Patent Application
20240376463
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 16, 2024, is named “Sequence_Listing_010498_01620_ST26” and is 102 KB in size.
The CRISPR type II system is a recent development that has been efficiently utilized in a broad spectrum of species. See Friedland, A. E., et al., Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods, 2013. 10(8): p. 741-3, Mali, P, et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6, Hwang, W. Y., et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013, Jiang, W., et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 2013, Jinek, M., et al., RNA-programmed genome editing in human cells. elife, 2013. 2: p. e00471, Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23, Yin, H., et al., Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol, 2014. 32(6): p. 551-3. CRISPR is particularly customizable because the active form consists of an invariant Cas9 protein and an easily programmable guide RNA (gRNA). See Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21. Of the various CRISPR orthologs, the Streptococcus pyogenes (Sp) CRISPR Cas9 is the most well-characterized and widely used. The Cas9-gRNA complex first probes DNA for the protospacer-adjacent motif (PAM) sequence (-NGG for Sp Cas9, or 5′-NRG with a cut site of 2-4 bp 5′ of the PAM, although predominantly 3 bp 5′ of the PAM) after which Watson-Crick base-pairing between the gRNA and target DNA proceeds in a ratchet mechanism to form an R-loop. Following formation of a ternary complex of Cas9, gRNA, and target DNA, the Cas9 protein generates two nicks in the target DNA, creating a blunt double-strand break (DSB) that is predominantly repaired by the non-homologous end joining (NHEJ) pathway or, to a lesser extent, template-directed homologous recombination (HR). It is through the DNA repair following induction of DSBs that enables generation of genetic modifications at defined genomic loci.
Aspects of the present disclosure are directed to the use of split Cas9 to perform CRISPR-based methods in cells. According to one aspect, two or more portions or segments of a Cas9 are provided to a cell, such as by being expressed from corresponding nucleic acids introduced into the cell. The two or more portions are combined within the cell to form the Cas9 which has an ability to colocalize with guide RNA at a target nucleic acid. It is to be understood that the Cas9 may have one or more modifications from a full length Cas9 known to those of skill in the art, yet still retain or have the capability of colocalizing with guide RNA at a target nucleic acid. Accordingly, the two or more portions or segments, when joined together, need only produce or result in a Cas9 which has an ability to colocalize with guide RNA at a target nucleic acid.
According to certain general aspects, when a foreign nucleic acid sequence or sequences are expressed by the cell, the two or more portions or segments of an RNA guided DNA binding protein, such as Cas9, are produced and joined together to produce the RNA guided DNA binding protein, such as Cas9. When a foreign nucleic acid sequence or sequences are expressed by the cell, one or more or a plurality of guide RNAs are produced. The RNA guided DNA binding protein, such as Cas9, and a guide RNA produces a complex of the RNA guided DNA binding protein, the guide RNA and a double stranded DNA target sequence. In this aspect, the RNA is said to guide the DNA binding protein to the double stranded DNA target sequence for binding thereto. This aspect of the present disclosure may be referred to as co-localization of the RNA and DNA binding protein to or with the double stranded DNA.
DNA binding proteins within the scope of the present disclosure may include those which create a double stranded break (which may be referred to as a DNA binding protein nuclease), those which create a single stranded break (referred to as a DNA binding protein nickase) or those which have no nuclease activity (referred to as a nuclease null DNA binding protein) but otherwise bind to target DNA. In this manner, a DNA binding protein-guide RNA complex may be used to create a double stranded break at a target DNA site, to create a single stranded break at a target DNA site or to localize a transcriptional regulator or function-conferring protein or domain, which may be expressed by the cell, at a target DNA site so as to regulate expression of target DNA. According to certain aspects, the foreign nucleic acid sequence may encode one or more of a DNA binding protein nuclease, a DNA binding protein nickase or a nuclease null DNA binding protein. The foreign nucleic acid sequence may also encode one or more transcriptional regulator or function-conferring proteins or domains or one or more donor nucleic acid sequences that are intended to be inserted into the genomic DNA. According to one aspect, the foreign nucleic acid sequence encoding an RNA guided nuclease-null DNA binding protein further encodes the transcriptional regulator or function-conferring protein or domain fused to the RNA guided nuclease-null DNA binding protein. According to one aspect, the foreign nucleic acid sequence encoding one or more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator or function-conferring protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator or function-conferring protein or domain.
Accordingly, expression of a foreign nucleic acid sequence by a germline cell may result in a double stranded break, a single stranded break and/or transcriptional activation or repression of the genomic DNA. Donor DNA may be inserted at the break site by cell mechanisms such as homologous recombination or nonhomologous end joining. It is to be understood that expression of a foreign nucleic acid sequence as described herein may result in a plurality of double stranded breaks or single stranded breaks at various locations along target genomic DNA, including one or more or a plurality of gene sequences, as desired.
Aspects of the present disclosure are directed a method of providing a cell with a Cas9 protein including providing to the cell one or more foreign nucleic acids encoding a plurality of separate portions or segments of the Cas9 protein, wherein the cell expresses the one or more foreign nucleic acids to produce the plurality of separate portions or segments of the Cas9 protein, and wherein the plurality of separate portions or segments of the Cas9 protein are joined together to form the Cas9 protein active to colocalize with guide RNA to a target nucleic acid.
According to one aspect, the one or more foreign nucleic acids are delivered to the cell by separate vectors. According to one aspect, the one or more foreign nucleic acids are delivered to the cell by separate plasmids or adeno-associated viruses. According to one aspect, the Cas9 is a Type II CRISPR system Cas9. According to one aspect, the plurality of separate portions or segments are connected or joined together by linker pairs. According to one aspect, the plurality of separate portions or segments are connected or joined together by split-intein pairs. According to one aspect, the cell is a eukaryotic cell or prokaryotic cell. According to one aspect, the cell is a bacteria cell, a yeast cell, a mammalian cell, a plant cell or an animal cell. According to one aspect, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein nickase or a nuclease null Cas9 protein (“Cas9Nuc”).
Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
Embodiments of the present disclosure are directed to a method of providing a cell with a Cas9 protein comprising providing to the cell a first nucleic acid encoding a first portion of the Cas9 protein and a second nucleic acid encoding a second portion of the Cas9 protein, wherein the cell expresses the first nucleic acid encoding the first portion of the Cas9 protein, wherein the cell expresses the second nucleic acid encoding the second portion of the Cas9 protein, wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein. According to one aspect, the first nucleic acid and the second nucleic acid are delivered to the cell by separate vectors. According to one aspect, the first nucleic acid is delivered to the cell by a plasmid or adeno-associated virus. According to one aspect, the second nucleic acid is delivered to the cell by a plasmid or an adeno-associated virus. According to one aspect, the Cas9 is a Type II CRISPR system Cas9.
According to one aspect, the first nucleic acid encodes a first portion of the Cas9 protein having a first split-intein and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a second split-intein complementary to the first split-intein and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
According to one aspect, the first nucleic acid encodes a first portion of the Cas9 protein having a Rhodothermus marinus N-split-intein RmaIntN and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a Rhodothermus marinus C-split-intein RmaIntC and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
According to one aspect, the first portion of the Cas9 protein is the N-terminal lobe of the Cas9 protein and the second portion of the Cas9 protein is the C-terminal lobe of the Cas9 protein.
According to one aspect, the first portion of the Cas9 protein is the N-terminal lobe of the Cas9 protein up to amino acid V713 and the second portion of the Cas9 protein is the C-terminal lobe of the Cas9 protein beginning at D714.
According to one aspect, the cell is a eukaryotic cell or a prokaryotic cell. According to one aspect, the cell is a bacteria cell, a yeast cell, a mammalian cell, a plant cell or an animal cell.
According to one aspect, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein nickase or a nuclease null Cas9 protein.
Embodiments of the present disclosure are directed to a method of altering a target nucleic acid in a eukaryotic cell comprising providing to the cell a first nucleic acid encoding a first portion of a Cas9 protein and a second nucleic acid encoding a second portion of the Cas9 protein, providing to the cell a third nucleic acid encoding RNA complementary to the target nucleic acid, wherein the cell expresses the RNA, the first portion of the Cas9 protein and the second portion of the Cas9 protein, wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein, wherein the RNA and the Cas9 protein form a co-localization complex with the target nucleic acid.
According to one aspect, the Cas9 protein is enzymatically active and the enzymatically active Cas9 protein cleaves the target nucleic acid in a site specific manner.
According to one aspect, the first nucleic acid and the second nucleic acid are delivered to the cell by separate vectors.
According to one aspect, the first nucleic acid is delivered to the cell by a plasmid or adeno-associated virus.
According to one aspect, the second nucleic acid is delivered to the cell by a plasmid or an adeno-associated virus.
According to one aspect, the Cas9 is a Type II CRISPR system Cas9.
According to one aspect, the first nucleic acid encodes a first portion of the Cas9 protein having a first split-intein and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a second split-intein complementary to the first split-intein and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
According to one aspect, the first nucleic acid encodes a first portion of the Cas9 protein having a N-split-intein RmaIntN and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a C-split-intein RmaIntC and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
According to one aspect, the first portion of the Cas9 protein is the N-terminal lobe of the Cas9 protein and the second portion of the Cas9 protein is the C-terminal lobe of the Cas9 protein.
According to one aspect, the first portion of the Cas9 protein is the N-terminal lobe of the Cas9 protein up to amino acid V713 and the second portion of the Cas9 protein is the C-terminal lobe of the Cas9 protein beginning at D714.
According to another aspect, the split Cas9 can also be from other Cas9 orthologs to include SpCas9, AnCas9, and SaCas9. All three orthologs have a bi-lobed structure. This consistent feature is likely present in other Cas9 orthologs with yet undetermined structures. See Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935-949 (2014), Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014), Nishimasu, H. et al. Crystal Structure of Staphylococcus aureus Cas9. Cell 162, 1113-1126 (2015).
According to one aspect, the cell is a eukaryotic cell or a prokaryotic cell. According to one aspect, the cell is a bacteria cell, a yeast cell, a mammalian cell, a plant cell or an animal cell.
According to one aspect, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein nickase or a nuclease null Cas9 protein.
Embodiments of the present disclosure are directed to a cell comprising a first foreign nucleic acid encoding a first portion of a Cas9 protein and a second nucleic acid encoding a second portion of the Cas9 protein, and a third foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes a target nucleic acid, wherein the one or more RNAs, the Cas9 protein are members of a co-localization complex for the target nucleic acid.
Embodiments of the present disclosure are directed to a method of delivering a Cas9 protein to cells within a subject comprising administering to the subject, such as systemically administering to the subject, such as by intravenous administration or injection, intraperitoneal administration or injection, intramuscular administration or injection, intracranial administration or injection, intraocular administration or injection, subcutaneous administration or injection, a first nucleic acid encoding a first portion of the Cas9 protein wherein the first nucleic acid is within a first vector and intravenously administering to the subject a second nucleic acid encoding a second portion of the Cas9 protein wherein the second nucleic acid is within a second vector, wherein the first vector delivers the first nucleic acid to a cell and wherein the second vector delivers the second nucleic acid to the cell, wherein the cell expresses the first nucleic acid encoding the first portion of the Cas9 protein, wherein the cell expresses the second nucleic acid encoding the second portion of the Cas9 protein, wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
According to one aspect, the first vector is a plasmid or adeno-associated virus.
According to one aspect, the second vector is a plasmid or adeno-associated virus.
According to one aspect, the Cas9 is a Type II CRISPR system Cas9.
According to one aspect, the first nucleic acid encodes a first portion of the Cas9 protein having a first split-intein and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a second split-intein complementary to the first split-intein and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
According to one aspect, the first nucleic acid encodes a first portion of the Cas9 protein having a N-split-intein RmaIntN and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a C-split-intein RmaIntC and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
According to one aspect, the first portion of the Cas9 protein is the N-terminal lobe of the Cas9 protein and the second portion of the Cas9 protein is the C-terminal lobe of the Cas9 protein.
According to one aspect, the first portion of the Cas9 protein is the N-terminal lobe of the Cas9 protein up to amino acid V713 and the second portion of the Cas9 protein is the C-terminal lobe of the Cas9 protein beginning at D714.
According to one aspect, the cell is a eukaryotic cell. According to one aspect, the cell is a mammalian cell.
According to one aspect, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein nickase or a nuclease null Cas9 protein.
The split Cas9 methods described herein are useful in CRISPR-related methods where Cas9 and a guide RNA are used to colocalize the Cas9 and the guide RNA to a target nucleic acid sequence.
Accordingly, embodiments of the present disclosure are based on the use of RNA guided DNA binding proteins, such as Cas9, to co-localize with guide RNA at a target DNA site. Such DNA binding proteins are readily known to those of skill in the art to bind to DNA for various purposes. Such DNA binding proteins may be naturally occurring. DNA binding proteins included within the scope of the present disclosure include those which may be guided by RNA, referred to herein as guide RNA. According to one aspect, the guide RNA is between about 10 to about 500 nucleotides. According to one aspect, the RNA is between about 20 to about 100 nucleotides. According to this aspect, the guide RNA and the RNA guided DNA binding protein form a co-localization complex at the DNA.
DNA binding proteins having nuclease activity are known to those of skill in the art, and include naturally occurring DNA binding proteins having nuclease activity, such as Cas9 proteins present, for example, in Type II CRISPR systems. Such Cas9 proteins and Type II CRISPR systems are well documented in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477 including all supplementary information hereby incorporated by reference in its entirety. Exemplary Cas include S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9) and S. thermophilus Cas9 (StCas9).
Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading foreign nucleic acid1. See Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012); Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic acids research 39, 9275-9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annual review of genetics 45, 273-297 (2011). A recent in vitro reconstitution of the S. pyogenes type II CRISPR system demonstrated that crRNA (“CRISPR RNA”) fused to a normally trans-encoded tracrRNA (“trans-activating CRISPR RNA”) is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA. Expressing a gRNA homologous to a target site results in Cas9 recruitment and degradation of the target DNA. See H. Deveau et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (February, 2008).
Three classes of CRISPR systems are generally known and are referred to as Type I, Type II or Type III). According to one aspect, a particular useful enzyme according to the present disclosure to cleave dsDNA is the single effector enzyme, Cas9, common to Type II. See K. S. Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nature reviews. Microbiology 9, 467 (June, 2011) hereby incorporated by reference in its entirety. Within bacteria, the Type II effector system consists of a long pre-crRNA transcribed from the spacer-containing CRISPR locus, the multifunctional Cas9 protein, and a tracrRNA important for gRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, initiating dsRNA cleavage by endogenous RNase III, which is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9. TracrRNA-crRNA fusions are contemplated for use in the present methods.
According to one aspect, the enzyme of the present disclosure, such as Cas9 unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a “protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Importantly, Cas9 cuts the DNA only if a correct protospacer-adjacent motif (PAM) is also present at the 3′ end. According to certain aspects, different protospacer-adjacent motif can be utilized. For example, the S. pyogenes system requires an NGG sequence, where N can be any nucleotide. S. thermophilus Type II systems require NGGNG (see P. Horvath, R. Barrangou, CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167 (Jan. 8, 2010) hereby incorporated by reference in its entirety and NNAGAAW (see H. Deveau et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of bacteriology 190, 1390 (February, 2008) hereby incorporatd by reference in its entirety), respectively, while different S. mutans systems tolerate NGG or NAAR (see J. R. van der Ploeg, Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology 155, 1966 (June, 2009) hereby incorporated by refernece in its entirety. Bioinformatic analyses have generated extensive databases of CRISPR loci in a variety of bacteria that may serve to identify additional useful PAMs and expand the set of CRISPR-targetable sequences (see M. Rho, Y. W. Wu, H. Tang, T. G. Doak, Y. Ye, Diverse CRISPRs evolving in human microbiomes. PLoS genetics 8, e1002441 (2012) and D. T. Pride et al., Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time. Genome research 21, 126 (January, 2011) each of which are hereby incorporated by reference in their entireties.
Exemplary DNA binding proteins having nuclease activity function to nick or cut double stranded DNA. Such nuclease activity may result from the DNA binding protein having one or more polypeptide sequences exhibiting nuclease activity. Such exemplary DNA binding proteins may have two separate nuclease domains with each domain responsible for cutting or nicking a particular strand of the double stranded DNA. Exemplary polypeptide sequences having nuclease activity known to those of skill in the art include the McrA-HNH nuclease related domain and the RuvC-like nuclease domain. Accordingly, exemplary DNA binding proteins are those that in nature contain one or more of the McrA-HNH nuclease related domain and the RuvC-like nuclease domain.
In S. pyogenes, Cas9 generates a blunt-ended double-stranded break 3 bp upstream of the protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand. See Jinek et al., Science 337, 816-821 (2012) hereby incorporated by reference in its entirety. Cas9 proteins are known to exist in many Type II CRISPR systems including the following as identified in the supplementary information to Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus maripaludis C7; Corynebacterium diphtheriae; Corynebacterium efficiens YS-314; Corynebacterium glutamicum ATCC 13032 Kitasato; Corynebacterium glutamicum ATCC 13032 Bielefeld; Corynebacterium glutamicum R; Corynebacterium kroppenstedtii DSM 44385; Mycobacterium abscessus ATCC 19977; Nocardia farcinica IFM10152; Rhodococcus erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus opacus B4 uid36573; Acidothermus cellulolyticus 11B; Arthrobacter chlorophenolicus A6; Kribbella flavida DSM 17836 uid43465; Thermomonospora curvata DSM 43183; Bifidobacterium dentium Bdl; Bifidobacterium longum DJO10A; Slackia heliotrinireducens DSM 20476; Persephonella marina EX H1; Bacteroides fragilis NCTC 9434; Capnocytophaga ochracea DSM 7271; Flavobacterium psychrophilum JIP02 86; Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM 13941; Roseiflexus RS1; Synechocystis PCC6803; Elusimicrobium minutum Peil91; uncultured Termite group 1 bacterium phylotype Rs D17; Fibrobacter succinogenes S85; Bacillus cereus ATCC 10987; Listeria innocua; Lactobacillus casei; Lactobacillus rhamnosus GG; Lactobacillus salivarius UCC118; Streptococcus agalactiae A909; Streptococcus agalactiae NEM316; Streptococcus agalactiae 2603; Streptococcus dysgalactiae equisimilis GGS 124; Streptococcus equi zooepidemicus MGCS10565; Streptococcus gallolyticus UCN34 uid46061; Streptococcus gordonii Challis subst CH1; Streptococcus mutans NN2025 uid46353; Streptococcus mutans; Streptococcus pyogenes M1 GAS; Streptococcus pyogenes MGAS5005; Streptococcus pyogenes MGAS2096; Streptococcus pyogenes MGAS9429; Streptococcus pyogenes MGAS10270; Streptococcus pyogenes MGAS6180; Streptococcus pyogenes MGAS315; Streptococcus pyogenes SSI-1; Streptococcus pyogenes MGAS10750; Streptococcus pyogenes NZ131; Streptococcus thermophiles CNRZ1066; Streptococcus thermophiles LMD-9; Streptococcus thermophiles LMG 18311; Clostridium botulinum A3 Loch Maree; Clostridium botulinum B Eklund 17B; Clostridium botulinum Ba4 657; Clostridium botulinum F Langeland; Clostridium cellulolyticum H10; Finegoldia magna ATCC 29328; Eubacterium rectale ATCC 33656; Mycoplasma gallisepticum; Mycoplasma mobile 163K; Mycoplasma penetrans; Mycoplasma synoviae 53; Streptobacillus moniliformis DSM 12112; Bradyrhizobium BTAil; Nitrobacter hamburgensis X14; Rhodopseudomonas palustris BisB18; Rhodopseudomonas palustris BisB5; Parvibaculum lavamentivorans DS-1; Dinoroseobacter shibae DFL 12; Gluconacetobacter diazotrophicus Pal 5 FAPERJ; Gluconacetobacter diazotrophicus Pal 5 JGI; Azospirillum B510 uid46085; Rhodospirillum rubrum ATCC 11170; Diaphorobacter TPSY uid29975; Verminephrobacter eiseniae EF01-2; Neisseria meningitides 053442; Neisseria meningitides alpha14; Neisseria meningitides Z2491; Desulfovibrio salexigens DSM 2638; Campylobacter jejuni doylei 269 97; Campylobacter jejuni 81116; Campylobacter jejuni; Campylobacter lari RM2100; Helicobacter hepaticus; Wolinella succinogenes; Tolumonas auensis DSM 9187; Pseudoalteromonas atlantica T6c; Shewanella pealeana ATCC 700345; Legionella pneumophila Paris; Actinobacillus succinogenes 130Z; Pasteurella multocida; Francisella tularensis novicida U112; Francisella tularensis holarctica; Francisella tularensis FSC 198; Francisella tularensis tularensis; Francisella tularensis WY96-3418; and Treponema denticola ATCC 35405. The Cas9 protein may be referred by one of skill in the art in the literature as Csn1. An exemplary S. pyogenes Cas9 protein sequence is shown below. See Deltcheva et al., Nature 471, 602-607 (2011) hereby incorporated b reference in its entirety.
According to one aspect, the specificity of gRNA-directed Cas9 cleavage is used as a mechanism for genome engineering. According to one aspect, hybridization of the gRNA need not be 100 percent in order for the enzyme to recognize the gRNA/DNA hybrid and affect cleavage. Some off-target activity could occur. For example, the S. pyogenes system tolerates mismatches in the first 6 bases out of the 20 bp mature spacer sequence in vitro. According to one aspect, greater stringency may be beneficial in vivo when potential off-target sites matching (last 14 bp) NGG exist within the human reference genome for the gRNAs.
According to certain aspects, specificity may be improved. When interference is sensitive to the melting temperature of the gRNA-DNA hybrid, AT-rich target sequences may have fewer off-target sites. Carefully choosing target sites to avoid pseudo-sites with at least 14 bp matching sequences elsewhere in the genome may improve specificity. The use of a Cas9 variant requiring a longer PAM sequence may reduce the frequency of off-target sites. Directed evolution may improve Cas9 specificity to a level sufficient to completely preclude off-target activity, ideally requiring a perfect 20 bp gRNA match with a minimal PAM. Accordingly, modification to the Cas9 protein is a representative embodiment of the present disclosure. CRISPR systems useful in the present disclosure are described in R. Barrangou, P. Horvath, CRISPR: new horizons in phage resistance and strain identification. Annual review of food science and technology 3, 143 (2012) and B. Wiedenheft, S. H. Sternberg, J. A. Doudna, RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331 (Feb. 16, 2012) each of which are hereby incorporated by reference in their entireties.
Guide RNAs useful in the disclosed methods include those having a spacer sequence, a tracr mate sequence and a tracr sequence, with the spacer sequence being between about 16 to about 20 nucleotides in length and with the tracr sequence being between about 60 to about 500 nucleotides in length and with a portion of the tracr sequence being hybridized to the tracr mate sequence and with the tracr mate sequence and the tracr sequence being linked by a linker nucleic acid sequence of between about 4 to about 6 nucleotides. crRNA-tracrRNA fusions are contemplated as exemplary guide RNA.
According to certain aspects, the DNA binding protein is altered or otherwise modified to inactivate the nuclease activity. Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. Such modification includes removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the DNA binding protein. Other modifications to inactivate nuclease activity will be readily apparent to one of skill in the art based on the present disclosure. Accordingly, a nuclease-null DNA binding protein includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The nuclease-null DNA binding protein retains the ability to bind to DNA even though the nuclease activity has been inactivated. Accordingly, the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease activity. Accordingly, the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may have one or more or all of the nuclease sequences exhibiting nuclease activity inactivated.
According to one aspect, a DNA binding protein having two or more nuclease domains may be modified or altered to inactivate all but one of the nuclease domains. Such a modified or altered DNA binding protein is referred to as a DNA binding protein nickase, to the extent that the DNA binding protein cuts or nicks only one strand of double stranded DNA. When guided by RNA to DNA, the DNA binding protein nickase is referred to as an RNA guided DNA binding protein nickase.
An exemplary DNA binding protein is an RNA guided DNA binding protein nuclease of a Type II CRISPR System, such as a Cas9 protein or modified Cas9 or homolog of Cas9. An exemplary DNA binding protein is a Cas9 protein nickase. An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II CRISPR System which lacks nuclease activity. An exemplary DNA binding protein is a nuclease-null Cas9 protein.
According to certain aspects of methods of RNA-guided genome regulation described herein, Cas9 is altered to reduce, substantially reduce or eliminate nuclease activity. According to one aspect, Cas9 nuclease activity is reduced, substantially reduced or eliminated by altering the RuvC nuclease domain or the HNH nuclease domain. According to one aspect, the RuvC nuclease domain is inactivated. According to one aspect, the HNH nuclease domain is inactivated. According to one aspect, the RuvC nuclease domain and the HNH nuclease domain are inactivated. According to an additional aspect, Cas9 proteins are provided where the RuvC nuclease domain and the HNH nuclease domain are inactivated. According to an additional aspect, nuclease-null Cas9 proteins are provided insofar as the RuvC nuclease domain and the HNH nuclease domain are inactivated. According to an additional aspect, a Cas9 nickase is provided where either the RuvC nuclease domain or the HNH nuclease domain is inactivated, thereby leaving the remaining nuclease domain active for nuclease activity. In this manner, only one strand of the double stranded DNA is cut or nicked.
According to an additional aspect, nuclease-null Cas9 proteins are provided where one or more amino acids in Cas9 are altered or otherwise removed to provide nuclease-null Cas9 proteins. According to one aspect, the amino acids include D10 and H840. See Jinek et al., Science 337, 816-821 (2012). According to an additional aspect, the amino acids include D839 and N863. According to one aspect, one or more or all of D10, H840, D839 and H863 are substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity. According to one aspect, one or more or all of D10, H840, D839 and H863 are substituted with alanine. According to one aspect, a Cas9 protein having one or more or all of D10, H840, D839 and H863 substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity, such as alanine, is referred to as a nuclease-null Cas9 (“Cas9Nuc”) and exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. According to this aspect, nuclease activity for a Cas9Nuc may be undetectable using known assays, i.e. below the level of detection of known assays.
According to one aspect, the Cas9 protein, Cas9 protein nickase or nuclease null Cas9 includes homologs and orthologs thereof which retain the ability of the protein to bind to the DNA and be guided by the RNA. According to one aspect, the Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from S. pyogenes and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA binding protein.
According to one aspect, an engineered Cas9-gRNA system is provided wherein one or more of function-conferring domains, such as FokI heterodimers (see Tsai, S. Q., et al., Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol, 2014. 32(6): p. 569-76, and Guilinger, J. P., D. B. Thompson, and D. R. Liu, Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol, 2014. 32(6): p. 577-82), transcriptional regulators (see Gilbert, L. A., et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013. 154(2): p. 442-51, Mali, P., et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol, 2013. 31(9): p. 833-8, Perez-Pinera, P., et al., RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods, 2013. 10(10): p. 973-6, and Cheng, A. W., et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res, 2013. 23(10): p. 1163-71), fluorescent proteins (see Gilbert, L. A., et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013. 154(2): p. 442-51 and Chen, B., et al., Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell, 2013. 155(7): p. 1479-91), protein-protein interacting-domains (see Tanenbaum, M. E., et al., A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell, 2014. 159(3): p. 635-46), and degradation tags are attached to either the Cas9 protein or the gRNA or both for delivery to a target nucleic acid.
According to one aspect, an engineered Cas9-gRNA system is provided which enables RNA-guided genome regulation in cells by tethering transcriptional activation domains to either a nuclease-null Cas9 or to guide RNAs. According to one aspect of the present disclosure, one or more transcriptional regulatory or function-conferring proteins or domains (such terms are used interchangeably) are joined or otherwise connected to a nuclease-deficient Cas9 or one or more guide RNA (gRNA). The transcriptional regulatory or function-conferring domains correspond to targeted loci. Accordingly, aspects of the present disclosure include methods and materials for localizing transcriptional regulatory or function-conferring domains to targeted loci by fusing, connecting or joining such domains to either Cas9Nuc or to the gRNA. According to certain aspects, methods are provided for regulating endogenous genes using Cas9Nuc, one or more gRNAs and a transcriptional regulatory or function-conferring protein or domain. According to one aspect, an endogenous gene can be any desired gene, referred to herein as a target gene.
According to one aspect, a Cas9Nuc-fusion protein capable of transcriptional activation is provided. According to one aspect, a VP64 activation domain (see Zhang et al., Nature Biotechnology 29, 149-153 (2011) hereby incorporated by reference in its entirety) is joined, fused, connected or otherwise tethered to the C terminus of Cas9Nuc. According to one method, the transcriptional regulatory or function-conferring domain is provided to the site of target genomic DNA by the Cas9N protein. According to one method, a Cas9Nuc fused to a transcriptional regulatory or function-conferring domain is provided within a cell along with one or more guide RNAs. The Cas9Nuc with the transcriptional regulatory or function-conferring domain fused thereto bind at or near target genomic DNA. The one or more guide RNAs bind at or near target genomic DNA. The transcriptional regulatory or function-conferring domain regulates expression of the target gene. According to a specific aspect, a Cas9Nuc-VP64 fusion activated transcription of reporter constructs when combined with gRNAs targeting sequences near the promoter, thereby displaying RNA-guided transcriptional activation.
According to one aspect, a gRNA-fusion protein capable of transcriptional activation is provided. According to one aspect, a VP64 activation domain is joined, fused, connected or otherwise tethered to the gRNA. According to one method, the transcriptional regulatory or function-conferring domain is provided to the site of target genomic DNA by the gRNA. According to one method, a gRNA fused to a transcriptional regulatory or function-conferring domain is provided within a cell along with a Cas9Nuc protein. The Cas9Nuc binds at or near target genomic DNA. The one or more guide RNAs with the transcriptional regulatory or function-conferring protein or domain fused thereto bind at or near target genomic DNA. The transcriptional regulatory or function-conferring domain regulates expression of the target gene. According to a specific aspect, a Cas9Nuc protein and a gRNA fused with a transcriptional regulatory or function-conferring domain activated transcription of reporter constructs, thereby displaying RNA-guided transcriptional activation.
According to one aspect, the transcriptional regulator protein or domain is a transcriptional activator. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or domain is a transcriptional repressor. According to one aspect, the transcriptional regulator protein or domain downregulates expression of the target nucleic acid. Transcriptional activators and transcriptional repressors can be readily identified by one of skill in the art based on the present disclosure.
According to one aspect, two or more guide RNAs are provided with each guide RNA being complementary to an adjacent site in the DNA target nucleic acid. At least one RNA guided DNA binding protein nickase is provided and being guided by the two or more RNAs, wherein the at least one RNA guided DNA binding protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks. According to certain aspects, the two or more adjacent nicks are on the same strand of the double stranded DNA. According to one aspect, the two or more adjacent nicks are on the same strand of the double stranded DNA and result in homologous recombination. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks resulting in nonhomologous end joining. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks resulting in nonhomologous end joining. According to one aspect, the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks resulting in fragmentation of the target nucleic acid thereby preventing expression of the target nucleic acid.
According to certain aspects, binding specificity of the RNA guided DNA binding protein may be increased according to methods described herein. According to one aspect, off-set nicks are used in methods of genome-editing. A large majority of nicks seldom result in NHEJ events, (see Certo et al., Nature Methods 8, 671-676 (2011) hereby incorporated by reference in its entirety) thus minimizing the effects of off-target nicking. In contrast, inducing off-set nicks to generate double stranded breaks (DSBs) is highly effective at inducing gene disruption. According to certain aspects, 5′ overhangs generate more significant NHEJ events as opposed to 3′ overhangs. Similarly, 3′ overhangs favor HR over NHEJ events, although the total number of HR events is significantly lower than when a 5′ overhang is generated. Accordingly, methods are provided for using nicks for homologous recombination and off-set nicks for generating double stranded breaks to minimize the effects of off-target Cas9-gRNA activity.
Target nucleic acids include any nucleic acid sequence to which a co-localization complex as described herein can be useful to either cut, nick or regulate. Target nucleic acids include genes. For purposes of the present disclosure, DNA, such as double stranded DNA, can include the target nucleic acid and a co-localization complex can bind to or otherwise co-localize with the DNA at or adjacent or near the target nucleic acid and in a manner in which the co-localization complex may have a desired effect on the target nucleic acid. Such target nucleic acids can include endogenous (or naturally occurring) nucleic acids and exogenous (or foreign) nucleic acids. One of skill based on the present disclosure will readily be able to identify or design guide RNAs and Cas9 proteins which co-localize to a DNA including a target nucleic acid. One of skill will further be able to identify transcriptional regulator proteins or domains which likewise co-localize to a DNA including a target nucleic acid. DNA includes genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA.
Foreign nucleic acids (i.e. those which are not part of a cell's natural nucleic acid composition) may be introduced into a cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like. One of skill in the art will readily understand and adapt such methods using readily identifiable literature sources.
Transcriptional regulator proteins or domains which are transcriptional activators or transcriptional repressors may be readily identifiable by those skilled in the art based on the present disclosure.
Vectors used to deliver the nucleic acids to cells as described herein include vectors known to those of skill in the art and used for such purposes. Certain exemplary vectors may be plasmids or adeno-associated viruses known to those of skill in the art. AAVs are highly prevalent within the human population (see Gao, G., et al., Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol, 2004. 78(12): p. 6381-8, and Boutin, S., et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther, 2010. 21(6): p. 704-12) and are useful as viral vectors. Many serotypes exist, each with different tropism for tissue types (see Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80), which allows specific tissues to be preferentially targeted with appropriate pseudotyping. Some serotypes, such as serotypes 8, 9, and rh10, transduce the mammalian body. See Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80, Inagaki, K., et al., Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 2006. 14(1): p. 45-53, Keeler, A. M., et al., Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. Mol Ther, 2012. 20(6): p. 1131-8, Gray, S. J., et al., Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011. 19(6): p. 1058-69, Okada, H., et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, and Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65. AAV9 has been demonstrated to cross the blood-brain barrier (see Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65, and Rahim, A. A., et al., Intravenous administration of AAV2/9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system. FASEB J, 2011. 25(10): p. 3505-18) that is inaccessible to many viral vectors and biologics. Certain AAVs have a payload of 4.7-5.0 kb (including viral inverted terminal repeats (ITRs), which are required in cis for viral packaging). See Wu, Z., H. Yang, and P. Colosi, Effect of genome size on AAV vector packaging. Mol Ther, 2010. 18(1): p. 80-6 and Dong, J. Y., P. D. Fan, and R. A. Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 1996. 7(17): p. 2101-12.
Delivery methods commonly used in research, such as lentiviruses, adenoviruses, or nucleic-acid-complexes, exhibit substantial immunogenic and cytotoxic properties, which can further compound the immunogenicity from ectopic transgene-expression. Furthermore, these approaches generally lack the capacity for targeting of specific tissues and for robust full-body delivery. To simultaneously minimize pathological impacts and enable systemic genome editing, CRISPR was delivered via adeno-associated viruses (AAVs). AAVs are prevalent within human populations (see Gao, G., et al., Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol, 2004. 78(12): p. 6381-8), and there have been no established cases of pathology associated with AAV infection, making them one of the most promising vectors currently used in clinical trials. Moreover, tissue-targeting is easily accomplished by pseudotyping to AAV serotypes with suitable tropism. Of particular interest is AAV serotype 9, which robustly transduces multiple cell types in the body (see Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80. and Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65.) and crosses endothelial barriers (e.g. blood-brain barrier, see Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65 and Zhang, H. et al. Several rAAV vectors efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system. Mol Ther, 2011 19(8): p. 1440-1448) that block access by other delivery vectors. Together, AAV and CRISPR present an enticing combination for achieving systemic gene-editing, but a key obstacle has been the limited capacity of AAV for packaging exogenous sequences (<4.7 kb). Dong, J. Y., P. D. Fan, and R. A. Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 1996. 7(17): p. 2101-12. Of the various Cas9 orthologs that have been co-opted for genome engineering (ST1, Nm, Sa) (see Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature, 2015. 520: p. 186-91 and Esvelt, K. M., et al., Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 2013. 10(11): p. 1116-21), Sp Cas9 has a least restrictive PAM and most consistent efficacy, but its size (4.2 kb) makes packaging into AAV challenging, necessitating use of a limited repertoire of compact regulatory elements (<500 bp) (see Swiech, L., et al., In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol, 2014 and Senis, E. et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnology journal, 2014, 9: p. 1402-1412), and precluding the fusion of function-conferring domains. The Cas9 protein was re-engineered to eliminate this obstacle.
According to certain aspects, two or more portions of a Cas9 protein are provided within a cell or are otherwise expressed within a cell and are combined together to form the Cas9 protein. This structure-guided design is essential since splitting Cas9 at ordered protein regions significantly impacts function. See Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935-949 (2014), Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014), Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nature biotechnology 33, 139-142 (2015), Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nature biotechnology 33, 755-760 (2015), Nishimasu, H. et al. Crystal Structure of Staphylococcus aureus Cas9. Cell 162, 1113-1126 (2015), and Fine, E. J. et al. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Scientific reports 5, 10777 (2015).
According to one aspect, two portions of a Cas9 protein are provided within a cell or are otherwise expressed within a cell and are combined together to form the Cas9 protein. The two portions of the Cas9 protein are sufficient in length such that when they are combined into the Cas9 protein, the Cas9 protein has the function of co-localizing at a target nucleic acid with a guide RNA as described above. According to certain aspects, various methods known to those of skill in the art may be used to combine the two or more portions of a Cas9 protein together. Exemplary methods and linkers include split-intein protein trans-splicing for reconstituting the Cas9 protein as is known in the art and as described herein. Other methods include protein-protein interacting domains (see Zakeri, B., et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. Proc Natl Acad Sci USA, 2012. 109(12): p. E690-7 and Fierer, J. O., G. Veggiani, and M. Howarth, SpyLigase peptide-peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture. Proc Natl Acad Sci USA, 2014. 111(13): p. E1176-81) or small molecule dependent interactions. See Los, G. V., et al., HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol, 2008. 3(6): p. 373-82 and Keppler, A., et al., A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol, 2003. 21(1): p. 86-9.
The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims.
U6-driven gRNA plasmids were constructed as previously described. See Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6. AAV plasmid backbone was derived from pZac2.1 (Penn Vector Core), while scAAV plasmid backbone from Addgene #32396 and Addgene #21894. Transgene cassettes were assembled by PCR, IDT gBlocks, and splicing-by-overlap-extension, and inserts were cloned into vector backbones by sticky-end ligation, blunt-end ligation, or Gibson Isothermal Assembly. See Gibson, D. G., et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 2009. 6(5): p. 343-5. Minicircles parental plasmids were cloned in ZYCY10P3S2T, and minicircles were generated as in. See Kay, M. A., C. Y. He, and Z. Y. Chen, A robust system for production of minicircle DNA vectors. Nat Biotechnol, 2010. 28(12): p. 1287-9. AAV plasmids were cloned with Stb13 (Life Technologies). All other plasmids were transformed into DH5α (NEB). All plasmids were verified with Sanger sequencing. Protein transgenes were expressed from ubiquitous hybrid promoters: SMVP promoter (generated by fusing SV40enhancer-CMV-promoter-chimeric intron), CASI promoter (see Balazs, A. B., et al., Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature, 2012. 481(7379): p. 81-4), or CAG promoter. See Matsuda, T. and C. L. Cepko, Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA, 2004. 101(1): p. 16-22. SMVP promoter was derived from pMAXGFP (Lonza).
gRNA Spacer Sequences:
All Sp. gRNAs were expressed under the U6 promoter, and utilized the chimeric gRNA scaffold (underlined) (see Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6):
TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGG
VSQQNFSVWALNPQTYRLERARVSRAFCTGIKPVYRLTTRLGRSIRATAN
HRFLTPQGWKRVDELQPGDYLALPRRIPTAS.
ATACTCTCATTACCCTGGCCGATGGACGACGAGTGCCTATTAGAGAACTG
GTGTCACAGCAGAATTTTTCCGTGTGGGCTCTGAATCCTCAGACTTACCG
CCTGGAGAGGGCTAGAGTGAGTAGAGCTTTCTGTACCGGCATCAAACCTG
TGTACCGCCTCACCACTAGACTGGGGAGATCCATTAGGGCCACTGCCAAC
CACCGATTTCTCACACCTCAGGGCTGGAAACGAGTCGATGAACTCCAGCC
TGGAGATTACCTGGCTCTGCCTAGGAGAATCCCTACTGCCTCCTGA
MAAACPELRQLAQSDVYWDPIVSIEPDGVEEVFDLTVPGPHNFVANDIIA
HNSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI
ATGGCGGCGGCGTGCCCGGAACTGCGTCAGCTGGCGCAGAGCGATGTGTA
TTGGGATCCGATTGTGAGCATTGAACCGGATGGCGTGGAAGAAGTGTTTG
ATCTGACCGTGCCGGGCCCGCATAACTTTGTGGCGAACGATATTATTGCG
CATAACTCTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGC
AAV were packaged via the triple-transfection method. See Zolotukhin, S., et al., Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther, 1999. 6(6): p. 973-85, Grieger, J. C., V. W. Choi, and R. J. Samulski, Production and characterization of adeno-associated viral vectors. Nat Protoc, 2006. 1(3): p. 1412-28, and Lock, M., et al., Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum Gene Ther, 2010. 21(10): p. 1259-71. 293AAV cells (Cell Biolabs) were plated in growth media (DMEM+glutaMAX+pyruvate+10% FBS+1×NEAA) 1-2 days before transfection, so that confluency at transfection is between 70-90%. Media was replaced with fresh pre-warmed growth media before transfection. For each 15-cm dish, 20 ug of pHelper (Cell Biolabs), 10 ug of pRepCap [encoding capsid proteins for AAV-DJ (Cell Biolabs) or AAV9 (Penn Vector Core)], and 10 ug of pAAV were mixed in 500 ul of DMEM, and 200 ug of PEI “MAX” (Polysciences)(40 kDa, 1 mg/ml in H2O, adjusted to pH 7.1) added for PEI:DNA mass ratio of 5:1. The mixture was incubated in the tissue culture hood for 15 mins, and transferred drop-wise to the 293AAV cell media. For large-scale AAV production, HYPERFlask ‘M’ (Corning) were used, and the transfection mixture consisted of 200 ug of pHelper, 100 ug of pRepCap, 100 ug of pAAV, and 2 mg of PEIMAX. The next day post-transfection, media was changed to DMEM+glutamax+pyruvate+2% FBS, and further incubated for 1-2 days. Cells were harvested 48-72 hrs post transfection by scrapping or dissociation with 1×PBS (pH7.2)+5 mM EDTA, and pelleted at 1500 g for 12 mins. Cell pellets were then resuspended in 1-5 ml of lysis buffer (TrisHCl pH7.5+2 mM MgCl+150 mM NaCl), and freeze-thawed 3 times between dry-ice-ethanol bath and 37° C. water bath. Cell debris was clarified via 4000 g for 5 minutes, and supernatant collected. Downstream processing differed depending on applications.
For preparation of AAV-containing lysates, the collected supernatant was first treated with 50 U/ml of Benzonase (Sigma-Aldrich) and 1 U/ml of Riboshredder (Epicentre) for 30 mins at 37° C. to remove unpackaged nucleic acids, filtered through a 0.45 um PVDF filter (Millipore), and used directly on cells or stored in −80° C.
For purification of AAV via chloroform-ammonium sulfate precipitation, 1/10th volume of chloroform and NaCl (1M final concentration) was added to the lysate and shaken vigorously at room temperature. Precipitated cell debris were removed by centrifugation (4000 g 30 mins), and supernatant collected. PEG-8000 (10% final w/v) was added to the supernatant, and incubated on ice for at least 1 hr or overnight. PEG-precipitated virions were then collected via centrifugation (4000 g 30 mins 4° C.), and resuspended in 50 mM HEPES buffer (pH8). 50 U/ml of Benzonase (Sigma-Aldrich) and 1 U/ml of Riboshredder (Epicentre) were added and incubated for 30 mins at 37° C. An equal volume of chloroform was then added, and vigorously vortexed. The precipitate was removed from the aqueous phase via centrifugation, and the aqueous phase collected and allowed to stand for 30 mins in the tissue culture hood for residual chloroform to evaporate. Ammonium sulfate was added to 0.5M, chilled on ice for at least 1 hour, centrifuged at 4000 g for 30 mins, and the supernatant collected. Additional ammonium sulfate was then added to 2M, chilled on ice overnight, and the precipitated virions collected via 4000 g 30 mins 4° C. AAVs were then resuspended and dialyzed in 1×PBS+35 mM NaCl, quantified for titers, and stored in −80° C.
For purification of AAV via iodixanol density gradient ultracentrifugation (see Zolotukhin, S., et al., Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther, 1999. 6(6): p. 973-85, and Grieger, J. C., V. W. Choi, and R. J. Samulski, Production and characterization of adeno-associated viral vectors. Nat Protoc, 2006. 1(3): p. 1412-28), the collected supernatant was first treated with 50 U/ml Benzonase and 1 U/ml Riboshredder for 30 mins at 37° C. After incubation, the lysate was concentrated to <3 ml by ultrafiltration with Amicon Ultra-15 (50 kDa MWCO)(Millipore), and loaded on top of a discontinuous density gradient consisting of 2 ml each of 15%, 25%, 40%, 60% Optiprep (Sigma-Aldrich) in an 11.2 ml Optiseal polypropylene tube (Beckman-Coulter). The volumes were topped up with lysis buffer. The tubes were centrifuged at 58000 rpm, at 18° C., for 1.5 hrs, on an NVT65 rotor. The 40% fraction was then extracted via a 18G needle, and dialyzed with 1×PBS (pH 7.2) supplemented with 35 mM NaCl, using Amicon Ultra-15 (50 kDa or 100 kDa MWCO)(Millipore). The purified AAVs were then quantified for titers, and stored in −80° C.
AAV titers (vector genomes) were quantified via hydrolysis-probe-based qPCR (see Aurnhammer, C., et al., Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods, 2012. 23(1): p. 18-28) against standard curves generated from linearized parental AAV plasmids and rAAV2RSM (ATCC VR-1616).
qPCR Probes and Primers:
All cells were incubated at 37° C. and 5% CO2. C2C12 cells were grown in growth media (DMEM+glutaMAX+10% FBS). Cells were split with TypLE Express (Invitrogen) every 2-3 days before confluency of 80% is reached to prevent terminal differentiation. Passage number was kept below 15 for all experiments. For transfection of C2C12 myoblasts, 105 cells were plated per well in a 24-well plate, in 500 ul of growth media. The following day, the media was replaced with fresh growth media, and 800 ng of total plasmid DNA was transfected with 2.4 ul of Lipofectamine 2000 (Life Technologies) according to manufacturer's protocol. 1:1 mass ratio of vectors encoding Cas9:gRNA(s) was used. Media was replaced with differentiation media (DMEM+glutaMAX+2% DHS) on the 1S and 3rd days post-lipofection.
For differentiation of C2C12 into myotubes, 2×104 cells were plated per well in a 96-well plate, in 100 ul of growth media. At confluency 1-2 day(s) post-plating, media was replaced with fresh differentiation media, and further incubated for 4-5 days. Fresh differentiation media was replaced before transduction. For transduction with AAV-containing lysates, 50 ul of each lysate was added per well. For transduction with purified AAV, the stated titers of vector genomes (vg) were added per well. Culture media was replaced with fresh differentiation media the next day, and cells were incubated for stated durations.
For puromycin selection, 3 ug of puromycin dihydrochloride (Sigma) per ml of cell culture media was applied 2 days post-transfection. 100 nM of dexamethasone (Sigma) was used for C2C12 atrophy experiments.
The LSL-tdTomato reporter cell line was derived from tail-tip fibroblasts of the Ai9 mouse strain (JAX 007909), and immortalized with lentiviruses encoding the large SV40 T-antigen. Cells were cultured in DMEM+pyruvate+glutaMAX+10% FBS. For transduction with AAVs, cells were plated at 2×104 per well in a 96-well plate, in 100 ul of growth media. AAV-containing lysates or purified AAVs were applied at confluency of 70-90%. Culture media was replaced with fresh growth media the next day, and cells were incubated for stated durations.
The GC-1 spg mouse spermatogonial cell line (CRL-2053) was obtained from ATCC, and cultured in DMEM+pyruvate+glutaMAX+10% FBS. Transduction of the cells with AAV was performed similarly to the LSL-tdTomato cell line.
C2C12 cells were harvested 4 days post-lipofection, with 100 ul of QuickExtract DNA Extraction Solution (Epicentre) per well of a 24-well plate; and C2C12 myotubes were harvested 7 days post-transduction, with 20 ul of DNA QuickExtract per well of a 96-well plate. The cell lysates were heated at 65° C. for 10 mins, 95° C. for 8 mins, and stored at −20° C. Each locus was amplified from 0.25-0.5 ul of cell culture lysate per 25 ul PCR reaction, for 15-25 cycles, to minimize amplification bias.
For barcoding for deep sequencing, 1 ul of each unpurified PCR reaction was added to 25 ul of barcoding PCR reaction, and thermocycled for 10 cycles. Amplicons were pooled, and the whole sequencing library was purified with home-made SPRI beads (9% PEG8000 final concentration), and sequenced on a Miseq (Illumina) for 2×251 cycles. FASTQ were analyzed with BLAT (with parameters -t=dna -q=dna -tileSize=11-stepSize=5-oneOff=1-repMatch=10000000-minMatch=4-minldentity=90-maxGap=3-noHead) and post-alignment analyses performed with custom MATLAB (MathWorks) scripts. Alignments due to primer dimers were excluded by filtering off sequence alignments that do not extend >2 bp into the targeted loci from the locus-specific primers. To minimize the impact of sequencing errors, conservative variant calling was performed by ignoring base substitutions, and considering only variants that overlap with a ±30 bp window from the designed CRISPR cut sites. Cas9-only controls were similarly analyzed.
For neonatal systemic injection (see Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65, Bostick, B., et al., Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration. Gene Ther, 2007. 14(22): p. 1605-9, Byrne, L. C., et al., The Expression Pattern of Systemically Injected AAV9 in the Developing Mouse Retina Is Determined by Age. Mol Ther, 2014, Gombash Lampe, S. E., B. K. Kaspar, and K. D. Foust, Intravenous injections in neonatal mice. J Vis Exp, 2014(93), and Sands, M. S. and J. E. Barker, Percutaneous intravenous injection in neonatal mice. Lab Anim Sci, 1999. 49(3): p. 328-30), the mother was first removed from the cage, and 1 to 4-day old neonates were individually placed on ice for sedation. For intravenous injections, 1×1011 to 5×1011 vector genomes (vg) of total AAV9 were injected into each neonate via the superficial temporal vein using a 3/10cc 30G insulin syringe. Higher dosages between 5×1011 to 5×1013 can be used. Same dosages of viruses were utilized for intraperitoneal injections. Vector volumes were kept below 50 ul, alternatively, volumes of 50 ul or more are acceptable. Injected neonates were gently cleaned, replaced into the cage, and rubbed with bedding. The mother was then returned to the cage after nose-numbing with ethanol pads.
For systemic injection into adult male or pregnant (E16) female mice, mice were anesthetized with isoflurane and the tail was swabbed with ethanol before being placed under a heated lamp. 1×1012 to 6×1012 vg of AAV9 were injected through the tail vein. The injected volume was kept under 150 ul. Higher dosages between 5×1011 to 5×1015 can be used. Also, calibration to body weight is desirable depending on the animal or patient size, for example 1010 to 1017 per kg of body weight.
Animals were sacrificed via CO2 asphyxia 2, 4, 8, or 10 weeks following injections. Histology was performed for the skeletal muscles (tibialis anterior, extensor digitorum longus, gastronomies, and quadriceps), heart, liver, diaphragm, brain, and gonads.
Male mice injected with AAV as neonates were allowed to reproductively mature (˜1 month of age), and crossed to uninjected Ai9 female mice. Male mice injected as adults were crossed to uninjected Ai9 female mice following AAV administration. Female mice were rotated weekly for multiple crosses, and also for tracking persistence of genetic modifications within spermatogonial stem cells.
Confocal images were taken using a Zeiss LSM780 inverted microscope. For live cell-imaging, each image consists of 3× z-stacks (7 um intervals) and 2×2 tiles. For fixed histological samples, the number of z-stacks and tiles are indicated in the figure legends. Fluorescent images were merged by maximum intensity projection. All images were then analyzed via Cellprofiler (see Carpenter, A. E., et al., CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol, 2006. 7(10): p. R100) and custom MATLAB (MathWorks) scripts.
The Sp Cas9 coding sequence is about 4.2 kb in length. The Sp Cas9 protein consists of a bi-lobed structure, with a disordered linker between amino acid residues V713 and D718. See Nishimasu, H., et al., Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 2014. 156(5): p. 935-49 and Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 2014, 343(6176): p. 1247997-1247997. Each lobe may be thought of as a portion of the Sp Cas9. The Sp cas9 has a first portion and a second portion, which if separate, can be linked together to form the Sp Cas9. This may be referred to herein as a split Cas9 design. According to one aspect, the first portion or sequence includes or has the Cas9 sequence up to and including V713. The second portion or sequence begins with S714 and includes the remaining portion or sequence of Cas9. According to the methods provided herein, each lobe is separately expressed and folded, and reconstituted in vivo by split-intein protein trans-splicing. See Li, J., et al., Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy. Hum Gene Ther, 2008. 19(9): p. 958-64, Wu, H., Z. Hu, and X. Q. Liu, Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci USA, 1998. 95(16): p. 9226-31, and Lohmueller, J. J., T. Z. Armel, and P. A. Silver, A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Nucleic Acids Res, 2012. 40(11): p. 5180-7. The N-terminal lobe is designed to be fused on its C-terminus with the Rhodothermus marinus N-split-intein (Cas9N), and the C-terminal lobe with C-split-intein (Cas9c). This reduces the coding sequences to 2.5 kb and 2.2 kb respectively, and providing >2 kb within each AAV for incorporation of transcriptional elements, gRNAs and fusion domains. See
The split-Cas9 design was tested by individually targeting three genes involved in muscular growth inhibition (mMstn, mActRIIB, mActRIIA). See McPherron, A. C. and S. J. Lee, Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA, 1997. 94(23): p. 12457-61, Lee, S. J. and A. C. McPherron, Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA, 2001. 98(16): p. 9306-11, Lee, S. J., Regulation of muscle mass by myostatin. Annu Rev Cell Dev Biol, 2004. 20: p. 61-86, Williams, M. S., Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med, 2004. 351(10): p. 1030-1; author reply 1030-1, Lee, S. J., et al., Regulation of muscle growth by multiple ligands signaling through activin type II receptors. Proc Natl Acad Sci USA, 2005. 102(50): p. 18117-22, and Lee, S. J., et al., Regulation of muscle mass by follistatin and activins. Mol Endocrinol, 2010. 24(10): p. 1998-2008. Plasmids encoding split-Cas9 (i.e., a plasmid encoding a first portion of the Sp Cas9 and a plasmid encoding a second portion of the Cas9, wherein the first portion and the second portion when linked together form the Sp Cas9) and gRNAs were transfected into proliferating C2C12 myoblasts with Cas9N:Cas9C ranging from 1:0 to 0:1 as shown in
CRISPR-mediated excision rates on a single-cell level, using the Ai9 excision-activated-tdTomato reporter fibroblasts as previously described were examined to compare efficiencies of split-Cas9 and full-length Cas9. Split-Cas9 and full-length Cas9 led to similar numbers of edited cells, across all four gRNA pairs tested as shown in
The split-Cas9 and gRNAs were packaged into AAV-DJs, a hybrid serotype evolved through capsid shuffling. See Grimm, D., et al., In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol, 2008. 82(12): p. 5887-911. AAVs were generated at high titers, as expected from the usage of optimal genome sizes. Cas9C expression from AAV transduction was detected via co-translating P2A-turboGFP. Since the majority of somatic tissues in adult organisms are terminally differentiated, transduction with differentiated C2C12 mouse myotubes was performed.
AAV-DJs encoding CRISPR targeting mMstn, mActRIIB, mActRIIA, and the LSL-tdTomato reporter were generated. Cells were transduced cells with the viruses and assayed for mutational activity and fluorescence-activation. A total of 100 ul of AAV-containing lysates or 1010 vector genomes (vg) of chloroform-ammonium sulfate purified AAVs were used. AAV-CRISPR induced on-target mutations on all three endogenous genes as shown in
Because elimination of empty capsids from the AAV preparations can result in enhanced transduction efficiency and reduced immunological burden in animals (see Gao, K., et al., Empty Virions In AAV8 Vector Preparations Reduce Transduction Efficiency And May Cause Total Viral Particle Dose-Limiting Side-Effects. Mol Ther Methods Clin Dev, 2014. 1(9): p. 20139, Ayuso, E., et al., High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther, 2010. 17(4): p. 503-10, and Zeltner, N., et al., Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors. Gene Ther, 2010. 17(7): p. 872-9), subsequent experiments were carried out with fully-packaged virions purified via density gradient ultracentrifugation. See Zolotukhin, S., et al., Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther, 1999. 6(6): p. 973-85, and Grieger, J. C., V. W. Choi, and R. J. Samulski, Production and characterization of adeno-associated viral vectors. Nat Protoc, 2006. 1(3): p. 1412-28. A total of 1×1012 (vg) AAV-DJ-CRISPR targeting the myostatin gene in C2C12 myotubes were transduced, while varying the ratios of Cas9N:Cas9C. See
Split-Cas9 and gRNAs are packaged into AAV9, a serotype that exhibits broad systemic transduction in mammals, with tropism preference to cardiac and skeletal muscles, and robust transduction of other organs such as the liver, nervous system, lungs, kidneys, spleen, and gonads. See Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80, Inagaki, K., et al., Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 2006. 14(1): p. 45-53, Keeler, A. M., et al., Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. Mol Ther, 2012. 20(6): p. 1131-8, Gray, S. J., et al., Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011. 19(6): p. 1058-69, Okada, H., et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, and Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65. Within the skeletal muscles, AAV9 exhibits transduction preference for fast-twitch myofibers (see Bostick, B., et al., Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration. Gene Ther, 2007. 14(22): p. 1605-9), which corresponds to the predominant myostatin-expressing fiber type. See Carlson, C. J., F. W. Booth, and S. E. Gordon, Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol, 1999. 277(2 Pt 2): p. R601-6. The biodistribution of CRISPR activity following systemic injection of AAV9-CRISPR in neonatal and adult mice may be determined. The mMstn gene is targeted to induce muscle hypertrophy, and the LSL-tdTomato locus is targeted for unbiased fluorescent detection of CRISPR activity. A functional CRISPR system using a split Cas9 design can be delivered systemically into a subject such as a plant or animal.
According to certain aspects, AAV9-CRISPR is injected systemically into neonatal and adult male mice to produce genetic modifications that are subsequently vertically transmitted. Spermatogenesis begins from the type A spermatogonial primitive stem cells (Aundiff), which give rise to all differentiating spermatogonia, spermatocytes, and spermatids that progressively migrate through a dynamic Sertoli cell layer into the seminiferous tubule lumen. See Yoshida, S., M. Sukeno, and Y. Nabeshima, A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science, 2007. 317(5845): p. 1722-6. The Aundiff spermatogonial stem cells reside between the vasculature and the blood-testis barrier as defined by Sertoli cell-Sertoli cell tight junctions, specifically in a microenvironment niche around vascular branchpoints (see Yoshida, S., M. Sukeno, and Y. Nabeshima, A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science, 2007. 317(5845): p. 1722-6), suggesting that these progenitors might be exposed to intravascularly delivered AAVs. As shown in
AAV9-CRISPR is injected systematically or locally into neonatal and adult male mice to target the LSL-tdTomato reporter and the myostatin gene. Genotype, transduction efficiency, fluorescence activation by CRISPR, body mass growth curve, serum myostatin isoforms, muscle fiber cross-sectional areas, histology of various organ types, germline transmission to F1 progeny following crosses to uninjected females is analyzed.
A key strength of CRISPR is its programmability, and facile multiplex gene targeting can be accomplished with a simple recoding of the gRNA spacer sequence. gRNAs can also be provided in separate AAVs, each targeting a different DNA site. The gRNAs can be encoded by self-complementary AAVs (scAAVs) (see
Various shorter promoters, Cas9 transgenes, and polyA sequences were tested. For the promoter sequence, the 173 bp truncated mCMV promoter (see Ostedgaard, L. S., et al., A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia. Proc Natl Acad Sci USA, 2005. 102(8): p. 2952-7), 312 bp synthetic muscle-specific promoter C5-12 (see Wang, B., et al., Construction and analysis of compact muscle-specific promoters for AAV vectors. Gene Ther, 2008. 15(22): p. 1489-99), 376 bp Tre3G promoter (7× TetO)(Clontech), and a truncated 231 bp Tre3Gtrun promoter (3×TetO) were tested. Notably, the Tre3G promoter offers tight temporal control of Cas9 expression by doxycycline induction, while allowing Cas9 expression to be dependent on a larger and more regulated tissue-specific promoter via trans-activation (driven by tTA or rtTA). Only the Tre3G promoter expressed Cas9 well enough to achieve mutational activity comparable to the strong ubiquitous hybrid promoters. The shorter ST1 CRISPR system was also tested, but the lower mutational frequency and restrictive PAM (NNAGAA) (see Esvelt, K. M., et al., Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 2013. 10(11): p. 1116-21) may make it less attractive. Truncation of the 133aa REC2 domain of Sp Cas9 abolishes targeting activity. Finally, both the SV40 polyA and the 49 bp synthetic polyA (see Levitt, N., et al., Definition of an efficient synthetic poly(A) site. Genes Dev, 1989. 3(7): p. 1019-25) resulted in similar mutational frequency when paired with strong ubiquitous hybrid promoters.
Transduction of Spermatogonial Stem Cells with
Purified scAAV-DJ and scAAV9 Conditioned Media
In parallel with transduction of GC-1 cells with crude and purified scAAV9, robust transduction with conditioned media from producer cells was also demonstrated. Unlike that of AAV2 variants, which are retained within the virus-producing HEK cells, serotype 9 viruses are both found within the producer cells and released into the cell culture media during production. See Lock, M., et al., Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum Gene Ther, 2010. 21(10): p. 1259-71 and Vandenberghe, L. H., et al., Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Hum Gene Ther, 2010. 21(10): p. 1251-7. Extraction of AAV9 from culture media bypasses the need for clarification of abundant cellular debris that is associated with producer cells lysis, potentially reducing the chance for protein contaminants in the final virus preparations. Furthermore, GC-1 cells are also robustly transduced with scAAV-DJ. Spermatologocal stem cells are permissive to AAV hybrid serotype DJ. scAAV-DJ encoding CMV-EGFP-SV40polyA-U6-gRNA (mMstn3 and mMstn4) was applied to GC-1 spg spermatogonial stem cell line.
The biological activity of split-Cas9 was investigated in transfected C2C12 myoblasts. Split-Cas9 was fully active, targeting all endogenous genes tested (Acvr2b, Acvr2a, and Mstn) at efficiencies 85% to 115% of Cas9FL. See
C2C12 myoblasts C2C12 cells were obtained from the American Tissue Collection Center (ATCC, Manassas, VA), and grown in growth media (DMEM+glutaMAX+10% FBS). Cells were split with TypLE Express (Invitrogen) every 2-3 days and before reaching 80% confluency, to prevent terminal differentiation. Passage number was kept below 15 for all experiments. For transfection of C2C12 myoblasts, 105 cells were plated per well in a 24-well plate, in 500 μl of growth media. The following day, the media was replaced with fresh growth media, and 800 ng of total plasmid DNA was transfected with 2.4 μl of Lipofectamine 2000 (Life Technologies) according to manufacturer's protocol. 1:1 mass ratio of vectors encoding Cas9:gRNA(s) was used. Media was replaced with differentiation media (DMEM+glutaMAX+2% donor horse serum) on the 1st and 3rd days post-lipofection. C2C12 cells were harvested 4 days post-lipofection, with 100 μl of QuickExtract DNA Extraction Solution (Epicentre) per well of a 24-well plate. The cell lysates were heated at 65° C. for 10 min., 95° C. for 8 min., and stored at −20° C.
Each locus was amplified from 0.5 μl of cell culture lysate per 25 μl PCR reaction, for 20-25 cycles. For barcoding for deep sequencing, 1 μl of each unpurified PCR reaction was added to 20 μl of barcoding PCR reaction, and thermocycled [95° C. for 3 min., and 10 cycles of (95° C. for 10 s, 72° C. for 65 s)]. Amplicons were pooled, and the whole sequencing library was purified with self-made SPRI beads (9% PEG final concentration), and sequenced on a Miseq (Illumina) for 2×251 cycles. FASTQ were analyzed with BLAT (with parameters -t=dna -q=dna -tileSize=11-stepSize=5-oneOff=1-repMatch=10000000-minMatch=4-minldentity=90-maxGap=3-noHead) and post-alignment analyses performed with custom MATLAB (MathWorks) scripts. Alignments due to primer dimers were excluded by filtering off sequence alignments that did not extend >2 bp into the loci from the locus-specific primers. To minimize the impact of sequencing errors, conservative variant calling was performed by ignoring base substitutions, and calling only variants that overlap with a ±30 bp window from the designated CRISPR cut sites. Controls were equally analyzed.
The 3×Stop-tdTomato reporter cell line was derived from tail-tip fibroblasts of Ai9 mouse, Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci, 2010. 13: p. 133-140, (JAX 007905), and immortalized with lentiviruses encoding the large SV40 T-antigen (GenTarget Inc, LVP016-Puro). Cells were cultured in DMEM+pyruvate+glutaMAX+10% FBS. Lipofectamine 2000 (Life Technologies) was used for transfection of plasmids, and images were taken 5 days after transfection. See
gRNA Spacer Sequences:
To assay the activity of split-Cas9 in terminally differentiated, post-mitotic cells, split-Cas9 and paired gRNAs (targeting Acvr2b, Acvr2a, and Mstn) were packaged into AAV serotype DJ (AAV-CRISPR), and applied the viruses to differentiated C2C12 myotubes,
For differentiation of C2C12 into myotubes, 2×104 cells were plated per well in a 96-well plate, in 100 μl of growth media. At confluency 1-2 day(s) after plating, media was replaced with fresh differentiation media (DMEM+glutaMAX+2% donor horse serum), and further incubated for 4 days. Fresh differentiation media was replaced before transduction. For transduction with AAV-containing lysates, 50 μl of each lysate was added per well. For transduction with purified AAV, the stated titers of vector genomes (vg) were added per well. Culture media was replaced with fresh differentiation media the next day, and cells were incubated for stated durations. See
The 3×Stop-tdTomato reporter cell line was derived from tail-tip fibroblasts of Ai9 mouse (JAX 007905), and immortalized with lentiviruses encoding the large SV40 T-antigen (GenTarget Inc, LVP016-Puro). Cells were cultured in DMEM+pyruvate+glutaMAX+10% FBS. For transduction with AAVs, cells were plated at 2×104 per well in a 96-well plate, in 100 μl of growth media. Iodixanol-purified AAVs were applied at confluency of 70-90%. Culture media was replaced with fresh growth media the next day. Images were taken 7 days post-transduction.
Transduction of Ai9 tail-tip fibroblasts with 1E12 (total vg) of AAV-CRISPR targeting the 3×Stop cassette induced excision-dependent fluorescence activation (n=2 transductions). gRNA pairs and Cas9N:Cas9C-P2A-turboGFP ratios are indicated. Td5 and TdL target 5′ of 3×Stop; Td3 and TdR target 3′ of 3×Stop. TdTomato was not observed in negative controls transduced with 6.7E11 (total vg) of Cas9C-P2A-turboGFP only. See
The GC-1 spg mouse spermatogonial cell line (CRL-2053) was obtained from the American Tissue Collection Center (ATCC, Manassas, VA), and cultured in DMEM+pyruvate+glutaMAX+10% FBS. Stated total volumes of AAV-CRISPR-containing lysates were applied to the cells 1 day post-plating. Fresh media was replaced the next day. AAV-CRISPRM3+M4 edits the Mstn gene in GC-1 spermatogonial cells (Cas9N:Cas9C, 1:1) (*, P<0.05, Welch's t-test, Bonferroni corrected). See
The viruses were pseudotyped to AAV serotype 9, and injected AAV9-CRISPR targeting Mstn (AAV9-CRISPRM3+M4) systemically into neonatal mice,
The CRISPR activity within each tissue can be predicted based on how well the AAV serotype transduces the said tissue(s). Many AAV serotypes exist, and they exhibit different infection profiles. Production of AAVs of desired serotype is easily done by using pRepCap expressing the desired capsid during AAV production. Hence, pseudotyping of the AAV-CRISPR to a serotype that efficiently transduces the organ/tissue of interest is critical for editing success. Conversely, to limit tissue-level off-targeting, one would use serotypes that do not transduce inappropriate tissue types.
Ai9 mice (JAX No. 007905) were used for all AAV experiments. All AAV9-CRISPR injections utilized a Cas9N-gRNAs:Cas9C-P2A-turboGFP ratio of 1:1. 3-day old neonates were each intraperitoneally injected with 4E12 or 5E11 vector genomes (vg) of total AAV9. Vector volumes were kept at 100 μl. Animals were euthanized via CO2 asphyxia and cervical dislocation 3 weeks following injections. For deep sequencing of whole tissues, samples were taken from the heart body wall, liver, gastrocnemius muscle, olfactory bulb, ovary, testis, and diaphragm.
Bulk tissues were each placed in 100 μl of QuickExtract DNA Extraction Solution, and heated at 65° C. for 15 min., 95° C. for 10 min. 0.5 μl of lysate was used per 25 μl PCR reaction, and thermocycled for 25 cycles.
For barcoding for deep sequencing, 1 μl of each unpurified PCR reaction was added to 20 μl of barcoding PCR reaction, and thermocycled [95° C. for 3 min., and 10 cycles of (95° C. for 10 s, 72° C. for 65 s)]. Amplicons were pooled, and the whole sequencing library was purified with self-made SPRI beads (9% PEG final concentration), and sequenced on a Miseq (Illumina) for 2×251 cycles. FASTQ were analyzed with BLAT (with parameters -t=dna -q=dna -tileSize=11-stepSize=5-oneOff=1-repMatch=10000000-minMatch=4-minldentity=90-maxGap=3-noHead) and post-alignment analyses performed with custom MATLAB (MathWorks) scripts. Alignments due to primer dimers were excluded by filtering off sequence alignments that did not extend >2 bp into the loci from the locus-specific primers. To minimize the impact of sequencing errors, conservative variant calling was performed by ignoring base substitutions, and calling only variants that overlap with a ±30 bp window from the designated CRISPR cut sites. Vehicle-injected controls were equally analyzed.
Each qPCR reaction consists of 1× FastStart Essential DNA Probes Master (Roche #06402682001), 100 nM of each hydrolysis probe (against the AAV ITR and the mouse Acvr2b locus), 340 nM of AAV ITR reverse primer, 100 nM each for all other forward and reverse primers, and 2.5 μl of input tissue lysate. For each qPCR run, a mastermix was first constituted before splitting 22.5 μl into each well, after which tissue lysates were added. The thermocycling conditions were: [95° C. 15 min.; 40 cycles of (95° C. 1 min., 60° C. 1 min.)]. FAM and HEX fluorescence were taken every cycle. AAV genomic copies per mouse diploid genome were calculated against standard curves.
qPCR Probes and Primers:
For each tissue sample, two repeated samplings were performed for qPCR and deep-sequencing, all on separate days, and the means plotted with s.e.m. Sequencing error and qPCR false positive rate were calculated similarly from two vehicle-injected negative control mice. See
Off-target sites for Mstn gRNAs were predicted using the online CRISPR Design Tool, Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology, 2013. 31: p. 827-832, (world wide website crispr.mit.edu). Off-target sites were ranked by the number of mismatches to the on-target sequence, and deep sequencing was performed on the top hits. Sequencing reads were analyzed equally between experimental samples (AAV9-CRISPRM3+M4) and control samples (AAV9-CRISPRTdL+TdR) using BLAT. Variant calls were performed for insertions and deletions that lie within a ±15 bp window from the potential off-target cut sites. The CRISPR off-target mutation frequency follows a similar inter-tissue bias. Chr16:+3906202 is the only detected bonafide off-target,
Because AAV9-CRISPR is delivered pervasively in the body, the biodistribution of CRISPR activity was examined in further detail. This is motivated in part by safety considerations for the impending human trials, where there is an urgent need to validate tissue-specificity of genetic edits by demarcating unintentionally targeted cells. Rare gene-edited cells can go undetected with deep-sequencing of bulk samples (sensitivity limits of ˜0.2%), an inadequacy in tissues where a few mutated cells could nonetheless have profound functional consequences, such as neurons, germ cells, or proto-oncogenic cells. Therefore, to determine if the sparsely transduced organs harbour mutant cells, CRISPR activity at single-cell resolution was tracked. Low (5E11) or high (4E12) doses of AAV9-CRISPR was injected targeting the 3×Stop cassette (AAV9-CRISPRTdL+TdR) into neonatal Ai9 mice,
The reporter allows functional analyses in situ, where genetically modified cells can be examined within the native tissue context. Furthermore, the reporter provides a tool for rapid and unbiased assessment of tissue-level off-targeting, where tissue-specificity of gene-targeting can be examined throughout the whole animal. The latter is especially useful when developing therapeutics meant for subsets of diseased organ(s), for validating that AAV-CRISPR is not inadvertently acting on the other organs within the subject.
Ai9 mice (JAX No. 007905) were used for all AAV experiments. All AAV9-CRISPR injections utilized a Cas9N-gRNAs:Cas9C-P2A-turboGFP ratio of 1:1. 3-day old neonates were each intraperitoneally injected with 4E12 or 5E11 vector genomes (vg) of total AAV9. Vector volumes were kept at 100 μl. Animals were euthanized via CO2 asphyxia and cervical dislocation 3 weeks following injections. Whole organ images were taken under a fluorescent stereomicroscope.
For histology, mouse organs and tissue samples were dissected, and fixed in 4% paraformaldehyde in 1× DPBS for 1.5 hr, followed by 3 washes with 1× DPBS for 5 min. each. Samples were then immersed in 30% sucrose until submersion, embedded in O.C.T. compound (Tissue-Tek), frozen in liquid-nitrogen-cold isopantane, and cryosectioned on a Microm HM550 (Thermo Scientific). Skeletal muscles were sectioned to a thickness of 12 μm, while the liver and heart were sectioned at 20 μm. Slides were then mounted with mounting media containing DAPI (Vector Laboratories, H1500). Confocal images were taken using a Zeiss LSM780 inverted microscope. See
Gene-editing tools delivered through AAV9 are delivered to the fetus when pregnant animals were injected intravenously. This is exemplified by the detection of mosaic tdTomato+ cells within the progeny when the pregnant Ai9 mothers were injected with AAV9-GFP-Cre (Cre being the gene-targeting recombinase that excises the 3×Stop cassette, activating tdTomato) AAV9 penetrates endothelial barriers, including the blood-placental barrier, see Picconi, J. L. et al. Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9. Molecular Therapy Methods & Clinical Development, 2014. 1: p. 14014 and Okada, H., et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, suggesting that gene-editing cargoes could be transmitted from mother to fetus. To assess vertical transmission of gene-editing viruses, pregnant mice (E16.5) were intravenously injected with AAV9-CRISPRTdL+TdR, but did not detect tdTomato+ cells within the offspring. In contrast, injecting the molar equivalent of AAV9-GFP-Cre into pregnant mice resulted in systemic loxP recombination within the mother, and mosaic genetic modifications in all progeny,
AAV9 viruses are known to penetrate the blood-placental barrier, as detected by reporter transgenes such as GFP or lacZ. Here, gene-editing tools were examined to determine if they are active within the progeny when AAV9s are injected intravenously into the pregnant mothers. The observation of mosaic tdTomato+ cells within the offspring indicates that AAV9 delivers active gene-editing tools (Cre) into the fetus. This also strongly suggests that CRISPR is likewise delivered. Gene-editing, however, has not been seen within the progeny with maternally injected AAV9-CRISPR—this is almost certainly due to CRISPR being less efficient than Cre at current efficiencies. It is likely that maternal transmission will be observed post-AAV9-CRISPR injection when AAV9-CRISPR dosage is increased, or when intrinsic efficiency of CRISPR increases with further optimization.
Pregnant dams were identified by visual inspection for vaginal plugs (E0.5). Pregnant female mice were injected via the tail vein at E16.5. 4E12 (vg) of AAV9-CRISPR or 2E12 (vg) of AAV9-GFP-Cre were injected. AAV2/9-CMV-hGHintron-GFP-Cre-WPRE-SV40 pA (Lot V4565MI-R, 3.91E13 GC/ml) was obtained from the University of Pennsylvania Vector Core. The injected volume was kept at 150 μl. Mothers and pups were euthanized at 1 month after birth. Whole-organ images were taken under a fluorescent stereomicroscope. See
To determine expression levels of Cas9 in vivo, protein lysates from bulk muscle tissues were quantified through Western blots. Each tibialis anterior muscle of 11-week old C57BL/6 mice was injected with 4E12 of AAV9-split-Cas9, intramuscularly electroporated with 30 g of plasmid-Cas9FL, or mock injected/electroporated with vehicle. Muscles were harvested 2 weeks post-injection, ˜10 mm3 tissue clippings were flash-frozen in liquid nitrogen, followed by lysis in 300-500 μl of T-PER Tissue Protein Extraction Solution (Thermo Scientific) supplemented with 1× Complete Protease Inhibitor (Roche), and homogenized in gentleMACS M tubes (Miltenyi Biotec). 10-15 μl of each tissue lysate was ran on 8% Bolt Bis-Tris Plus gels (Life Technologies) in 1× Bolt MOPS SDS running buffer at 165 V for 50 mins. Protein transfer was performed with iBlot (Life Technologies) onto PVDF membranes, using program 3 for 13 mins. Western blots were conducted with 1:200 of anti-Cas9 polyclonal antibody (Clontech 632607), 1:400 of anti-GAPDH polyclonal antibody (Santa Cruz sc-25778), and 1:2500 of anti-rabbit IgG-HRP secondary antibody (Santa Cruz sc-2004), using an iBind device (Life Technologies). Stained membranes were developed with SuperSignal West Femto Maximum Sensitivity Subtrate (Thermo Scientific) and imaged on Chemidoc MP (Bio-Rad). Band intensities were quantified with ImageJ software.
As shown in
Split Cas9 can be Packaged into Self-Complementary AAV (scAAV)
Split-Cas9 can be packaged into self-complementary AAV (scAAV), a robust delivery platform that expresses the payload stronger and faster. Correspondingly, the higher transduction efficiency would enable administration of lower dosages (5-100 fold lower), while achieving similar expression levels as single-stranded AAVs (ssAAV). Useful scAAV are disclosed in McCarty, D. M., P. E. Monahan, and R. J. Samulski. “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.” Gene therapy 8.16 (2001): 1248-1254; McCarty, D. M., et al. “Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.” Gene therapy 10.26 (2003): 2112-2118.
Gao, Guang-Ping, et al. “High-level transgene expression in nonhuman primate liver with novel adeno-associated virus serotypes containing self-complementary genomes.” Journal of virology 80.12 (2006): 6192-6194; Wang, Z., et al. “Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo.” Gene therapy 10.26 (2003): 2105-2111 and Jianqing, et al. “Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity.” Human gene therapy 18.2 (2007): 171-182 each of which are hereby incorporated by reference in their entireties. The key feature of scAAV is the mutation in one of its ITRs, which generates dsDNA viral genomes instead of the normal ssDNA genomes. This means the payload exists in a transcriptionally active state, without the need for double-strand conversion within the host that is a major rate-limiting step of AAV gene delivery. However, because AAV viruses are spatially compact, the packaging of dsDNA would reduce the payload capacity by half (2.2-2.4 kb). A report (Wu, 2007) has claimed that the upper limit is higher than expected, at 3.3 kb, but importantly, because all known Cas9 orthologs are larger than 2.95 kb, this means none can be trivially packaged as a full-length transgene. Split-Cas9 reduces the S. pyogenes Cas9 coding sequences to 2.5 kb and 2.2 kb respectively, smaller than that of all known orthologs. Splitting or truncating the other Cas9 orthologs would be necessary for this route of administration.
Each half of the split-Cas9 was cloned into a scAAV plasmid vector. scAAV viruses were produced via the triple-transfection method, and purified by density gradient ultracentrifugation. To determine the integrity of the viral genomes, purified viruses were lysed with proteinase K, and gel electrophoresis was performed with the lysates.
As shown in
scAAV-Promoter-Cas9N-RmaIntN-synpA (3.2 kb):
CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG
GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCG
CAGAGAGGGAGTGGGGTTAAACGTTGACATTGATTATTGACTAGCCGCT
GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAAC
AACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGG
TCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACG
CCATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTT
AAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAA
CTGAATTCGCCGCCACCATGGCCCCAAAGAAGAAGCGGAAGGTCGGTAT
TTGGTTTTTTGTGTTAGCTAGCGCGTAGATAAGTAGCATGGCGGGTTAA
GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGC
CCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCCAG
scAAV-Promoter-RmaIntC-Cas9C-synpA (2.8 kb):
CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG
GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCG
CAGAGAGGGAGTGGGGTTAAACGTTGACATTGATTATTGACTAGCCGCT
GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAAC
AACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGG
TCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACG
CCATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTT
AAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAA
CTGAATTCGCCGCCACCATGGCGGCGGCGTGCCCGGAACTGCGTCAGCT
TGTGTTGGTTTTTTGTGTTAGCTAGCGCGTAGATAAGTAGCATGGCGGG
CTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG
ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCCAG
scAAV-Promoter-GFP-SV40pA-U6-gRNA (2.0 kb):
CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCC
GGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCG
CGCAGAGAGGGAGTGGGGTTAAACGTTGACATTGATTATTGACTAGCC
ATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTC
GTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT
GGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCT
GGAGACGCCATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTAT
AATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAA
CCTTTGGAACTGAATTCCGCGGGCCCGGGATCCACCGGTCGCCACCAT
GTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAG
TTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGG
TGTGGGAGGTTTTTTAAACTAGTTGTACAAAAAAGCAGGCTTTAAAGG
AACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGG
GCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTG
TTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAG
TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAG
TTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT
GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAA
ACACCG
[spacer]GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG
CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
TT
AGCTAGCGCGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACA
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT
CGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG
CCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCCAG
This application is a continuation application which claims priority to U.S. application Ser. No. 15/541,859, filed Jul. 6, 2017; which is a National Stage Application under 35 U.S.C. 371 of co-pending PCT application PCT/US2016/012570 designating the United States and filed Jan. 8, 2016; which claims the benefit of U.S. Provisional Application No. 62/246,321, filed Oct. 26, 2015 and U.S. Provisional Application No. 62/101,043, filed Jan. 8, 2015, each of which are hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 62246321 | Oct 2015 | US | |
| 62101043 | Jan 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15541859 | Jul 2017 | US |
| Child | 18775301 | US |
