CAMTA family
LFY family
SBP family
Alfin-like family
CCAAT family
LIM family
Sigma70-like family
AP2-EREBP family
CPP family
LOB family
SRS family
ARF family
CSD family
MADS family
TAZ family
ARR-B family
DBP family
mTERF family
TCP family
BBR/BPC family
E2F-DP family
MYB family
Tify family
BES1 family
EIL family
MYB-related family
TIG family
bHLH family
FARI family
NAC family
Trihelix family
BSD family
FHA family
NOZZLE family
TUB family
bZIP family
G2-like family
OFP family
ULT family
C2C2-CO-like family
GeBP family
Orphans family
VARL family
C2C2-Dof family
GRAS family
PBF-2-like family
VOZ family
C2C2-GATA family
GRF family
PLATZ family
WRKY family
C2C2-YABBY family
HB family
RWP-RK family
zf-HD family
C2H2 family
HRT family
S1Fa-like family
Zn-clus family
C3H family
HSF family
SAP family

Endonucleases

In some embodiments, the DNA-binding polypeptide is derived from an endonuclease. The DNA-binding domain may be a catalytically inactive endonuclease, e.g., may have a substitution in or deletion of the domain that catalyzes DNA cleavage. If the endonuclease has other activities such as DNA modification activity, one may introduce mutations into the other active domains as well.

The restriction endonuclease may be, e.g., of Type I; Type II, e.g., Type IIR, Type IIS, or Type IIG; Type III; or Type IV.

In some embodiments where the endonuclease has a short recognition sequence, it may be used in combination with other DNA-binding polypeptides, e.g., other endonuclease-derived polypeptides, to achieve higher affinity binding to a longer recognition site.

In some embodiments, the endonuclease recognizes modified DNA, e.g., methylated DNA, and the template binding domain partner comprises modified DNA.

Exemplary restriction endonucleases are given in the table below. The template-binding domain may comprise one or more of these endonucleases or polypeptides derived therefrom.

TABLE V.3
Endonucleases
Restriction endonucleases
AatII
AbaSI
Acc65I
AccI
AciI
AclI
AcuI
AfeI
AflII
AflIII
AgeI
AhdI
AleI
AluI
AlwI
AlwNI
ApaI
ApaLI
ApeKI
ApoI
AscI
AseI
AsiSI
AvaI
AvaII
AvrII
BaeGI
BaeI
BamHI
BanI
BanII
BbsI
BbvCI
BbvI
BccI
BceAI
BcgI
BciVI
BclI
BcoDI
BfaI
BfuAI
BfuCI
BglI
BglII
BlpI
BmgBI
BmrI
BmtI
BpmI
Bpu10I
BpuEI
BsaAI
BsaBI
BsaHI
BsaI
BsaJI
BsaWI
BsaXI
BseRI
BseYI
BsgI
BsiEI
BsiHKAI
BsiWI
BslI
BsmAI
BsmBI
BsmFI
BsmI
BsoBI
Bsp1286I
BspCNI
BspDI
BspEI
BspHI
BspMI
BspQI
BsrBI
BsrDI
BsrFI
BsrGI
BsrI
BssHII
BssKI
BssSI
BstAPI
BstBI
BstEII
BstNI
BstUI
BstXI
BstYI
BstZ17I
Bsu36I
BtgI
BtgZI
BtsCI
BtsI
BtsIMutI
Cac8I
ClaI
CspCI
CviAII
CviKI-1
CviQI
DdeI
DpnI
DpnII
DraI
DraIII
DrdI
EaeI
EagI
EarI
EciI
Eco53kI
EcoNI
EcoO109I
EcoP15I
EcoRI
EcoRV
FatI
FauI
Fnu4HI
FokI
FseI
FspEI
FspI
HaeII
HaeIII
HgaI
HhaI
HincII
HindIII
HinfI
HinP1I
HpaI
HpaII
HphI
Hpy166II
Hpy188I
Hpy188III
Hpy99I
HpyAV
HpyCH4III
HpyCH4IV
HpyCH4V
I-CeuI
I-SceI
KasI
KpnI
LpnPI
MboI
MboII
MfeI
MluCI
MluI
MlyI
MmeI
MnlI
MscI
MseI
MsII
MspA1I
MspI
MspJI
MwoI
NaeI
NarI
Nb.BbvCI
Nb.BsmI
Nb.BsrDI
Nb.BtsI
NciI
NcoI
NdeI
NgoMIV
NheI
NlaIII
NlaIV
NmeAIII
NotI
NruI
NsiI
NspI
Nt. AlwI
Nt.BbvCI
Nt.BsmAI
Nt.BspQI
Nt.BstNBI
Nt. CviPII
PacI
PaeR7I
PciI
PfIFI
PfIMI
PI-PspI
PI-SceI
PleI
PluTI
PmeI
PmlI
PpuMI
PshAI
PsiI
PspGI
PspOMI
PspXI
PstI
PvuI
PvuII
RsaI
RsrII
SacI
SacII
SalI
SapI
Sau3AI
Sau96I
SbfI
ScaI
ScrFI
SexAI
SfaNI
SfcI
SfiI
SfoI
SgrAI
SmaI
SmlI
SnaBI
SpeI
SphI
Sphl
SspI
StuI
StyD4I
StyI
SwaI
TaqαI
Tfil
TliI
TseI
Tsp45I
Tsp509I
TspMI
TspRI
Tth111I
XbaI
XcmI
XhoI
XmaI
XmnI
ZraI

TAL Effectors

In some embodiments, the DNA-binding polypeptide is, or is derived from, a TAL (transcription activator-like) effector. TAL effectors bind specifically to DNA through a series of 34-amino acid repeats, and engineering of these repeats tailors the specificity of the TAL effector to bind a desired DNA sequence. Details on how to engineer specificity are given in, e.g., U.S. Pat. No. 8,440,431. Briefly, each repeat in the TAL effector has a direct, linear correspondence with one nucleotide in the target site. Accordingly, one can readily engineer a TAL effector by selecting a first residue at position 12 and a second residue at position 13, in order to have that repeat bind to A, C, G, or T. Different repeats can be assembled to create a binding domain that is customized to recognize the desired target sequence. Table V.4 lists different combinations of amino acid residues that can be used to create repeats with specificity for a given nucleotide in the target binding sequence.

TABLE V.4
Code for designing a specific TAL effector
	1st residue	2nd residue	Nucleotide
	N	*	C or T
	H	*	T
	H	A	C
	N	A	G
	H	D	C
	N	D	C
	H	G	T
	I	G	T
	N	G	T
	Y	G	T
	N	I	A
	H	I	C
	N	K	G
	H	N	G
	S	N	G or A
	N	N	G or A1
	N	S	A or C or G1

In some embodiments, the DNA-binding polypeptide is derived from a TALEN (TAL effector nuclease), and is mutated to lack nuclease activity. For example, there may be a substitution in or deletion of the domain that catalyzes DNA cleavage.

In some embodiments, the TAL effector is from, or is derived from, a TAL effector in a Xanthomonas bacterium, Ralstonia solanacearum, or Burkholderia rhizoxinica.

Exemplary TAL effectors and TALENs are given in the table below. The template-binding domain may comprise one or more of these TAL effectors and TALENs or polypeptides derived therefrom.

TABLE V.5
Publications describing TAL effectors and TALENs
Morbitzer, R. et al. (2010) ″Regulation of selected genome loci using de novo-engineered
transcription activator-like effector (TALE)-type transcription factors,″ PROCEEDINGS OF
THE NATIONAL ACADEMY OF SCIENCES 107 (50): 21617-22. Bibcode:
2010PNAS..10721617M. doi:10.1073/pnas. 1013133107. PMC 3003021. PMID 21106758
Boch J. et al. (2009) ″Breaking the code of DNA binding specificity of TAL-type III
effectors,″ SCIENCE 326 (5959): 1509-12. Bibcode:2009Sci...326.1509B.
doi:10.1126/science.1178811
Li, T. et al. (2011) ″Modularly assembled designer TAL effector nucleases for targeted
gene knockout and gene replacement in eukaryotes,″ NUCLEIC ACIDS RESEARCH
39:6315-25. doi:10.1093/nar/gkr188
Mahfouz, M.M. et al. (2011) ″De novo-engineered transcription activator-like effector
(TALE) hybrid nuclease with novel DNA binding specificity creates double-strand
breaks,″ PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES 108 (6):
2623-8. doi:10.1073/pnas. 1019533108
Cermak, T. et al. (2011) ″Efficient design and assembly of custom TALEN and other TAL
effector-based constructs for DNA targeting,″ NUCLEIC ACIDS RESEARCH 39 (12): e82.
doi:10.1093/nar/gkr218. PMC 3130291
Huang, P. et al. (2011) ″Heritable gene targeting in zebrafish using customized TALENs,″
NATURE BIOTECHNOLOGY 29 (8): 699-700. doi:10.1038/nbt. 1939
Sander, J.D. et al. (2011) ″Targeted gene disruption in somatic zebrafish cells using
engineered TALENs,″ NATURE BIOTECHNOLOGY 29 (8): 697-8. doi:10.1038/nbt. 1934
Tesson, L. et al. (2011) ″Knockout rats generated by embryo microinjection of TALENs,″
NATURE BIOTECHNOLOGY 29 (8): 695-6. doi:10.1038/nbt. 1940

V.2 Template Binding Domains that are Protein-Binding Polypeptides

In some embodiments, the template binding domain is a protein, and the template binding domain partner is a protein, and the template binding domain and the template binding domain partner have affinity for each other. Generally, when the template binding domain is a protein, it lacks substantial affinity for other proteins present in the cell to be altered. This helps to avoid nonspecific binding. In some embodiments, the template-binding domain is derived from a protein in another species than the species of the cell to be altered. In some embodiments, the template-binding domain is derived from a protein that has no binding partners that are expressed in the cell type to be altered.

In some embodiments, the protein-binding polypeptide comprises one of more of the following domains: SH2, SH3, PTB, 14-3-3, FHA, WW, WD40, bromo, chromo, EVH1, PDZ, DD, DED, CARD, BH1-4, CSD, F-box, Hect, RING, ANK, ARM, LIM, EF-hand, MH2.

In some embodiments, the template-binding domain comprises an antibody with affinity for the template binding domain partner. Conversely, in some embodiments, the template-binding domain is a protein and the template binding domain partner is an antibody with affinity for the template binding domain. The antibody may be, e.g., an scFv or any antibody having sufficient CDR sequences to bind its target.

In some embodiments, the template-binding domain carries one or more deletions relative to the wild-type protein from which it was derived. For example, there may be a deletion of a catalytic domain. In some embodiments, the wild-type protein has multiple protein-binding domains, one or more of these domains, e.g., all but one of these domains, is deleted.

Exemplary protein-binding domains are given in the table below. The template-binding domain may comprise one or more of these protein-binding domains or polypeptides derived therefrom. It is understood that in some embodiments, the template binding domain is, or is derived from, the protein in the left column and the template binding domain partner is, or is derived from, the protein in the right column. In other embodiments, the template binding domain is, or is derived from, the protein in the right column and the template binding domain partner is, or is derived from, the protein in the left column.

TABLE V.6
Protein-protein interaction domains
Protein or domain                Binding partner
TE33 Fab L chain (BBa_K126000 from the B subunit of cholera
Registry of Standard Biological Parts) toxin
protein ZSPA-1 (BBa_K103004 from the Staphylococcal
Registry of Standard Biological Parts) protein A
RGD (BBa_K133059 from the Registry of integrins
Standard Biological Parts)
Cdc4 (found in yeast; comprises F-box Sic1 CDK inhibitor;
domain)                          Skp1, Rbx1
Grr1 (found in yeast; comprises F-box Cyclin (CLN) 1, 2;
domain)                          Skp1, Rbx1
TrCp (found in yeast; comprises F-box IkB(NFKB regulator);
domain)                          Skp1, Rbx1

V.3 Template Binding Domains that are Small Molecule-Binding Polypeptides

In some embodiments, the template binding domain is a protein, and the template binding domain partner is a small molecule. Generally, when the template binding domain has affinity for a small molecule, the small molecule is rare or absent in the cell being altered. This helps to avoid nonspecific binding.

In some embodiments, the template-binding domain carries one or more deletions or substitutions relative to the wild-type protein from which it was derived. For example, there may be a deletion of or substitution within a catalytic domain, a DNA-binding domain, a protein-protein interaction domain, and/or a domain necessary for transcriptional regulation.

Exemplary small molecule-binding domains are given in the table below. The template-binding domain may comprise one or more of these small molecule-binding domains or polypeptides derived therefrom.

TABLE V.7
Proteins that bind small molecules
Protein                          Small molecule
Avidin or Streptavidin (BBa K283010 from biotin
the Registry of Standard Biological Parts)
gyrEC (BBa_K133070 from the Registry of coumermycin
Standard Biological Parts)
RI7 (BBa K211001 from the Registry of octanal, heptanal or
Standard Biological Parts)       hexanal
VirA receptor (BBa_K389001 from the acetosyringone
Registry of Standard Biological Parts)
Penicillin-binding proteins (PBPs), e.g., penicillin or
serine type D-alanyl-D-alanine   cephalosporin
carboxypeptidase/transpeptidase
TetR                             tetracycline
ASGPR                            N-Acetylgalactosamine
                                 or galactose

VI. Template Binding Domain Partner

VI.1 Template binding domain partners that are nucleic acids

Double Stranded DNA

In some embodiments, the template binding domain partner is double-stranded DNA. For instance, in some embodiments, the template binding domain partner is double-stranded DNA that is recognized by a DNA-binding protein described above in Section V.1.

The template binding domain partner may be, e.g., identical to or derived from a DNA sequence that is bound by a protein in a wild-type context. In some embodiments, the template binding domain partner comprises all or part of a transcription factor binding site from an organism other than the organism of the cell being altered. In some embodiments, the template binding domain partner comprises all or part of a transcriptional regulation site from an operon, e.g., a bacterial operon.

In some embodiments, the template binding domain partner is at least 10 nucleotides long, e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, or 200 nucleotides long. In some embodiments, the template binding domain partner is at most 200 nucleotides long, e.g., at most 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, or 200 nucleotides long. In some embodiments, the template binding domain partner is 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, or 150-200 nucleotides long.

In some embodiments, the template binding domain partner comprises palindromic sequences.

In some embodiments, the template binding domain partner comprises a plurality of shorter sequences, wherein each shorter sequence is bound by a distinct DNA-binding domain. In some embodiments, the plurality of shorter sequences are identical, e.g., the template binding domain partner comprises repeats. In other embodiments, one or more of, e.g., all of the plurality of shorter sequences are not identical to each other.

In some embodiments, the template binding domain partner is chemically modified DNA, e.g., as set out in Section XI below. The modification may be, e.g., to one or more bases and/or to the backbone. The chemical modification may do one or more of the following: improve the stability of the DNA, reduce the innate immune response against the DNA, and improve the binding of the template binding domain to the template binding domain partner.

The template binding domain partner need not always be the same type of molecule as the template nucleic acid. For instance, in some embodiments, the template binding domain partner is double stranded, while the template nucleic acid is single stranded. In some such embodiments, a long single-stranded DNA comprises a hairpin at one end, and the double stranded region of the hairpin comprises the template binding domain partner. In other embodiments, the template binding domain partner and the template nucleic acid are both double stranded. In some embodiments, the template binding domain partner is derived from a wild-type template binding domain partner. For example, the template binding domain partner may be a fragment of a naturally occurring nucleic acid, a mutagenized nucleic acid, a synthetic nucleic acid modeled after a naturally-occurring nucleic acid. In some embodiments, the template binding domain partner is mutagenized to increase its affinity for a template binding domain. In some embodiments, the template binding domain partner is mutagenized to decrease its affinity for a template binding domain.

Single Stranded DNA

In some embodiments, the template binding domain partner is single-stranded DNA.

VI. 2 Template Binding Domain Partners that are Polypeptides

In some embodiments, the template binding domain partner is a polypeptide. Any of the proteins and/or polypeptides discussed above in Sections V, may be used in the present invention, as a template binding domain partner. For example, protein-protein interacting pairs are discussed above, e.g., in Table V.6.

VII. Functional Analysis of Candidate Molecules

Candidate Cas9 molecules, candidate Cas9 fusion molecules, candidate gRNA molecules, and candidate Cas9 fusion molecule/gRNA molecule complexes, can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 fusion molecule are described, e.g., in Jinek et al. (2012) SCIENCE 337(6096): 816-821.

The methods in this section may be used, e.g., to test various portions of a gRNA, for example, the targeting domain, the first complementarity domain, the linking domain, the second complementarity domain, the proximal domain, or the tail domain. In some embodiments, the methods in this section are tested to determine whether modifications made in one or more of these domains interfere with targeting efficacy. A gRNA with a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system of this section.

Binding and Cleavage Assay: Testing the Endonuclease Activity of Cas9 Fusion Molecule

The ability of a Cas9 fusion molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in a plasmid cleavage assay. In this assay, synthetic or in vitro-transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95° C. and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (˜8 nM)) is incubated for 60 min at 37° C. with purified Cas9 protein molecule (50-500 nM) and gRNA (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl₂. The reactions are stopped with 5×DNA loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands. For example, linear DNA products indicate the cleavage of both DNA strands. Nicked open circular products indicate that only one of the two strands is cleaved.

Alternatively, the ability of a Cas9 fusion molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in an oligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides (10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotide kinase and ˜3-6 pmol (˜20-40 mCi) [γ-32P]-ATP in 1×T4 polynucleotide kinase reaction buffer at 37° C. for 30 min., in a 50 μL reaction. After heat inactivation (65° C. for 20 min), reactions are purified through a column to remove unincorporated label. Duplex substrates (100 nM) are generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95° C. for 3 min., followed by slow cooling to room temperature. For cleavage assays, gRNA molecules are annealed by heating to 95° C. for 30 s, followed by slow cooling to room temperature. Cas9 fusion molecule (500 nM final concentration) is pre-incubated with the annealed gRNA molecules (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 5% glycerol) in a total volume of 9 μl. Reactions are initiated by the addition of 1 μl target DNA (10 nM) and incubated for 1 h at 37° C. Reactions are quenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95° C. for 5 min. Cleavage products are resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging. The resulting cleavage products indicate that whether the complementary strand, the non-complementary strand, or both, are cleaved.

One or both of these assays can be used to evaluate the suitability of a candidate gRNA molecule, a candidate Cas9 molecule, or a candidate Cas9 fusion molecule.

Binding Assay: Testing the Binding of Cas9 Fusion Molecule to Target DNA

Exemplary methods for evaluating the binding of a Cas9 fusion molecule to target DNA are described, e.g., in Jinek et al. (2012) SCIENCE 337(6096):816-821.

For example, in an electrophoretic mobility shift assay, target DNA duplexes are formed by mixing of each strand (10 nmol) in deionized water, heating to 95° C. for 3 min. and slow cooling to room temperature. All DNAs are purified on 8% native gels containing 1×TBE. DNA bands are visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H2O. Eluted DNA is ethanol precipitated and dissolved in DEPC-treated H2O. DNA samples are 5′ end labeled with [γ-32P]-ATP using T4 polynucleotide kinase for 30 min. at 37° C. Polynucleotide kinase is heat denatured at 65° C. for 20 min., and unincorporated radiolabel is removed using a column. Binding assays are performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT and 10% glycerol in a total volume of 10 Cas9 fusion molecule is programmed with equimolar amounts of pre-annealed gRNA molecule and titrated from 100 pM to 1 μM. Radiolabeled DNA is added to a final concentration of 20 pM. Samples are incubated for 1 h at 37° C. and resolved at 4° C. on an 8% native polyacrylamide gel containing 1×TBE and 5 mM MgCl₂. Gels are dried and DNA visualized by phosphorimaging.

Differential Scanning Flourimetry (DSF)

The thermostability of Cas9 molecule-gRNA ribonucleoprotein (RNP) complexes, e.g., a Cas9 fusion molecule-gRNA RNP complex, can be measured via DSF. This technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding RNA molecule, e.g., a gRNA.

The assay is performed using two different protocols, one to test the best stoichiometric ratio of gRNA:Cas9 protein and another to determine the best solution conditions for RNP formation.

To determine the best solution to form RNP complexes, a 2 uM solution of Cas9 in water+10×SYPRO Orange® (Life Technologies cat #S-6650) and dispensed into a 384 well plate. An equimolar amount of gRNA diluted in solutions with varied pH and salt is then added. After incubating at room temperature for 10 min. and brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20° C. to 90° C. with a 1° C. increase in temperature every 10 seconds.

The second assay consists of mixing various concentrations of gRNA with 2 uM Cas9 molecule in optimal buffer from the assay above and incubating at RT for 10 min in a 384 well plate. An equal volume of optimal buffer+10×SYPRO Orange® (Life Techonologies cat #S-6650) is added and the plate sealed with Microseal® B adhesive (MSB-1001). Following brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20° C. to 90° C. with a 1° increase in temperature every 10 seconds.

VIII. Genome Editing Approaches

Mutations may be corrected, and undesirable nucleic acid sequences may be cleaved, using one of the approaches discussed herein. In an embodiment, a mutation in a target nucleic acid is corrected by homology directed repair (HDR) using a template nucleic acid (see Section VIII.1).

VIII.1 HDR Repair and Template Nucleic Acids

As described herein, nuclease-induced homology directed repair (HDR) can be used to alter a target sequence and correct (e.g., repair or edit) a mutation in the genome. While not wishing to be bound by theory, it is believed that alteration of the target sequence occurs by homology-directed repair (HDR) with a donor template or template nucleic acid. For example, the donor template or the template nucleic acid provides for alteration of the target position. It is contemplated that a plasmid donor can be used as a template for homologous recombination. It is further contemplated that a single stranded donor template can be used as a template for alteration of the target position by alternate methods of homology directed repair (e.g., single strand annealing) between the target position and the donor template.

Donor template-effected alteration of a target position depends on cleavage by a Cas9 molecule. Cleavage by Cas9 can comprise a nick, a double strand break, or two single strand breaks, e.g., one on each strand of the target nucleic acid. In an embodiment, a mutation can be corrected by either a single double-strand break or two single strand breaks. In an embodiment, a mutation can be corrected by (1) a single double-strand break, (2) two single strand breaks, (3) two double stranded breaks with a break occurring on each side of the target position, (4) one double stranded break and two single strand breaks with the double strand break and two single strand breaks occurring on each side of the target position or (5) four single stranded breaks with a pair of single stranded breaks occurring on each side of the target position.

Additional details on template nucleic acids are provided in Section IV entitled “Template Nucleic Acids” in PCT Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference.

Double Strand Break Mediated Correction

In an embodiment, double strand cleavage is effected by a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, e.g., an N-terminal RuvC-like domain, e.g., a wild type Cas9 molecule. Such embodiments require only a single gRNA.

Single Strand Break Mediated Correction

In other embodiments, two single strand breaks, or nicks, are effected by a Cas9 molecule having nickase activity, e.g., cleavage activity associated with an HNH-like domain or cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments usually require two gRNAs, one for placement of each single strand break. In an embodiment, the Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is complementary to the strand to which the gRNA hybridizes. In an embodiment, the Cas9 molecule having nickase activity does not cleave the strand to which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand to which the gRNA hybridizes.

In an embodiment, the nickase has HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation. D10A inactivates RuvC; therefore, the Cas9 nickase has (only) HNH activity and will cut on the strand to which the gRNA hybridizes (e.g., the complementary strand, which does not have the NGG PAM on it). In other embodiments, a Cas9 molecule having an H840, e.g., an H840A, mutation can be used as a nickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (e.g., the strand that has the NGG PAM and whose sequence is identical to the gRNA). In other embodiments, a Cas9 molecule having an N863, e.g., the N863A mutation, mutation can be used as a nickase. N863A inactivates HNH therefore the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (the strand that has the NGG PAM and whose sequence is identical to the gRNA). In other embodiments, a Cas9 molecule having an N580, e.g., the N580A mutation, mutation can be used as a nickase. N580A inactivates HNH therefore the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (the strand that has the NGG PAM and whose sequence is identical to the gRNA). In an embodiment, in which a nickase and two gRNAs are used to position two single strand nicks, one nick is on the +strand and one nick is on the −strand of the target nucleic acid. The PAMs can be outwardly facing. The gRNAs can be selected such that the gRNAs are separated by, from about 0-50, 0-100, or 0-200 nucleotides. In an embodiment, there is no overlap between the target sequences that are complementary to the targeting domains of the two gRNAs. In an embodiment, the gRNAs do not overlap and are separated by as much as 50, 100, or 200 nucleotides. In an embodiment, the use of two gRNAs can increase specificity, e.g., by decreasing off-target binding (Ran et al. 2013, CELL 154:1380-1389).

In an embodiment, a single nick can be used to induce HDR. It is contemplated herein that a single nick can be used to increase the ratio of HR to NHEJ at a given cleavage site. In an embodiment, a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of said gRNA is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which the targeting domain of said gRNA is complementary.

Placement of the Double Strand or Single Strand Breaks Relative to the Target Position

The double strand break or single strand break in one of the strands should be sufficiently close to target position such that an alteration is produced in the desired region, e.g., correction of a mutation occurs. In an embodiment, the distance is not more than 50, 100, 200, 300, 350 or 400 nucleotides. While not wishing to be bound by theory, in some embodiments, it is believed that the break should be sufficiently close to target position such that the target position is within the region that is subject to exonuclease-mediated removal during end resection. If the distance between the target position and a break is too great, the mutation or other sequence desired to be altered may not be included in the end resection and, therefore, may not be corrected, as donor sequence, either exogenously provided donor sequence or endogenous genomic donor sequence, in some embodiments is only used to correct sequence within the end resection region.

In an embodiment, the targeting domain is configured such that a cleavage event, e.g., a double strand or single strand break, is positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or 200 nucleotides of the region desired to be altered, e.g., a mutation. The break, e.g., a double strand or single strand break, can be positioned upstream or downstream of the region desired to be altered, e.g., a mutation. In some embodiments, a break is positioned within the region desired to be altered, e.g., within a region defined by at least two mutant nucleotides. In some embodiments, a break is positioned immediately adjacent to the region desired to be altered, e.g., immediately upstream or downstream of a mutation.

In an embodiment, a single strand break is accompanied by an additional single strand break, positioned by a second gRNA molecule, as discussed below. For example, the targeting domains bind configured such that a cleavage event, e.g., the two single strand breaks, are positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or 200 nucleotides of a target position. In an embodiment, the first and second gRNA molecules are configured such, that when guiding a Cas9 nickase, a single strand break will be accompanied by an additional single strand break, positioned by a second gRNA, sufficiently close to one another to result in alteration of the desired region. In an embodiment, the first and second gRNA molecules are configured such that a single strand break positioned by said second gRNA is within 10, 20, 30, 40, or 50 nucleotides of the break positioned by said first gRNA molecule, e.g., when the Cas9 is a nickase. In an embodiment, the two gRNA molecules are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, e.g., essentially mimicking a double strand break.

In an embodiment, in which a gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 fusion molecule induce a double strand break for the purpose of inducing HDR-mediated correction, the cleavage site is between 0-200 bp (e.g., 0-175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position. In an embodiment, the cleavage site is between 0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.

In an embodiment, the targeting domain of a gRNA molecule is configured to position a cleavage event sufficiently far from a preselected nucleotide, e.g., the nucleotide of a coding region, such that the nucleotide is not altered. In an embodiment, the targeting domain of a gRNA molecule is configured to position an intronic cleavage event sufficiently far from an intron/exon border, or naturally occurring splice signal, to avoid alteration of the exonic sequence or unwanted splicing events. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule, as described herein.

Placement of a First Break and a Second Break Relative to Each Other

In an embodiment, a double strand break can be accompanied by an additional double strand break, positioned by a second gRNA molecule, as is discussed below.

In an embodiment, a double strand break can be accompanied by two additional single strand breaks, positioned by a second gRNA molecule and a third gRNA molecule.

In an embodiment, a first and second single strand breaks can be accompanied by two additional single strand breaks positioned by a third gRNA molecule and a fourth gRNA molecule.

When two or more gRNAs are used to position two or more cleavage events, e.g., double strand or single strand breaks, in a target nucleic acid, it is contemplated that the two or more cleavage events may be made by the same or different Cas9 molecules. For example, when two gRNAs are used to position two double stranded breaks, a single Cas9 molecule may be used to create both double stranded breaks. When two or more gRNAs are used to position two or more single stranded breaks (nicks), a single Cas9 nickase may be used to create the two or more nicks. When two or more gRNAs are used to position at least one double stranded break and at least one single stranded break, two Cas9 proteins may be used, e.g., one Cas9 nuclease and one Cas9 nickase. It is contemplated that when two or more Cas9 proteins are used that the two or more Cas9 proteins may be delivered sequentially to control specificity of a double stranded versus a single stranded break at the desired position in the target nucleic acid.

In some embodiments, the targeting domain of the first gRNA molecule and the targeting domain of the second gRNA molecules are complementary to opposite strands of the target nucleic acid molecule. In some embodiments, the gRNA molecule and the second gRNA molecule are configured such that the PAMs are oriented outward.

In an embodiment, in which two gRNAs (independently, unimolecular (or chimeric) or modular gRNA) complexing with Cas9 nickases induce two single strand breaks for the purpose of inducing HDR-mediated correction, the closer nick is between 0-200 bp (e.g., 0-175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position and the two nicks will ideally be within 25-65 bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 55, 40 to 50, 40 to 45 bp, 45 to 50 bp, 50 to 55 bp, 55 to 60 bp, 60 to 65 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 bp away from each other). In an embodiment, the cleavage site is between 0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.

In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a Cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position. In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position. The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are, in some embodiments, within 25-65 bp of each other (e.g., between 25 to 55, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, 40 to 45 bp, 45 to 50 bp, 50 to 55 bp, 55 to 60 bp, or 60 to 65 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). When two gRNAs are used to target Cas9 molecules to breaks, different combinations of Cas9 molecules are envisioned. In some embodiments, a first gRNA is used to target a first Cas9 molecule to a first target position, and a second gRNA is used to target a second Cas9 molecule to a second target position. In some embodiments, the first Cas9 molecule creates a nick on the first strand of the target nucleic acid, and the second Cas9 molecule creates a nick on the opposite strand, resulting in a double stranded break (e.g., a blunt ended cut or a cut with overhangs).

Different combinations of nickases can be chosen to target one single stranded break to one strand and a second single stranded break to the opposite strand. When choosing a combination, one can take into account that there are nickases having one active RuvC-like domain, and nickases having one active HNH domain. In an embodiment, a RuvC-like domain cleaves the non-complementary strand of the target nucleic acid molecule. In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. Generally, if both Cas9 molecules have the same active domain (e.g., both have an active RuvC domain or both have an active HNH domain), one will choose two gRNAs that bind to opposite strands of the target. In more detail, in some embodiments, a first gRNA is complementary with a first strand of the target nucleic acid and binds a nickase having an active RuvC-like domain and causes that nickase to cleave the strand that is non-complementary to that first gRNA, i.e., a second strand of the target nucleic acid; and a second gRNA is complementary with a second strand of the target nucleic acid and binds a nickase having an active RuvC-like domain and causes that nickase to cleave the strand that is non-complementary to that second gRNA, i.e., the first strand of the target nucleic acid. Conversely, in some embodiments, a first gRNA is complementary with a first strand of the target nucleic acid and binds a nickase having an active HNH domain and causes that nickase to cleave the strand that is complementary to that first gRNA, i.e., a first strand of the target nucleic acid; and a second gRNA is complementary with a second strand of the target nucleic acid and binds a nickase having an active HNH domain and causes that nickase to cleave the strand that is complementary to that second gRNA, i.e., the second strand of the target nucleic acid. In another arrangement, if one Cas9 molecule has an active RuvC-like domain and the other Cas9 molecule has an active HNH domain, the gRNAs for both Cas9 molecules can be complementary to the same strand of the target nucleic acid, so that the Cas9 molecule with the active RuvC-like domain will cleave the non-complementary strand and the Cas9 molecule with the HNH domain will cleave the complementary strand, resulting in a double stranded break.

Exemplary Template Nucleic Acids

A template nucleic acid, as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with a Cas9 fusion molecule and a gRNA molecule to alter the structure of a target position.

Target position, as used herein, refers to a site on a target nucleic acid (e.g., the chromosome) that is modified by a Cas9 molecule-dependent process, e.g., a Cas9 fusion molecule-dependent process. For example, the target position can be a modified Cas9 fusion molecule cleavage of the target nucleic acid and template nucleic acid directed modification, e.g., correction, of the target position. In an embodiment, a target position can be a site between two nucleotides, e.g., adjacent nucleotides, on the target nucleic acid into which one or more nucleotides are added. The target position may comprise one or more nucleotides that are altered, e.g., corrected, by a template nucleic acid. In an embodiment, the target position is within a target sequence (e.g., the sequence to which the gRNA binds). In an embodiment, a target position is upstream or downstream of a target sequence (e.g., the sequence to which the gRNA binds).

In an embodiment, the target nucleic acid is modified to have the some or all of the sequence of the template nucleic acid, typically at or near cleavage site(s). In an embodiment, the template nucleic acid is single stranded. In an alternate embodiment, the template nucleic acid is double stranded. In an embodiment, the template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the template nucleic acid is single stranded DNA. In an embodiment, the template nucleic acid is encoded on the same vector backbone, e.g., AAV genome or plasmid DNA, as the Cas9 fusion molecule and gRNA. In an embodiment, the template nucleic acid is excised from a vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences. In an embodiment, the template nucleic acid comprises endogenous genomic sequence.

In an embodiment, the template nucleic acid alters the structure of the target position by participating in a homology directed repair event. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid.

Typically, the template sequence undergoes a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid includes sequence that corresponds to a site on the target sequence that is cleaved by an eaCas9 mediated cleavage event. In an embodiment, the template nucleic acid includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas9 mediated event, and a second site on the target sequence that is cleaved in a second Cas9 mediated event.

In an embodiment, the template nucleic acid can include sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation.

In other embodiments, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an exon or in a 5′ or 3′ non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.

A template nucleic acid having homology with a target position in a gene, e.g., a gene described herein, can be used to alter the structure of a target sequence. The template sequence can be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.

In an embodiment, the template nucleic acid is a single stranded nucleic acid. In another embodiment, the template nucleic acid is a double stranded nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide sequence, e.g., of one or more nucleotides, that will be added to or will template a change in the target nucleic acid. In other embodiments, the template nucleic acid comprises a nucleotide sequence that may be used to modify the target position. In other embodiments, the template nucleic acid comprises a nucleotide sequence, e.g., of one or more nucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position.

The template nucleic acid may comprise a replacement sequence. In some embodiments, the template nucleic acid comprises a 5′ homology arm. In other embodiments, the template nucleic acid comprises a 3′ homology arm.

In some embodiments, the template nucleic acid is linear double stranded DNA. The length may be, e.g., about 50-500 base pairs. The length may be, e.g., about 150-200 base pairs, e.g., about 150, 160, 170, 180, 190, or 200 base pairs. The length may be, e.g., at least 150, 160, 170, 180, 190, or 200 base pairs. In some embodiments, the length is no greater than 150, 160, 170, 180, 190, or 200 base pairs. In some embodiments, a double stranded template nucleic acid has a length of about 160 base pairs, e.g., about 155-165, 150-170, 140-180, 130-190, 120-200, 110-210, 100-220, 90-230, or 80-240 base pairs.

The template nucleic acid can be linear single stranded DNA. In some embodiments, the template nucleic acid is (i) linear single stranded DNA that can anneal to the nicked strand of the target nucleic acid, (ii) linear single stranded DNA that can anneal to the intact strand of the target nucleic acid, (iii) linear single stranded DNA that can anneal to the transcribed strand of the target nucleic acid, (iv) linear single stranded DNA that can anneal to the non-transcribed strand of the target nucleic acid, or more than one of the preceding. The length may be, e.g., 50-500 nucleotides. The length may be, e.g., about 50-500 nucleotides. The length may be, e.g., about 150-200 nucleotides, e.g., about 150, 160, 170, 180, 190, or 200 nucleotides. The length may be, e.g., at least 150, 160, 170, 180, 190, or 200 nucleotides. In some embodiments, the length is no greater than 150, 160, 170, 180, 190, or 200 nucleotides. In some embodiments, a single stranded template nucleic acid has a length of about 160 nucleotides, e.g., about 155-165, 150-170, 140-180, 130-190, 120-200, 110-210, 100-220, 90-230, or 80-240 nucleotides.

In some embodiments, the template nucleic acid is circular double stranded DNA, e.g., a plasmid. In some embodiments, the template nucleic acid comprises about 500 to 1000 base pairs of homology on either side of the replacement sequence and/or the nick. In some embodiments, the template nucleic acid comprises about 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence.

In some embodiments, the template nucleic acid is an adenovirus vector, e.g., an AAV vector, e.g., a ssDNA molecule of a length and sequence that allows it to be packaged in an AAV capsid. The vector may be, e.g., less than 5 kb and may contain an ITR sequence that promotes packaging into the capsid. The vector may be integration-deficient. In some embodiments, the template nucleic acid comprises about 150 to 1000 nucleotides of homology on either side of the replacement sequence and/or the nick. In some embodiments, the template nucleic acid comprises about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at most 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence.

In some embodiments, the template nucleic acid is a lentiviral vector, e.g., an IDLV (integration deficiency lentivirus). In some embodiments, the template nucleic acid comprises about 500 to 1000 base pairs of homology on either side of the replacement sequence and/or the nick. In some embodiments, the template nucleic acid comprises about 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence. In some embodiments, the template nucleic acid comprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequence.

In an embodiment, the template nucleic acid comprises one or more mutations, e.g., silent mutations, that prevent Cas9 from recognizing and cleaving the template nucleic acid. The template nucleic acid may comprise, e.g., at least 1, 2, 3, 4, 5, 10, 20, or 30 silent mutations relative to the corresponding sequence in the genome of the cell to be altered. In an embodiment, the template nucleic acid comprises at most 2, 3, 4, 5, 10, 20, 30, or 50 silent mutations relative to the corresponding sequence in the genome of the cell to be altered.

In an embodiment, the template nucleic acid alters the structure of the target position by participating in a homology directed repair event. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid.

Typically, the template sequence undergoes a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid includes sequence that corresponds to a site on the target sequence that is cleaved by an eaCas9 mediated cleavage event. In an embodiment, the template nucleic acid includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas9 mediated event, and a second site on the target sequence that is cleaved in a second Cas9 mediated event.

In an embodiment, the template nucleic acid can include sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation. In an embodiment, the template nucleic acid can include sequence which results in an alteration in a coding sequence, e.g., an alteration in an exon.

In other embodiments, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an intron or in a 5′ or 3′ non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.

A template nucleic acid having homology with a target position can be used to alter the structure of a target sequence. The template sequence can be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.

Length of the Homology Arms

The homology arm should extend at least as far as the region in which end resection may occur, e.g., in order to allow the resected single stranded overhang to find a complementary region within the donor template. The overall length could be limited by parameters such as plasmid size or viral packaging limits. In an embodiment, a homology arm does not extend into repeated elements, e.g., ALU elements or LINE elements.

Exemplary homology arm lengths include a least 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, or 5000 nucleotides. In some embodiments, the homology arm length is 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

A template nucleic acid typically comprises the following components:

• • [5′ homology arm]-[replacement sequence]-[3′ homology arm].

The homology arms provide for recombination into the chromosome, thus replacing the undesired element, e.g., a mutation or signature, with the replacement sequence. In an embodiment, the homology arms flank the most distal cleavage sites.

In an embodiment, the 3′ end of the 5′ homology arm is the position next to the 5′ end of the replacement sequence. In an embodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 5′ from the 5′ end of the replacement sequence.

In an embodiment, the 5′ end of the 3′ homology arm is the position next to the 3′ end of the replacement sequence. In an embodiment, the 3′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 3′ from the 3′ end of the replacement sequence.

In an embodiment, to correct a mutation, the homology arms, e.g., the 5′ and 3′ homology arms, may each comprise about 1000 base pairs (bp) of sequence flanking the most distal gRNAs (e.g., 1000 bp of sequence on either side of the mutation).

It is contemplated herein that one or both homology arms may be shortened to avoid including certain sequence repeat elements, e.g., Alu element or LINE elements. For example, a 5′ homology arm may be shortened to avoid a sequence repeat element. In other embodiments, a 3′ homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5′ and the 3′ homology arms may be shortened to avoid including certain sequence repeat elements.

It is contemplated herein that template nucleic acids for correcting a mutation may be designed for use as a single-stranded oligonucleotide, e.g., a single-stranded oligodeoxynucleotide (ssODN). When using a ssODN, 5′ and 3′ homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in oligonucleotide synthesis continue to be made. In some embodiments, a longer homology arm is made by a method other than chemical synthesis, e.g., by denaturing a long double stranded nucleic acid and purifying one of the strands, e.g., by affinity for a strand-specific sequence anchored to a solid substrate.

Exemplary Arrangements of Linear Nucleic Acid Template Systems

In an embodiment, the nucleic acid template system is double stranded. In an embodiment, the nucleic acid template system is single stranded. In an embodiment, the nucleic acid template system comprises a single stranded portion and a double stranded portion. For example, the template binding domain partner is double stranded and the template nucleic acid is single stranded.

In an embodiment, the template nucleic acid comprises about 50 to 500 base pairs. In an embodiment, the template nucleic acid comprises about 50 to 100, e.g., 55 to 95, 60 to 90, 65 to 85, or 70 to 80, base pairs, homology on either side of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequences.

In an embodiment, the template nucleic acid comprises about 150 to 200, e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180, base pairs homology 3′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 base pairs homology 3′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs homology 5′ of the nick or replacement sequence.

In an embodiment, the template nucleic acid comprises about 150 to 200, e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180, base pairs homology 5′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 base pairs homology 5′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs homology 3′ of the nick or replacement sequence.

In an embodiment, the 5′ of the homology region is the position next or adjacent (e.g., within 10, 20, 30, 40, or 50 base pairs) to the 3′ of the template binding domain partner. In an embodiment, the 3′ of the homology region is the position next or adjacent (e.g., within 10, 20, 30, 40, or 50 base pairs) to the 5′ of the template binding domain partner. For example, the template binding domain partner can be placed at the right of the nucleic acid template system, or at the left of the nucleic acid template system, as shown in FIG. 3.

Exemplary Arrangements of Circular Nucleic Acid Template Systems

In an embodiment, the nucleic acid template system is double stranded. In an embodiment, the nucleic acid template system is double stranded comprises a single stranded portion and a double stranded portion. In an embodiment, the nucleic acid template system is single stranded.

In an embodiment, the template nucleic acid comprises about 500 to 2000, e.g., 700 to 1900, 800 to 1800, 900 to 1700, 900 to 1600, 1000 to 1500, 1100 to 1400, or 1200 to 1300 base pairs, homology on either side of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 base pairs homology 5′ of the nick or replacement sequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick or replacement sequences.

In an embodiment, the template nucleic acid comprises about 500 to 2000, e.g., 700 to 1900, 800 to 1800, 900 to 1700, 900 to 1600, 1000 to 1500, 1100 to 1400, or 1200 to 1300, base pairs homology 3′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 base pairs homology 3′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 500, 400, 300, 200, 100, or 50 base pairs homology 5′ of the nick or replacement sequence.

In an embodiment, the template nucleic acid comprises about 500 to 2000, e.g., 700 to 1900, 800 to 1800, 900 to 1700, 900 to 1600, 1000 to 1500, 1100 to 1400, or 1200 to 1300, base pairs homology 5′ of the nick and/or replacement sequence. In an embodiment, the template nucleic acid comprises about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 base pairs homology 5′ of the nick or replacement sequence. In an embodiment, the template nucleic acid comprises less than about 500, 400, 300, 200, 100, or 50 base pairs homology 3′ of the nick or replacement sequence.

In an embodiment, the 5′ of the homology region is the position next or adjacent (e.g., within 10, 20, 30, 40, or 50 base pairs) to the 3′ of the template binding domain partner. In an embodiment, the 3′ of the homology region is the position next or adjacent (e.g., within 10, 20, 30, 40, or 50 base pairs) to the 5′ of the template binding domain partner. In an embodiment, the 5′ of the homology region is the position at least 100, 250, 500, 1000, 1500, 2000, or 2500 base pairs downstream from the 3′ of the template binding domain partner. In an embodiment, the 3′ of the homology region is the position at least 100, 250, 500, 1000, 1500, 2000, or 2500 base pairs upstream from the 5′ of the template binding domain partner.

In an embodiment, the 5′ of the homology region is the position next or adjacent (e.g., within 10, 20, 30, 40, or 50 base pairs) to the 3′ of the template binding domain partner, and the 3′ of the homology region is the position at least 100, 250, 500, 1000, 1500, 2000, or 2500 base pairs upstream from the 5′ of the template binding domain partner. In an embodiment, the 3′ of the homology region is the position next or adjacent (e.g., within 10, 20, 30, 40, or 50 base pairs) to the 5′ of the template binding domain partner, and the 5′ of the homology region is the position at least 100, 250, 500, 1000, 1500, 2000, or 2500 base pairs downstream from the 3′ of the template binding domain partner.

In an embodiment, the 5′ of the homology region is the position at least 100, 250, 500, 1000, 1500, 2000, or 2500 base pairs downstream from the 3′ of the template binding domain partner, and the 3′ of the homology region is the position at least 100, 250, 500, 1000, 1500, 2000, or 2500 base pairs upstream from the 5′ of the template binding domain partner.

For example, the template nucleic acid can comprise a homology region to the right of the template binding domain partner, to the left of the template binding domain partner, or on the opposite side of the circular nucleic acid molecule from the template binding domain partner, as shown in FIG. 4.

Methods of Promoting Break Repair by an HDR Pathway

In another aspect, disclosed herein is a method of altering a cell, e.g., altering the structure, e.g., altering the sequence, of a target nucleic acid of a cell, comprising contacting the cell with: (a) a gRNA that targets a target position, e.g., a gRNA as described herein; (b) a Cas9 molecule, e.g., a Cas9 fusion molecule as described herein; (c) a template nucleic acid, (d) a template binding domain, and (e) a template binding domain partner.

• • In some embodiments, the method comprises contacting the cell with (a) and (b), or with (a) and (c), or with (a) and (d), or with (a) and (e), or with (b) and (c), or with (b) and (d), or with (b) and (e), or with (c) and (d), or with (c) and (e), or with (d) and (e).
  • In some embodiments, the method comprises contacting the cell with (a), (b), and (c); with (a), (b), and (d); with (a), (b), and (e); with (b), (c), and (d); or with (b), (c), and (e); with (c), (d), and (e).

In some embodiments, the method comprises contacting said cell with (a), (b), (c) and (d); with (a), (b), (c), and (e); with (a), (b), (d), and (e); with (a), (c), (d), and (e); or with (b), (c), (d), and (e).

The contacting may be performed ex vivo and the contacted cell may be returned to the subject's body after the contacting step. In other embodiments, the contacting step may be performed in vivo.

In some embodiments, contacting a cell with a Cas9 fusion molecule comprises contacting the cell with a nucleic acid encoding the Cas9 fusion molecule and allowing the cell to produce the Cas9 fusion molecule. In some embodiments, contacting a cell with a gRNA comprises contacting the cell with DNA that can direct transcription of the gRNA, and allowing the cell to produce the gRNA.

In some embodiments, the method of altering a cell as described herein comprises acquiring knowledge of the presence of an undesired sequence in said cell, prior to the contacting step. Acquiring knowledge of the sequence of the undesired sequence in the cell may be by DNA sequencing.

In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises at least one of (a), (b), (c), (d), and (e). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises each of (a), (b), (c), (d), and (e). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises two, three, or four of (a), (b), (c), (d), and (e). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses or comprises each of (a) and (b).

In an embodiment, contacting comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector or an AAV9 vector.

In an embodiment, contacting comprises delivering to the cell a Cas9 fusion molecule of (b) and a template binding domain of (d), as a protein or an mRNA, and a nucleic acid which encodes or comprises (a), (c) and (e).

In an embodiment, contacting comprises delivering to the cell a Cas9 fusion molecule of (b), as a protein or an mRNA, said gRNA of (a), as an RNA, and optionally (c) as a nucleic acid.

In some embodiments, a subject is treated by inducing a Cas9-mediated break at a target position, wherein the target position causes or exacerbates a disease or disorder, and administering one or more of (a), (b), (c), (d), and (e), wherein the break is repaired by HDR.

The method of treating a subject may comprise contacting the subject (or a cell from the subject) with (c) a template nucleic acid. A template nucleic acid is used when the method of treating a subject uses HDR to alter the sequence of the target nucleic acid of the subject.

In an embodiment, the method comprises acquiring knowledge of an undesired sequence in said subject, e.g., by DNA sequencing.

In an embodiment, the method comprises correcting an undesired sequence by HDR.

When the method comprises correcting an undesired sequence by HDR, a Cas9 fusion molecule of (b), at least one guide RNA, e.g., a guide RNA of (a) and a template nucleic acid (c) can be included in the contacting step.

In an embodiment, a cell of the subject is contacted ex vivo with (a), (b), (c), (d), and (e). In an embodiment, said cell is returned to the subject's body.

In an embodiment, a cell of the subject is contacted is in vivo with one or more of e.g., all of, (a), (b) (c), (d), and (e).

In an embodiment, the cell of the subject is contacted in vivo by intravenous delivery of one or more of, e.g., all of, (a), (b), (c), (d), and (e).

In an embodiment, contacting comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes or comprises at least one of, e.g., all of, (a), (b), (c), (d), and (e).

In an embodiment, contacting comprises delivering to said subject said Cas9 fusion molecule of (b), as a protein or mRNA, and one or more nucleic acid which encodes or comprises at least one of, e.g., all of (a), (c), (d), and (e)

In an embodiment, contacting comprises delivering to the subject the Cas9 fusion molecule of (b), as a protein or mRNA, the gRNA of (a), as an RNA, a nucleic acid of (c) as a DNA, the template binding domain of (d) as an RNA, and a nucleic acid of (e) as a DNA.

In an embodiment, contacting comprises delivering to the subject the gRNA of (a), as an RNA, a nucleic acid that encodes the Cas9 fusion molecule of (b), and a nucleic acid of (c).

In an embodiment, a cell of the subject is contacted ex vivo with (a), (b), (c), (d), and (e). In an embodiment, said cell is returned to the subject's body.

In an embodiment, contacting comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes or comprises at least one of, e.g., all of (a), (b), (c), (d), and (e).

In an embodiment, contacting comprises delivering to said subject the Cas9 fusion molecule of (b), as a protein or mRNA, and a nucleic acid which encodes or comprises one or more of (a), (c), (d), and (e).

In an embodiment, contacting comprises delivering to the subject the Cas9 fusion molecule of (b), as a protein or mRNA, the gRNA of (a), as an RNA, and the template nucleic acid of (c) as a DNA.

VIII.2 Examples of gRNAs in Genome Editing Methods

gRNA molecules as described herein can be used with Cas9 fusion molecules that generate a double strand break or a single strand break to alter the sequence of a target nucleic acid, e.g., a target position or target genetic signature. gRNA molecules useful in these methods are described below.

In some embodiments, the gRNA is used in making double stranded breaks. In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties:

• • a) it can position, e.g., when targeting a Cas9 fusion molecule that makes double strand breaks, a double strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
  • b) it has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and
  • c)
• (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. aureus, S. thermophilus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. aureus, S. thermophilus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail domain; or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
• (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail domain.

In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).

In an embodiment, the gRNA is configured such that it comprises properties: a and c.

In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).

In some embodiments, the gRNA is used in making single stranded breaks. In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties:

• • a) it can position, e.g., when targeting a Cas9 fusion molecule that makes single strand breaks, a single strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
  • b) it has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and
  • c)
• (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. aureus, S. thermophilus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. aureus, S. thermophilus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail domain; or, a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
• (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail domain.

In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).

In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).

In an embodiment, the gRNA is configured such that it comprises properties: a and c.

In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).

In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).

In an embodiment, the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.

In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

In an embodiment, a pair of gRNAs, e.g., a pair of chimeric gRNAs, comprising a first and a second gRNA, is configured such that they comprises one or more of the following properties:

• • a) one or both of the gRNAs can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break within (i) 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
  • b) one or both have a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17 or (ii) 18 nucleotides;
  • c) for one or both:
• (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. aureus, S. thermophilus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. aureus, S. thermophilus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
• iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail domain; or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
• (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. aureus, S. thermophilus, or N. meningitidis tail domain;
  • d) the gRNAs are configured such that, when hybridized to target nucleic acid, they are separated by 0-50, 0-100, 0-200, at least 10, at least 20, at least 30 or at least 50 nucleotides;
  • e) the breaks made by the first gRNA and second gRNA are on different strands; and
  • f) the PAMs are facing outwards.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(i).

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(ii).

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(iii).

In an embodiment, one or both of the gRNAs configured such that it comprises properties: a and c.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a, b, and c.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), and c(i).

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), and c(ii).

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and d.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and e.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and f.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, d, and e.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, d, and f.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, d, e, and f.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), and c(i).

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), and c(ii).

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), c, and d.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), c, and e.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), c, and f.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), c, d, and e.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), c, d, and f.

In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), c, d, e, and f.

In an embodiment, the gRNAs are used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.

In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

IX. Target Cells

Cas9 molecules and gRNA molecules, e.g., a Cas9 fusion molecule/gRNA molecule complex, as disclosed herein, can be used to manipulate, e.g., to edit a target nucleic acid, in a wide variety of cells. Additional details on types of cells that can be manipulated may be found in the section entitled “VIIA. TARGETS: CELLS” of PCT Application WO 2015/048577, the entire contents of which are expressly incorporated herein by reference.

In some embodiments, a cell is manipulated by editing (e.g., introducing a mutation or correcting) one or more target genes, e.g., as described herein. In an embodiment, a cell is manipulated by editing one or more non-coding sequences, e.g., an alteration in an intron or in a 5′ or 3′ non-translated or non-transcribed region. In an embodiment, a cell is manipulated by editing the sequence of a control element, e.g., a promoter, enhancer, or a cis-acting or trans-acting control element. In an embodiment, a cell is manipulated by editing one or more coding sequences, e.g., an alteration in an exon.

In some embodiments, a cell is manipulated in vitro. In other embodiments, a cell is manipulated ex vivo. In some embodiments, a cell is manipulated in vivo. In some embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., in vivo. In other embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., ex vivo. In other embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., in vitro.

In some embodiments, the cells are manipulated (e.g., converted or differentiated) from one cell type to another. In some embodiments, a pancreatic cell is manipulated into a beta islet cell. In some embodiments, a fibroblast is manipulated into an iPS cell. In some embodiments, a preadipocyte is manipulated into a brown fat cell. Other exemplary cells include, e.g., muscle cells, neural cells, leukocytes, and lymphocytes.

In some embodiments, a nucleic acid at one or more target positions in a cell, or a population of cells, is altered e.g., as described herein. In some embodiments, a nucleic acid at one or more target positions in a cell, or a population of cells (e.g., target positions at one or more genes described herein) is altered, e.g., in vivo. In other embodiments, a nucleic acid at one or more target positions in a cell, or a population of cells (e.g., target positions at one or more genes described herein) is altered, e.g., ex vivo. The Cas9 fusion molecule, nucleic acid template system, and/or gRNA molecules described herein can be delivered to a cell or to a population of cells.

In some embodiments, the cell, or the population of cells, is a T cell, a CD8+ T cell, a CD8+ naïve T cell, a central memory T cell, an effector memory T cell, a CD4+ T cell, a stem cell memory T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a hematopoietic stem cell, a long term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a pancreatic progenitor cell, an endocrine progenitor cell, an exocrine progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a monocytic precursor cell, an endocrine precursor cell, an exocrine cell, a fibroblast, a hepatoblast, a myoblast, a macrophage, an islet beta-cell, a cardiomyocyte, a blood cell, a ductal cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a cholangiocyte, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epithelial cell, a pulmonary epithelial progenitor cell, a striated muscle cell, a cardiac muscle cell, a muscle satellite cell, a myocyte, a neuron, a neuronal stem cell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, an embryonic stem cell, a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g. a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell, a gastrointestinal epithelial cell, a biliary epithelial cell, a pancreatic ductal epithelial cell, an intestinal stem cell, a hepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, an osteoclast, an adipocyte (e.g., a brown adipocyte, or a white adipocyte), a preadipocyte, a pancreatic precursor cell, a pancreatic islet cell, a pancreatic beta cell, a pancreatic alpha cell, a pancreatic delta cell, a pancreatic exocrine cell, a Schwann cell, or an oligodendrocyte, or a population of such cells.

In some embodiments, the cell, or the population of cells, is a mammalian cell, e.g., a human cell, a mouse cell, a rat cell, a sheep cell, a cow cell, a pig cell, a horse cell, a goat cell, a dog cell or a cat cell, or a population of mammalian cells. In one embodiment, the cell is a human cell.

In an embodiment, the cell, or population of cells, is manipulated ex vivo by altering a nucleic acid at one or more target positions, and administered to a subject. A cell, or population of cells, to be altered according to the methods disclosed herein, may include a stem cell such as, by way of example, an embryonic stem cell, an induced pluripotent stem cell or a neuronal stem cell, or a population of such cells. In an embodiment, the cell, or population of cells, is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, or a population of such cells, altered to correct a mutation and differentiated into a clinically relevant cell, or population of cells.

In some embodiments, the cell is a cell from a disease-causing organism, e.g., a bacterium, fungus, protozoan, or parasite. In some embodiments, the cell is a cell infected with a disease-causing organism (e.g., a virus, fungus, protozoan, or parasite).

In some embodiments, the cell is situated in the body of a subject. In such instances, the cell might be the subject's own cells or might be a cell of a disease-causing organism. In this case, a gRNA molecule, a Cas9 fusion molecule, and a nucleic acid template system, may be administered to the subject as pharmaceutical compositions. In some embodiments, the subject is a mammal, e.g., a human, a farm animal (e.g., a cow, a pig, a horse, or a goat), or a companion animal (e.g., a dog or a cat).

In some embodiments, the subject suffers from a disease caused by a target position in a nucleic acid, e.g., a particular mutation, of a cell, or population of cells.

In some embodiments, the cell, or population of cells, is a diseased or mutant-bearing cell, or population of cells. Such cells can be altered to treat the disease, e.g., to correct a mutation, or to alter the phenotype of the cell, or population of cells, e.g., to inhibit the growth of a cancer cell or a population of cancer cells, e.g., a tumor. For example, a cell, or a population of cells, is associated with one or more diseases or conditions describe herein. In some embodiments, the cell is a cancer stem cell. In some embodiments, the cancer cell is selected from lung cancer cells, breast cancer cells, skin cancer cells, brain cancer cells, pancreatic cancer cells, hematopoietic cancer cells, liver cancer cells, kidney cancer cells, and ovarian cancer cells.

In some embodiments, the cell is characterized by a disorder caused by aberrant mtDNA. This disorder may be, e.g., a mtDNA depletion syndrome (e.g., Alpers or early infantile hepatocerebral syndromes) or a mtDNA deletion disorder (e.g., progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)).

In some embodiments, the cell, or population of cells, is a normal cell or a population of normal cells.

In some embodiments, the cell, or population of cells, is a stem cell or a progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells), or a population of such cells.

The cells may also be treated at a time when they are not situated in the body of a subject. In some embodiments, a cell, or a population of cells, is treated ex vivo to avoid exposing a patient to an agent or agents that cause undesirable side effects. In some embodiments, treating cells ex vivo allows a user to select a sub-population of cells to administer to the patient. The sub-population may be, e.g., cells having a nucleic acid that was successfully altered, or cells having a desired phenotype, such as minimal undesired alterations to DNA, or a phenotype that indicates the nucleic acid was successfully altered.

In some embodiments, the cell, or population of cells, is not situated in a subject's body and the cell, or population of cells, is modified for research or manufacturing purposes. In some embodiments, the cell, or population of cells, is suitable for producing a recombinant biological product. For example, the cell, or population of cells, can be a CHO cell or a fibroblast. In one embodiment, the cell, or population of cells, is a cell, or population of cells, that has been engineered to express a protein.

In some embodiments, the cell, or population of cells, is actively dividing. In some embodiments, the cell is in G2 phase. In some embodiments, the population of cells comprises cells that are in G2 phase. In some embodiments, the cell is in G1 phase. In some embodiments, the population of cells comprises cells that are in G1 phase. In some embodiments, the cell is in S phase. In some embodiments, the population of cells comprises cells that are in S phase.

The technology described herein can be used to edit numerous types of genomes, including plant genomes. The CRISPR/Cas system has been used for plant genome editing, as has been described in, e.g., Belhaj et al., PLANT METHODS 9:39, 2013. Accordingly, in certain embodiments, the cell, or the population of cells, is a plant cell, e.g., a monocot plant cell, or a dicot plant cell, or a population of plant cell. In certain embodiments, the plant is a crop, e.g., a food crop. In certain embodiments, the plant is rice (e.g., Orzya sativa), maize (e.g., Zea mays), wheat (e.g., Triticum aestivum), soy (e.g., Glycine max), potato (e.g., Solanum tuberosum), a species of Nicotiana, a species of Arabidopsis e.g., Arabidopsis thaliana, cassava, sweet potato, sorghum, yam, plantain, or a citrus plant. In some embodiments, the plant is a pesticide-resistant plant, e.g., a plant that expresses one or more genes that confer resistance to a pesticide. In some embodiments, the plant is herbicide-resistant plant, e.g., a plant that expresses one or more genes that confer resistance to a herbicide. The herbicide may be, e.g., Roundup® (also known as glyphosate or N-(phosphonomethyl)glycine). In some embodiments, the plant produces a pesticide, e.g., Bt.

In some embodiments, the components used in the methods described herein (e.g., a Cas9 fusion molecule, a nucleic acid template system, and/or a gRNA) are introduced into the plant cell via protoplast transformation or agroinfiltration.

In some embodiments, after genome editing using the methods described herein, seeds are screened and a desired sub-population of seeds are selected. The sub-population may be, e.g., cells having a nucleic acid that was successfully altered, or cells having a desired phenotype such as minimal undesired alterations to DNA, or a phenotype that indicates the nucleic acid was successfully altered.

X. Delivery, Formulations and Routes of Administration

The components, e.g., a Cas9 fusion molecule, a nucleic acid template system, and/or a gRNA molecule can be delivered or formulated in a variety of forms, see, e.g., Tables 4-5. When a Cas9 fusion molecule, nucleic acid template system component, and/or a gRNA component is encoded by DNA for delivery, the DNA will typically but not necessarily include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for Cas9 fusion molecule sequences include CMV, EF-1a, MSCV, PGK, CAG control promoters. Useful promoters for gRNAs include H1, EF-1a and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 fusion molecule can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment a promoter for a Cas9 fusion molecule or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific.

Table 4 provides non-limiting examples of the form in which the components can be delivered to a target cell.

TABLE 4
Elements
		Template
Cas9		Nucleic
Fusion	gRNA	Acid
Molecule(s)	Molecule(s)	System	Comments
DNA	DNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			and a gRNA are transcribed from DNA. In
			this embodiment, they are encoded on
			separate molecules. In this embodiment, the
			donor template is provided as a separate
			DNA molecule. In some embodiments, the
			template binding domain is encoded by
			DNA, optionally on the same nucleic acid
			as the Cas9 fusion molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
DNA	DNA	In an embodiment, a Cas9 fusion molecule,
			e.g., an eaCas9 fusion molecule, and a
			gRNA are transcribed from DNA. In this
			embodiment, they are encoded on separate
			molecules. In this embodiment, the donor
			template is provided on the same DNA
			molecule that encodes the gRNA. In some
			embodiments, the template binding domain
			is encoded in DNA, optionally on the same
			nucleic acid as the Cas9 fusion molecule. In
			some embodiments, the template binding
			domain partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
DNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			and a gRNA are transcribed from DNA,
			here from a single molecule. In this
			embodiment, the donor template is provided
			as a separate DNA molecule. In some
			embodiments, the template binding domain
			is encoded in DNA, optionally on the same
			nucleic acid as the Cas9 fusion molecule. In
			some embodiments, the template binding
			domain partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
DNA	DNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			and a gRNA are transcribed from DNA. In
			this embodiment, they are encoded on
			separate molecules. In this embodiment, the
			donor template is provided on the same
			DNA molecule that encodes the Cas9 fusion
			molecule. In some embodiments, the
			template binding domain is encoded in
			DNA, optionally on the same nucleic acid
			as the Cas9 fusion molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
DNA	RNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			is transcribed from DNA, and a gRNA is
			provided as in vitro transcribed or
			synthesized RNA. In this embodiment, the
			donor template is provided as a separate
			DNA molecule. In some embodiments, the
			template binding domain is encoded in
			DNA, optionally on the same nucleic acid
			as the Cas9 fusion molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
DNA	RNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			is transcribed from DNA, and a gRNA is
			provided as in vitro transcribed or
			synthesized RNA. In this embodiment, the
			donor template is provided on the same
			DNA molecule that encodes the Cas9 fusion
			molecule. In some embodiments, the
			template binding domain is encoded in
			DNA, optionally on the same nucleic acid
			as the Cas9 fusion molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
mRNA	RNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			is translated from in vitro transcribed
			mRNA, and a gRNA is provided as in vitro
			transcribed or synthesized RNA. In this
			embodiment, the donor template is provided
			as a DNA molecule. In some embodiments,
			the template binding domain is translated
			form in vitro transcribed mRNA, and is
			optionally part of the same nucleic acid as
			the Cas9 fusion molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
mRNA	DNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			is translated from in vitro transcribed
			mRNA, and a gRNA is transcribed from
			DNA. In this embodiment, the donor
			template is provided as a separate DNA
			molecule. In some embodiments, the
			template binding domain is translated form
			in vitro transcribed mRNA, and is
			optionally part of the same nucleic acid as
			the Cas9 fusion molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
mRNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			is translated from in vitro transcribed
			mRNA, and a gRNA is transcribed from
			DNA. In this embodiment, the donor
			template is provided on the same DNA
			molecule that encodes the gRNA. In some
			embodiments, the template binding domain
			is translated form in vitro transcribed
			mRNA, and is optionally part of the same
			nucleic acid as the Cas9 fusion molecule. In
			some embodiments, the template binding
			domain partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
Protein	DNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			is provided as a protein, and a gRNA is
			transcribed from DNA. In this embodiment,
			the donor template is provided as a separate
			DNA molecule. In some embodiments, the
			template binding domain is provided as a
			protein, and is optionally covalently linked
			to, e.g., fused to, the Cas9 fusion molecule.
			In some embodiments, the template binding
			domain partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
Protein	DNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule,
			is provided as a protein, and a gRNA is
			transcribed from DNA. In this embodiment,
			the donor template is provided on the same
			DNA molecule that encodes the gRNA. In
			some embodiments, the template binding
			domain is provided as a protein, and is
			optionally covalently linked to, e.g., fused
			to, the Cas9 molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.
Protein	RNA	DNA	In some embodiments, a Cas9 fusion
			molecule, e.g., an eaCas9 fusion molecule is
			provided as a protein, and a gRNA is
			provided as transcribed or synthesized
			RNA. In this embodiment, the donor
			template is provided as a DNA molecule. In
			some embodiments, the template binding
			domain is provided as a protein, and is
			optionally covalently linked to, e.g., fused
			to, the Cas9 fusion molecule. In some
			embodiments, the template binding domain
			partner is provided as DNA, and is
			optionally part of the same nucleic acid as
			the donor template.

Table 5 summarizes various delivery methods for the components of a Cas9 system, e.g., the Cas9 fusion molecule, the nucleic acid template system, and a gRNA molecule, as described herein.

TABLE 5
	Delivery
	into Non-	Duration		Type of
	Dividing	of	Genome	Molecule
Delivery Vector/Mode	Cells	Expression	Integration	Delivered
Physical (e.g., electro-	YES	Transient	NO	Nucleic Acids
poration, particle gun,				and Proteins
Calcium Phosphate
transfection)
Viral	Retrovirus	NO	Stable	YES
	Lentivirus	YES	Stable	YES/NO with	RNA
				modifications
	Adenovirus	YES	Transient	NO	DNA
	Adeno-	YES	Stable	NO	DNA
	Associated
	Virus (AAV)
	Vaccinia Virus	YES	Very	NO	DNA
			Transient
	Herpes Simplex	YES	Stable	NO	DNA
	Virus
Non-Viral	Cationic	YES	Transient	Depends on	Nucleic Acids
	Liposomes			what is	and Proteins
				delivered
	Polymeric	YES	Transient	Depends on	Nucleic Acids
	Nanoparticles			what is	and Proteins
				delivered
Biological	Attenuated	YES	Transient	NO	Nucleic Acids
Non-Viral	Bacteria
Delivery	Engineered	YES	Transient	NO	Nucleic Acids
Vehicles	Bacteriophages
	Mammalian	YES	Transient	NO	Nucleic Acids
	Virus-like
	Particles
	Biological	YES	Transient	NO	Nucleic Acids
	liposomes:
	Erythrocyte
	Ghosts and
	Exosomes

DNA-Based Delivery of a Cas9 Fusion Molecule, Nucleic Acid Template System and/or a gRNA Molecule

DNA encoding Cas9 fusion molecules (e.g., eaCas9 fusion molecules), gRNA molecules, template nucleic acids, template binding domains, and/or template binding domain partners can be administered to subjects or delivered into cells by any appropriate method, e.g., by art-known methods or as described herein. For example, Cas9 fusion molecule-encoding and/or gRNA-encoding DNA, a template binding domain partner, and a template nucleic acid can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.

In some embodiments, the nucleic acid, e.g., Cas9 fusion molecule- and/or gRNA-encoding DNA is delivered by a vector (e.g., viral vector/virus or plasmid).

In one embodiment, a vector can comprise a sequence that encodes a Cas9 fusion molecule, a gRNA molecule, and a nucleic acid template system. In one embodiment, a vector can comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused, e.g., to a Cas9 fusion molecule sequence. For example, a vector can comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 fusion molecule.

One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and splice acceptor or donor, can be included in the vectors. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV promoter).

In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., dsDNA or ssDNA virus). In other embodiments, the virus is an RNA virus (e.g., an ssRNA virus). Exemplary viral vectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.

In some embodiments, the virus infects dividing cells. In other embodiments, the virus infects non-dividing cells. In some embodiments, the virus infects both dividing and non-dividing cells. In some embodiments, the virus can integrate into the host genome. In some embodiments, the virus is engineered to have reduced immunity, e.g., in human. In some embodiments, the virus is replication-competent. In other embodiments, the virus is replication-defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In some embodiments, the virus causes transient expression of the Cas9 fusion molecule and/or the gRNA molecule. In other embodiments, the virus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 fusion molecule and/or the gRNA molecule. The packaging capacity of the viruses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

In some embodiments, the Cas9 fusion molecule-, gRNA-, and/or template binding domain-encoding DNA and/or the nucleic acid template system is delivered by a recombinant adenovirus. In some embodiments, the adenovirus is engineered to have reduced immunity in human.

In some embodiments, the Cas9 fusion molecule-, gRNA-, and/or template binding domain-encoding DNA and/or the nucleic acid template system is delivered by a recombinant AAV. In some embodiments, the AAV can incorporate its genome into that of a host cell, e.g., a target cell as described herein. In some embodiments, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods, include AAV1, AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y731F and/or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8, AAV 8.2, AAV9, AAV rh10, and pseudotyped AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods.

In some embodiments, the Cas9 fusion molecule-gRNA-, and/or template binding domain-encoding DNA, and/or the nucleic acid template system is delivered by a non-vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.

In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe₃MnO₂) and silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.

Exemplary lipids for gene transfer are shown below in Table 6.

TABLE 6
Lipids Used for Gene Transfer
Lipid	Abbreviation	Feature
1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine	DOPC	Helper
1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine	DOPE	Helper
Cholesterol		Helper
N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium chloride	DOTMA	Cationic
1,2-Dioleoyloxy-3-trimethy lammonium-propane	DOTAP	Cationic
Dioctadecylamidoglycylspermine	DOGS	Cationic
N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-	GAP-DLRIE	Cationic
propanaminium bromide
Cetyltrimethylammonium bromide	CTAB	Cationic
6-Lauroxyhexyl ornithinate	LHON	Cationic
1-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium	2Oc	Cationic
2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-dimethyl-1-	DOSPA	Cationic
propanaminium trifluoroacetate
1,2-Dioleyl-3-trimethylammonium-propane	DOPA	Cationic
N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-	MDRIE	Cationic
propanaminium bromide
Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide	DMRI	Cationic
3B-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol	DC-Chol	Cationic
Bis-guanidium-tren-cholesterol	BGTC	Cationic
1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide	DOSPER	Cationic
Dimethyloctadecylammonium bromide	DDAB	Cationic
Dioctadecylamidoglicylspermidin	DSL	Cationic
rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]-	CLIP-1	Cationic
dimethylammonium chloride
rac-[2(2,3-Dihexadecyloxypropyl-	CLIP-6	Cationic
oxymethyloxy)ethyl]trimethylammonium bromide
Ethyldimyristoy lphosphatidylcholine	EDMPC	Cationic
1,2-Distearyloxy-N,N-dimethyl-3-aminopropane	DSDMA	Cationic
1,2-Dimyristoyl-trimethylammonium propane	DMTAP	Cationic
O,O′-Dimyristyl-N-lysyl aspartate	DMKE	Cationic
1,2-Distearoyl-sn-glycero-3-ethylphosphocholine	DSEPC	Cationic
N-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine	CCS	Cationic
N-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine	diC14-amidine	Cationic
Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl]	DOTIM	Cationic
imidazolinium chloride
N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine	CDAN	Cationic
2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N-	RPR209120	Cationic
ditetradecylcarbamoylme-ethyl-acetamide
1,2-dilinoleyloxy-3-dimethylaminopropane	DLinDMA	Cationic
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane	DLin-KC2-DMA	Cationic
dilinoleyl-methyl-4-dimethylaminobutyrate	DLin-MC3-DMA	Cationic

Exemplary polymers for gene transfer are shown below in Table 7.

TABLE 7
Polymers Used for Gene Transfer
Polymer                          Abbreviation
Poly(ethylene)glycol             PEG
Polyethylenimine                 PEI
Dithiobis(succinimidylpropionate) DSP
Dimethyl-3,3′-dithiobispropionimidate DTBP
Poly(ethylene imine )biscarbamate PEIC
Poly(L-lysine)                   PLL
Histidine modified PLL
Poly(N-vinylpyrrolidone)         PVP
Poly(propylenimine)              PPI
Poly(amidoamine)                 PAMAM
Poly(amidoethylenimine)          SS-PAEI
Triethylenetetramine             TETA
Poly(β-aminoester)
Poly(4-hydroxy-L-proline ester)  PHP
Poly(allylamine)
Poly(a-[4-aminobutyl]-L-glycolic acid) PAGA
Poly(D,L-lactic-co-glycolic acid) PLGA
Poly(N-ethyl-4-vinylpyridinium bromide)
Poly(phosphazene)s               PPZ
Poly(phosphoester)s              PPE
Poly(phosphoramidate)s           PPA
Poly(N-2-hydroxypropylmethacrylamide) pHPMA
Poly (2-(dimethylamino)ethyl methacrylate) pDMAEMA
Poly(2-aminoethyl propylene phosphate) PPE-EA
Chitosan
Galactosylated chitosan
N-Dodacylated chitosan
Histone
Collagen
Dextran-spermine                 D-SPM

In an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle.

In an embodiment, one or more nucleic acid molecules (e.g., a DNA molecule or a nucleic acid template system) other than the components of a Cas9 system, e.g., the Cas9 fusion molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the components of the Cas9 system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas9 system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas9 system, e.g., the Cas9 fusion molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 fusion molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNA) is reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein. In some embodiments, the nucleic acid is a nucleic acid template system capable of participating in HDR.

Delivery of RNA Encoding a Cas9 Fusion Molecule

RNA encoding Cas9 fusion molecules (e.g., mRNA encoding an eaCas9 fusion molecules), gRNA molecules, and/or template binding domains can be delivered into cells, or populations of cells, described herein, by any appropriate method, including art-known methods or methods described herein. For example, Cas9 fusion molecule-, gRNA-, and/or template binding domain-encoding RNA, e.g., mRNA can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof

Delivery of Cas9 Fusion Molecule Protein

Cas9 fusion molecules (e.g., eaCas9 fusion molecules) and/or template binding domains can be delivered into cells by any appropriate method, including art-known methods or methods described herein. For example, the protein molecules can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA, or by a gRNA, a nucleic acid template system, and/or DNA encoding a template binding domain partner.

Routes of Administration

Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intrarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to the desired cell type.

Local modes of administration include, by way of example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate or into the putamen)), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CM region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum or substantia nigra intraocular, intraorbital, subconjuctival, intravitreal, subretinal or transscleral routes. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intraparenchymal or intravitreal) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.

Administration may be provided as a periodic bolus or as continuous infusion from an internal reservoir or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device.

Bi-Modal or Differential Delivery of Components

Separate delivery of the components of a Cas9 system, e.g., the Cas9 fusion molecule component, the gRNA molecule component, the template binding domain, the template binding domain partner, and/or the nucleic acid template system, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety.

In an embodiment, one or more of the Cas9 fusion molecule, the gRNA molecule, the template binding domain, the template binding domain partner, and/or the nucleic acid template system, are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer to modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., a Cas9 fusion molecule, gRNA molecule, nucleic acid template system, template binding domain, and/or template binding domain partner. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ. In many embodiments, the components are delivered so that one or more of; e.g., all of a Cas9 fusion molecule, gRNA molecule, nucleic acid template system, template binding domain, and template binding domain partner will be present in the same cell at the same time.

In some embodiments, two gRNAs are delivered to a cell so that a first nickase will make a first single stranded break and a second nickase will make a second single stranded break. In such embodiments, the two gRNAs and other components (e.g., the Cas9 fusion molecule) are delivered such that the two breaks are made at substantially the same time. In some embodiments this comprises the second break being formed before the first break engages with machinery specific to the SSBR (single stranded break repair) pathway, and in some embodiments, it comprises the second break being formed before the first break is repaired. More generally, when one desires to make two or more breaks in a target nucleic acid, the gRNAs and other components can be delivered such that the two or more breaks are made at substantially the same time.

Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., adeno-associated virus or lentivirus, delivery.

By way of example, the components, e.g., a Cas9 fusion molecule, a gRNA molecule, nucleic acid template system, template binding domain, and template binding domain partner can be delivered by modes that differ in terms of resulting half-life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, one or more of, e.g., all of, a gRNA molecule, nucleic acid template system, template binding domain, and template binding domain partner can be delivered by such modes. The Cas9 fusion molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.

More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.

In an embodiment, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.

In an embodiment, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

In an embodiment, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

In an embodiment, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.

In an embodiment, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.

In an embodiment, the first component comprises a gRNA, template nucleic acid, template binding domain, or template binding domain partner and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes generally do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, a Cas9 fusion molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full Cas9 fusion molecule/gRNA molecule complex is only present and active for a short period of time.

Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.

Use of differential delivery modes can enhance performance, safety and efficacy. For example, the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 fusion molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MEW molecules. A two-part delivery system can alleviate these drawbacks.

Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA molecule is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a Cas9 fusion molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In embodiment, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.

When the Cas9 fusion molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the Cas9 fusion molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only formed in the tissue that is targeted by both vectors.

Ex Vivo Delivery

In some embodiments, components described in Table 4 are introduced into cells which are then introduced into the subject. Methods of introducing the components can include, e.g., any of the delivery methods described in Table 5.

In some embodiments, the cells are contacted with a Cas9 fusion molecule (or a nucleic acid encoding it) ex vivo. In some embodiments, the cells are contacted with a gRNA (or a nucleic acid encoding it) ex vivo. In some embodiment, the cells are contacted with a template nucleic acid ex vivo. In some embodiment, the cells are contacted with a template binding domain (or a nucleic acid encoding it) ex vivo. In some embodiment, the cells are contacted with a template binding domain partner ex vivo. In some embodiments, the cells are contacted with the nucleic acid template system ex vivo. In some embodiments, the cells are contacted with two, three, four, or all of the preceding compositions (or nucleic acids encoding them) ex vivo. In some embodiments, the cells are contacted with one or more of the preceding components (or nucleic acids encoding them), and one or more remaining components are administered to the patient.

XI. Modified Nucleosides, Nucleotides, and Nucleic Acids

Modified nucleosides and modified nucleotides can be present in nucleic acids, e.g., particularly gRNA, a template binding domain partner, and/or a template nucleic acid, but also other forms of DNA or RNA, e.g., mRNA. As described herein, “nucleoside” is defined as a compound containing a five-carbon sugar molecule (a pentose such as deoxyribose or ribose) or derivative thereof; and an organic base (purine or pyrimidine, or a derivative thereof). As described herein, “nucleotide” is defined as a nucleoside further comprising a phosphate group.

Modified nucleosides and nucleotides can include one or more of:

• • (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;
  • (ii) alteration, e.g., replacement, of a constituent of the ribose or deoxyribose sugar, e.g., of the 2′ hydroxyl on the sugar;
  • (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers;
  • (iv) modification or replacement of a naturally occurring nucleobase;
  • (v) replacement or modification of the ribose- or deoxyribose-phosphate backbone;
  • (vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and
  • (vii) modification of the sugar.

The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In an embodiment, every base of a gRNA, a template binding domain partner, or template nucleic acid is modified, e.g., all bases have a modified phosphate group, e.g., all are phosphorothioate groups. In an embodiment, all, or substantially all, of the phosphate groups of a unimolecular or modular gRNA molecule, a template binding domain partner, or template nucleic acid are replaced with phosphorothioate groups.

In an embodiment, modified nucleotides, e.g., nucleotides having modifications as described herein, can be incorporated into a nucleic acid, e.g., a “modified nucleic acid.” In some embodiments, the modified nucleic acids comprise one, two, three or more modified nucleotides. In some embodiments, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g., cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases.

In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo, and also disrupt binding of a major groove interacting partner with the nucleic acid.

In some embodiments, a template nucleic acid comprises modifications, e.g., modified nucleotides, modifications to the backbone, and other modifications described herein. In some embodiments, the modification improves the stability of the template nucleic acid, e.g., by increasing its resistance to endonucleases and/or exonucleases.

In some embodiments, a template nucleic acid that comprises modifications is double stranded, e.g., is double stranded DNA. In some such embodiments, all the modifications are confined to one strand. In other embodiments, modifications are present on both strands. Modifications may be present in the 5′ homology arm, the 3′ homology arm, or the replacement sequence, or any combination thereof. In some embodiments, modifications are present in one or both homology arms but not the replacement sequence.

In some embodiments, a template nucleic acid that comprises modifications is single stranded, e.g., is single stranded DNA.

Definitions of Chemical Groups

As used herein, “alkyl” is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.

As used herein, “alkenyl” refers to an aliphatic group containing at least one double bond.

As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl.

As used herein, “arylalkyl” or “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

As used herein, “cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.

As used herein, “heterocyclyl” refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.

As used herein, “heteroaryl” refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

Phosphate Backbone Modifications

The Phosphate Group

In some embodiments, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleotide, e.g., modified nucleotide present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR₃ (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR₂ (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide (or oligodeoxyribonucleotide) diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).

The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charge phosphate group can be replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

Replacement of the Ribophosphate Backbone

Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

Sugar Modifications

The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. The 2′-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.

Examples of “oxy”-2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the “oxy”-2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C_1-6 alkylene or C_1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)_n-amino, (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the “oxy”-2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).

“Deoxy” modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid);

• • NH(CH₂CH₂NH)_nCH₂CH₂-amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide “monomer” can have an alpha linkage at the 1′ position on the sugar, e.g., alpha-nucleoside s. The modified nucleic acids can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L-nucleosides.

Generally, RNA includes the sugar group ribose, and DNA includes the sugar group deoxyribose, each of which is a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include, without limitation, replacement of the oxygen in the ribose or deoxyribose ring (e.g., with sulk (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or 5-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with α-L-threofuranosyl-(3′→2′)).

Modifications on the Nucleobase

The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

Uracil

In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s2U), 5-aminomethyl-2-thio-uridine (nm⁵s2U), 5-methylaminomethyl-uridine (mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s2U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τcm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ), 5-methyl-2-thio-uridine (m⁵s2U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-methyl-uridine (mcm ⁵Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm ⁵Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵Um), 3,2′-O-dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm ⁵Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-0H-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

Thymine

In some embodiments, the modified nucleobase is a modified thymine. Thymine differs from uracil in that thymine has a methyl group on carbon 5 of the 6-carbon ring, while uracil has a hydrogen in that position. In some embodiments, the modified thymine is derived from one of the modified uracils described in the previous paragraph, but having said methyl group instead of a hydrogen.

Cytosine

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m³C), N4-acetyl-cytidine (act), 5-formyl-cytidine (f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudo isocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k²C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm), N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm), N4,N4,2′-O-trimethyl-cytidine (m⁴₂Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

Adenine

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A), 2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms2 m⁶A), N6-isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adeno sine (ms²i⁶A), N6-(cis-hydroxyisopentenyl) adenosine (io⁶A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io⁶A), N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine (t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶2 A), N6-hydroxynorvalylcarbamoyl-adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn⁶A), N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N⁶,2′-O-dimethyl-adenosine (m⁶Am), N⁶-Methyl-2′-deoxyadenosine, N6,N6,2′-O-trimethyl-adenosine (m⁶ 2 Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

Guanine

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybuto sine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀), 7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m′G), N2-methyl-guanosine (m²G), N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²,7G), N2, N2,7-dimethyl-guanosine (m²,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m²Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm), 1-methyl-2′-O-methyl-guanosine (m′Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m²,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m′Im), O⁶-phenyl-2′-deoxyinosine, 2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O⁶-methyl-guanosine, O⁶-Methyl-2′-deoxyguanosine, 2′-F-ara-guanosine, and 2′-F-guanosine.

Modified gRNAs

In some embodiments, the modified nucleic acids can be modified gRNAs. In some embodiments, gRNAs can be modified at the 3′ end. In this embodiment, the gRNAs can be modified at the 3′ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:

• • wherein “U” can be an unmodified or modified uridine.

In another embodiment, the 3′ terminal U can be modified with a 2′3′ cyclic phosphate as shown below:

• • wherein “U” can be an unmodified or modified uridine.

In some embodiments, the gRNA molecules may contain 3′ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2′ OH-group is replaced by a group selected from H, —OR, —R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (—CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2-F 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5 Ceo), and any combinations thereof.

In an embodiment, one or more or all of the nucleotides in single stranded RNA molecule, e.g., a gRNA molecule, are deoxynucleotides.

Modified Nucleic Acid Template Systems

In some embodiments, the nucleic acid template system comprises chemical modifications. These modifications may, e.g., increase the stability or half-life of the nucleic acid or reduce the innate immune response to the nucleic acid. In some embodiments, the template binding domain partner comprises chemical modifications; in some embodiments the template nucleic acid comprises chemical modifications; and in some embodiments, both the template binding domain partner and the template nucleic acid comprise chemical modifications.

In some embodiments, the nucleic acid template system can be modified at one or two 3′ ends. In this embodiment, the nucleic acid template system can be modified at the 3′ nucleotide. For example, the two terminal hydroxyl groups of the 3′-most sugar can be oxidized to aldehyde groups and a concomitant opening of the ring to afford a modified nucleoside, analogous to the first ribonucleotide shown in the previous section entitled “Modified gRNAs”.

In another embodiment, the 3′ terminal sugar can be modified with a 2′3′ cyclic phosphate, analogous to the second ribonucleotide shown in the previous section entitled “Modified gRNAs”.

In some embodiments, the nucleic acid template system may contain 3′ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., thymines can be replaced with any of the modified thymines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the nucleic acid template system. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the nucleic acid template system. In some embodiments, sugar-modified deoxyribonucleotides can be incorporated, e.g., wherein the 2′ H-group is replaced by a group selected from OH, —OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (—CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleic acid template system comprises an overhang region, and the nucleotides in the overhang region can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2-F 2′-O-methyl, 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

miRNA Binding Sites

MicroRNAs (miRNAs) are naturally occurring cellular 19-25 nucleotide long noncoding RNAs. They bind to nucleic acid molecules having an appropriate miRNA binding site, e.g., in the 3′ UTR of an mRNA, and down-regulate gene expression. While not wishing to be bound by theory it is believed that the down regulation is either by reducing nucleic acid molecule stability or by inhibiting translation. An RNA species disclosed herein, e.g., an mRNA encoding Cas9 fusion molecule can comprise an miRNA binding site, e.g., in its 3′UTR. The miRNA binding site can be selected to promote down regulation of expression is a selected cell type. By way of example, the incorporation of a binding site for miR-122, a microRNA abundant in liver, can inhibit the expression of the gene of interest in the liver.

XII. Nucleic Acids; Kits; Methods of Production

In some aspects, disclosed herein is a nucleic acid, e.g., an isolated or non-naturally occurring nucleic acid, e.g., DNA, that comprises a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain as disclosed herein. In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a region desired to be altered to allow alteration, e.g., alteration associated with HDR of the region desired to be altered.

A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain as disclosed herein; (b) a sequence that encodes a Cas9 fusion molecule; and further comprises (c) (i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain, and optionally, (ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain; and optionally, (iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain.

In some embodiments, when a region that is desired to be altered is corrected by HDR, the nucleic acid encodes (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain gene as disclosed herein; (b) a sequence that encodes a Cas9 fusion molecule; (c) a template nucleic acid, (d) a template binding domain, and (e) a template binding domain partner.

In some embodiments, one or more of e.g., all of (a), (b), (c), (d), and (e) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno-associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector. Exemplary AAV vectors that may be used in any of the described compositions and methods include an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector and an AAV9 vector.

In other embodiments, a subset of (a), (b), (c), (d), and (e) is present on a first nucleic acid molecule, e.g. a first vector, e.g., a first viral vector, e.g., a first AAV vector; and the remainder of (a), (b), (c), (d), and (e) is present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecules may be AAV vectors. In many embodiments, the Cas9 fusion molecule of (b) and the template binding domain of (d) are present on the same nucleic acid molecule, e.g., a viral vector, e.g., an AAV vector. In many embodiments, the template nucleic acid of (c) and the template binding domain partner of (e) are present on the same nucleic acid molecule, e.g., a viral vector, e.g., an AAV vector.

In some embodiments, all of (a), (b), (c), (d), and (e) are on the same vector, e.g., the same AAV vector. In some embodiments, all of (a), (b), (c), (d), and (e) are on different vectors, e.g., one or more AAV vectors. In some embodiments, two or more, e.g., two, three, or four of (a), (b), (c), (d), and (e) are on the same vector, e.g., an AAV vector, and the remainder are on one or more other vectors, e.g., one or more AAV vectors. In some embodiments, (a) and (b) are on the same vector, e.g., an AAV vector; (a) and (c) are on the same vector, e.g., an AAV vector, (a) and (d) are on the same vector, e.g., an AAV vector, (b) and (c) are on the same vector, e.g., an AAV vector, (b) and (d) are on the same vector, e.g., an AAV vector, or (c) and (d) are on the same vector, e.g., an AAV vector.

The nucleic acids described herein may comprise a promoter operably linked to the sequence that encodes the gRNA molecule of (a), e.g., a promoter described herein. The nucleic acid may further comprise a second promoter operably linked to the sequence that encodes the second, third and/or fourth gRNA molecule of (e), e.g., a promoter described herein. The promoter and second promoter differ from one another. In some embodiments, the promoter and second promoter are the same.

The nucleic acids described herein may further comprise a promoter operably linked to the sequence that encodes the Cas9 fusion molecule of (b), e.g., a promoter described herein.

The nucleic acids described herein may further comprise a promoter operably linked to the sequence that encodes the template binding domain of (d), e.g., a promoter described herein. In some embodiments, the template binding domain of (d) and the Cas9 fusion molecule of (b) are controlled by the same promoter, e.g., they can form a fusion protein.

In another aspect, disclosed herein is a kit comprising one or more, e.g., all of the following:

• • (a) gRNA molecule described herein, or nucleic acid that encodes the gRNA;
  • (b) a Cas9 fusion molecule, e.g., a Cas9 fusion molecule described herein, or a nucleic acid or mRNA that encodes the Cas9 fusion molecule;
  • (c) a template nucleic acid; and
  • (d) a template binding domain, and
  • (e) a template binding domain partner.

In an embodiment, the kit comprises a nucleic acid comprising both (c) and (e).

In an embodiment, the kit comprises nucleic acid, e.g., an AAV vector, that encodes one or more of (a), (b), (c), (d), and (e).

XIII. Methods of Treatment

A genetic disease is caused by a mutation in the patient's genome. Often, the mutation results in a change in a protein, e.g., an amino acid substitution or a truncation. Genetic diseases can be dominant, i.e., one mutant gene is sufficient to cause the disease, or recessive, where a patient with one copy of the mutant gene is an asymptomatic carrier, and two copies of the mutant gene are necessary for the disease to result.

Disclosed herein are the approaches to treat or prevent genetic diseases, using the compositions and methods described herein.

One approach to treat or prevent genetic diseases is to repair (i.e., correct) one or more mutations in the disease-causing gene by HDR. In this approach, mutant allele(s) are corrected and restored to wild type state. While not wishing to be bound by theory, it is believed that correction of the mutation to the corresponding wild-type sequence restores wild type protein production within the relevant cell type. The method described herein can be performed in all cell types.

In an embodiment, one mutant allele is repaired in the subject. For example, in a patient with an autosomal dominant genetic disease, the sole mutant allele in the cell is corrected so that the cell becomes wild-type at both loci. As another example, in a patient with an autosomal recessive genetic disease, one of the two mutant alleles in the cell is corrected, and so the cell becomes heterozygous, which is sufficient for normal functioning. As a recessive genetic disease only displays a phenotype when both alleles are mutated, repair of a single allele is adequate for a cure. In another embodiment, both mutant alleles are repaired in the subject. In either situation, the subjects can be cured of disease.

Correction of a mutation in the relevant gene may be performed prior to disease onset (e.g., prior to the appearance of symptoms) or after disease onset, for instance, early in the disease course.

In an embodiment, the method comprises initiating treatment of a subject prior to disease onset. In an embodiment, the method comprises initiating treatment of a subject after disease onset. In an embodiment, the method comprises initiating treatment of a subject well after disease onset, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, or 36 months after onset of the disease. While not wishing to be bound by theory it is believed that this may be effective if subjects did not present to physician until well into the course of illness.

In an embodiment, the method comprises initiating treatment of a subject in an advanced stage of disease.

Overall, initiation of treatment for subjects at all stages of disease is expected to prevent negative consequences of disease and be of benefit to subjects.

In an embodiment, the method comprises initiating treatment of a subject prior to disease expression. In an embodiment, the method comprises initiating treatment of a subject in an early stage of disease, e.g., when a subject has tested positive for the disease but has no signs or symptoms associated with the disease.

In an embodiment, the method comprises initiating treatment of a subject who has tested positive for the mutation underlying the disease, based on diagnosis via electrophoresis, genotyping, family history or other diagnostic criteria.

EXAMPLES

The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Example 1: Enhancing HDR-Mediated Alteration of a Target Nucleic Acid in Cultured Cells Using a Cas9 Fusion Protein

This study demonstrates an increased rate of HDR-mediated nucleic acid modification at a target site in cultured cells by administering to the cells a Cas9 fusion protein, one or more gRNAs, and an HDR donor nucleic acid template. The Cas9 fusion protein is a Cas9 protein fused to a polypeptide comprising the DNA binding domain derived from a sequence-specific DNA binding protein, e.g., one of the DNA binding proteins listed in Tables V.1-V.4 of Section V, above. The Cas9 fusion protein (or, in control cells, Cas9 protein lacking the fusion domain) is administered as a DNA expression vector, an mRNA or a protein. The one or more gRNAs are administered as DNA expression vectors or RNA molecules. The donor template is provided as a plasmid DNA, a linear double-stranded DNA, a single-stranded oligonucleotide or viral genomic DNA, as described above, e.g., in FIGS. 1-4. The donor template contains one or more copies of a nucleotide sequence that can be bound by the sequence-specific DNA binding domain that is fused to the Cas9 polypeptide. The Cas9 fusion protein (or control Cas9), gRNAs and donor template are introduced into the cells by methods known in the art, such as viral transduction, non-viral transfection or electroporation.

At various time points, cells are harvested in order to assess the rate of nucleic acid modification by HDR. This is achieved by 1) isolating genomic DNA from the Cas9 control and Cas9 fusion protein treated cells; 2) PCR amplifying the DNA encompassing the region targeted for modification; 3) sequencing the amplified DNA products; and 4) determining the frequency of HDR-mediated alteration by dividing the number of sequence reads containing the donor template-specified sequence by the total number of sequence reads comprising the targeted region. Treatment of cells with a Cas9 fusion protein is shown to increase the rate of HDR-mediated nucleic acid modification.

Example 2: Enhancing HDR-Mediated Alteration of a Target Nucleic Acid in a Tissue of an Animal Using a Cas9 Fusion Protein

This study demonstrates an increased rate of HDR-mediated nucleic acid modification at a target site in a tissue of an animal by administering to the animal a Cas9 fusion protein, one or more gRNAs, and a nucleic acid template system, e.g., an HDR donor nucleic acid template. The Cas9 fusion protein is a Cas9 protein fused to a polypeptide comprising the DNA binding domain derived from a sequence-specific DNA binding protein, e.g. one of the DNA binding proteins listed in Tables V.1-V.4 of Section V, above. The Cas9 fusion protein (or, in a control cohort of animals, Cas9 protein lacking the fusion domain) is administered as a DNA expression vector, an mRNA or a protein. The one or more gRNAs are administered as DNA expression vectors or RNA molecules. The donor template is provided as a plasmid DNA, a linear double-stranded DNA, a single-stranded oligonucleotide or viral genomic DNA, as described above, e.g., in FIGS. 1-4. The donor template contains one or more copies of a nucleotide sequence that can be bound by the sequence-specific DNA binding domain that is fused to the Cas9 polypeptide. The Cas9 fusion protein (or control Cas9), gRNAs and nucleic acid template system are introduced into the animal by local or systemic administration of viral vectors or non-viral delivery vehicles.

At various time points, tissues are harvested in order to assess the rate of nucleic acid modification by HDR. This is achieved by 1) isolating genomic DNA from the Cas9 control and Cas9 fusion protein treated cells; 2) PCR amplifying the DNA encompassing the region targeted for modification; 3) sequencing the amplified DNA products; and 4) determining the frequency of HDR-mediated alteration by dividing the number of sequence reads containing the donor template-specified sequence by the total number of sequence reads comprising the targeted region. Treatment of cells with a Cas9 fusion protein is shown to increase the rate of HDR-mediated nucleic acid modification.

Example 3: Constructing a Cas9 Fusion Protein

To generate a Cas9 fusion protein, established molecular biology techniques are used to ligate a nucleotide sequence encoding a DNA binding domain derived from a sequence-specific DNA binding protein, e.g., one of the DNA binding proteins listed in Tables V.1-V.4 of Section V, above, in-frame to a nucleotide sequence encoding a Cas9 protein. A nucleotide sequence encoding a linker peptide may be inserted in-frame between the nucleotide sequences encoding the Cas9 protein and the DNA binding domain. For expression in bacteria, cultured cells, or animal tissues, the nucleotide sequence encoding the Cas9-DNA binding domain fusion molecule is operably linked to one or more transcriptional control elements, e.g., promoter and/or enhancer elements, which enable expression in the relevant bacteria, cultured cells, or animal tissue. The Cas9 fusion protein can be purified from the bacteria, cultured cells, or animal tissue using established biochemical techniques. To generate mRNA encoding the Cas9 fusion protein, the nucleotide sequence encoding the Cas9-DNA binding domain fusion is operably linked to a promoter, e.g., a bacteriophage promoter, e.g., a T7 RNA polymerase promoter enabling in vitro transcription of mRNA encoding the Cas9-DNA binding domain fusion molecule.

Example 4: Optimization of the Tet Repressor Binding Domain

In this study, the Tet Repressor (TetR) Operon system was selected the optimal composition of the template binding domain partner assessed to achieve strong binding and ensure sufficient length of a locus-specific DNA donor.

The nucleotide sequence to which the Tet Repressor protein binds, typically consists of several repeats (5 or more) of the Tet Operator (TetO)19 base pair sequence, each followed by a 17 bp spacer. Here, we evaluated the binding of the Tet repressor protein to several Tet Operator sequence-containing variants that included spacer sequences of differing sizes, or no spacer sequences. Specifically, we investigated whether only 3 repeats of the Tet Operator sequence were sufficient for binding of the Tet repressor protein, and whether shrinking the spacer sequence would still allow for efficient Tet repressor protein binding. The conditions tested (including the sequences) are indicated in Table 8, and visually depicted in FIG. 8A.

TABLE 8
Template		SEQ ID
DNA ID	Description	NO	Sequence
ssODN-179	ssODN-179	162	TGCTTCTGACACAACTGTGTTCACTAGCAA
(control)			CCTCAAACAGACACCATGGTGCATCTGACT
			CGTGTGGAGAAGTCGGCCGTTACTGCCCTG
			CAGGGCAAGCTTAACGTGGATGAAGTTCGT
			GGTGAGGCCCTGGGCAGGTTGGTATCAAGG
			TTACAAGACAGGTTTAAGGAGACCAATAG
ssODN-109	ssODN-109	163	AACAGACACCATGGTGCATCTGACTCGTGT
(control)			GGAGAAGTCGGCCGTTACTGCCCTGCAGGG
			CAAGCTTAACGTGGATGAAGTTCGTGGTGA
			GGCCCTGGGCAGGTTGGTA
V1.1	(3xTetO, 17 bp	164	TCCCTATCAGTGATAGAGAACGTATGTCGA
	spacer)-ssODN-109		GTTTACTCCCTATCAGTGATAGAGAACGTA
			TGTCGAGTTTACTCCCTATCAGTGATAGAG
			AAACAGACACCATGGTGCATCTGACTCGTG
			TGGAGAAGTCGGCCGTTACTGCCCTGCAGG
			GCAAGCTTAACGTGGATGAAGTTCGTGGTG
			AGGCCCTGGGCAGGTTGGTA
V1.2	ssODN-109-(3xTetO,	165	AACAGACACCATGGTGCATCTGACTCGTGT
	17 bp spacer)		GGAGAAGTCGGCCGTTACTGCCCTGCAGGG
			CAAGCTTAACGTGGATGAAGTTCGTGGTGA
			GGCCCTGGGCAGGTTGGTATCCCTATCAGT
			GATAGAGAACGTATGTCGAGTTTACTCCCT
			ATCAGTGATAGAGAACGTATGTCGAGTTTA
			CTCCCTATCAGTGATAGAGA
V2	(3xTetO, 8 bp	166	TCCCTATCAGTGATAGAGAACGTATGTTCC
	spacer)-ssODN-109		CTATCAGTGATAGAGAACGTATGTTCCCTA
			TCAGTGATAGAGAACGTATGTAACAGACAC
			CATGGTGCATCTGACTCGTGTGGAGAAGTC
			GGCCGTTACTGCCCTGCAGGGCAAGCTTAA
			CGTGGATGAAGTTCGTGGTGAGGCCCTGGG
			CAGGTTGGTA
V3	(3xTetO, no spacer)-	167	TCCCTATCAGTGATAGAGATCCCTATCAGT
	ssODN-109		GATAGAGATCCCTATCAGTGATAGAGAAAC
			AGACACCATGGTGCATCTGACTCGTGTGGA
			GAAGTCGGCCGTTACTGCCCTGCAGGGCAA
			GCTTAACGTGGATGAAGTTCGTGGTGAGGC
			CCTGGGCAGGTTGGTA

To test the binding capacity of the Tet Operator sequence-containing variants experimentally, 500 nM of recombinant TetR protein (Imgen BioSciences, Inc.; Cat. No. P-1002-0.5 mg) was incubated with 50 nM of template DNA in binding buffer (50 mM HEPES, 20% glycerol, 300 mM NaCl, pH 7.7) and protein binding to the Tet Operator sequence-containing variants was analyzed using an electrophoretic mobility shift assay using a 10% polyacrylamide gel (FIG. 8B).

As expected, no gel shift was observed for control templates lacking the Tet operator sequence (ssODN-179, ssODN109). In contrast, complete gel shift (indicating binding of Tet repressor protein to the Tet Operator sequence) was observed for templates V1.1, V1.2, and V2. Moreover, partial gel shift was observed for template V3, indicating some, but not complete, binding of TetR protein. These data indicate that Tet Operator sequences separated by a shorter 8 bp spacer are indeed functional, and that three Tet Operator repeats are sufficient for Tet Repressor protein binding to occur. Based on these results, novel templates, with shortened spacer sequences (8 bp spacer sequences), were designed, which allowed for increased donor template sequence length (by 10 bp, see V4.1 and V4.2), or for the inclusion of an additional Tet Operator sequence attached to the donor template sequence (V5.1 and V5.2), as shown in Table 9 and FIG. 9.

Based on these findings, Cas9 fusion molecules comprising a Cas9 molecule, e.g., wild type Cas9 molecule, linked to a Tet Repressor molecule, may be generated using established molecular biology techniques. In addition, a nucleic acid template system comprising a template binding domain partner comprising, e.g., three Tet Operator sequences, organized in one or more configurations described above, and a template nucleic acid, may also be generated. U20S cells may be electroporated with 200 ng of gRNA targeting the HBB endogenous locus (gRNA #8 (GTAACGGCAGACTTCTCCTC)(SEQ ID NO: 168) and gRNA #15 (AAGGTGAACGTGGATGAAGT) (SEQ ID NO: 169)), 750 ng of plasmid encoding a Cas9 fusion molecule, comprising, e.g., a wild type Cas9 or mutant Cas9 (D10A or N863A) fused to wild type Tet Repressor molecule, or to a mutant Tet Repressor molecule as a negative control. Simultaneously, the cells may also be electroporated with 25 pmol of the aforementioned optimized single stranded oligo (SSODN). Cells are collected 6 days after electroporation and genomic DNA extracted. PCR amplification of the HBB locus is performed and amplicons are subcloned into Topo Blunt Vector. For each condition, 96 colonies are sequenced using Sanger sequencing and homology-directed repair efficacy is assessed.

TABLE 9
Template ID	Description	SEQ ID NO	Sequence
V4.1	3xTetO (8bp spacer)-	170	TCCCTATCAGTGATAGAGAACGTATGTTCCC
	ssODN-119		TATCAGTGATAGAGAACGTATGTTCCCTATC
			AGTGATAGAGAACGTATGTCCTCAAACAGA
			CACCATGGTGCATCTGACTCGTGTGGAGAAG
			TCGGCCGTTACTGCCCTGCAGGGCAAGCTTA
			ACGTGGATGAAGTTCGTGGTGAGGCCCTGG
			GCAGGTTGGTATCAAG
V4.2	ssODN-119-3xTetO	171	CCTCAAACAGACACCATGGTGCATCTGACTC
	(8bp spacer)		GTGTGGAGAAGTCGGCCGTTACTGCCCTGCA
			GGGCAAGCTTAACGTGGATGAAGTTCGTGG
			TGAGGCCCTGGGCAGGTTGGTATCAAGTCCC
			TATCAGTGATAGAGAACGTATGTTCCCTATC
			AGTGATAGAGAACGTATGTTCCCTATCAGTG
			ATAGAGAACGTATGT
V5.1	(4xTetO (8bp spacer)-	172	TCCCTATCAGTGATAGAGAACGTATGTTCCC
	ssODN-100		TATCAGTGATAGAGAACGTATGTTCCCTATC
			AGTGATAGAGAACGTATGTTCCCTATCAGTG
			ATAGAGAACACCATGGTGCATCTGACTCGTG
			TGGAGAAGTCGGCCGTTACTGCCCTGCAGG
			GCAAGCTTAACGTGGATGAAGTTCGTGGTG
			AGGCCCTGGGCAGGT
V5.2	ssODN-100-4xTetO	173	ACACCATGGTGCATCTGACTCGTGTGGAGAA
	(8bp spacer)		GTCGGCCGTTACTGCCCTGCAGGGCAAGCTT
			AACGTGGATGAAGTTCGTGGTGAGGCCCTG
			GGCAGGTTCCCTATCAGTGATAGAGAACGT
			ATGTTCCCTATCAGTGATAGAGAACGTATGT
			TCCCTATCAGTGATAGAGAACGTATGTTCCC
			TATCAGTGATAGAGA

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Other embodiments are within the following claims.

Claims

1. A Cas9 system comprising a Cas9 fusion molecule comprising a Cas9 molecule linked to a template binding domain, and a nucleic acid template system comprising a template binding domain partner and a template nucleic acid,wherein the template binding domain comprises an affinity for the template binding domain partner and does not bind an endogenous nucleic acid of a cell, wherein the template binding domain partner is exogenous to the cell,wherein the template binding domain is selected from the group consisting of a TetR repressor protein, or a fragment thereof; a LacI repressor protein, or a fragment thereof; a Gal4 repressor protein, or a fragment thereof; and C1 repressor protein, or a fragment thereof; andwherein the template binding domain partner is a DNA sequence recognized by a DNA binding protein selected from the group consisting of a Tet-O sequence, a Lac operon O1 sequence, a UAS sequence, and an Operator L and R sequence.

2. The Cas9 system of claim 1, wherein the template binding domain of the Cas9 fusion molecule is bound to the template binding domain partner.

3. The Cas9 system of claim 2, wherein the template binding domain of the Cas9 fusion molecule is covalently or non-covalently bound to the template binding domain partner.

4. The Cas9 system of claim 1, wherein the template binding domain partner is linked to the template nucleic acid.

5. The Cas9 system of claim 1, wherein the DNA binding protein comprises a TetR repressor, or a fragment of the TetR repressor, and the DNA comprises at least one Tet-O sequence.

6. The Cas9 system of claim 1, wherein the nucleic acid template system comprises a double stranded nucleic acid sequence or a single stranded nucleic acid sequence.

7. The Cas9 system of claim 1, wherein the template nucleic acid comprises about 50-500 nucleotides of homology with a target nucleic acid.

8. The Cas9 system of claim 1, further comprising a gRNA.

9. A cell, or a population of cells, comprising the Cas9 system of claim 1.

10. An in vitro or ex vivo method of altering a nucleic acid at a target position in a cell, or a population of cells, the method comprising contacting the cell or the population of cells with the Cas9 system of claim 8, wherein the Cas9 molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-89, wherein the gRNA molecule and Cas9 fusion molecule interact with the nucleic acid, resulting in a cleavage event, wherein the cleavage event is repaired by at least one DNA repair pathway, andwherein the sequence of the nucleic acid after the cleavage event is different than the sequence of the nucleic acid prior to the cleavage event,thereby altering the nucleic acid at the target position in the cell, or in the population of cells.

11. The method of claim 10, further comprising contacting the cell, or the population of cells, with a second gRNA molecule, wherein the second gRNA molecule and the Cas9 fusion molecule interact with the nucleic acid, resulting in a second cleavage event.

12. The method of claim 10, wherein the cell, or population of cells, is from a subject suffering from a disease or disorder selected from the group consisting of a blood disease, an immune disease, a neurological disease, a cancer, an infectious disease, a genetic disease, a disorder caused by aberrant mtDNA, a metabolic disease, a disorder caused by aberrant cell cycle, a disorder caused by aberrant angiogenesis, a disorder cause by aberrant DNA damage repair, or a pain disorder.

13. The method of claim 10, wherein the cell, or population of cells, is from a subject having at least one mutation at the target position.

14. A cell, or a population of cells, altered by the method of claim 10.

15. A pharmaceutical composition comprising the cell, or the population of cells, of claim 14.

16. A pharmaceutical composition comprising the Cas9 system of claim 8.

17. An in vitro or ex vivo method of treating a subject suffering from a disease or disorder, the method comprising contacting a cell, or a population of cells, from the subject with the Cas9 system of claim 8, wherein the Cas9 molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-89, wherein the gRNA molecule and the Cas9 fusion molecule interact with a nucleic acid at a target position, resulting in a cleavage event,wherein the cleavage event is repaired by at least one DNA repair pathway, andwherein the sequence of the nucleic acid after the cleavage event is different than the sequence of the nucleic acid prior to the cleavage event,thereby treating the subject suffering from the disease or disorder.