Patent Application
20230265405
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 file, created on Dec. 20, 2022, is named 083474-033PC_ST2650177518.1 and is 387 kilobytes in size.
Genome editing with CRISPR-Cas nucleases harnesses cellular double-strand break (DSB) repair pathways, such as non-homologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ), and homology-directed repair (HDR), to mutate targeted loci. The Cas nuclease is programmed to create a DNA break, such as a DSB, at a specific target DNA sequence using an associated guide RNA (gRNA). NHEJ produces semi-random insertion/deletion mutations (indels) that are generally small. In contrast, MMEJ makes sequence-specific indels that can be small or large by using small homologous sequences flanking the DSB. Meanwhile, HDR utilizes a homologous DNA repair template that recombines with the DSB site to create specific mutations called precise edits. These repair pathways compete to repair each DSB due to multi-faceted regulation, though NHEJ is the dominant mechanism. The relative frequencies of these repair pathways and the mutations they produce follow a predictable distribution that is thought to be defined by the sequence of the targeted locus and the cell state. A central goal of the field is to develop means for controlling the relative frequencies of particular repair pathways, primarily with the aim of making precise editing by HDR or specific indels by MMEJ more frequent. This requires understanding what underlies competition between repair pathways and accordingly designing strategies to redirect DNA repair.
CRISPR-Cas nucleases can also be utilized in complex genome editing tools where one or more proteins are fused to a Cas, such as prime editing systems. Prime editors are Cas nucleases paired with extended gRNAs featuring homologous DNA synthesis templates (pegRNAs) whose sequences can be copied into broken DNA ends by a fused polymerase. The sequence of the DNA synthesis template is therefore directly written into the target DNA by extension of the broken target DNA end; thus, it does not require repair mechanisms such as HDR. Yet prime editing systems frequently produce indels when the polymerase fails to extend the target DNA ends and/or DNA repair pathways reject the newly extended target DNA strand, through pathways such as NHEJ and mismatch repair (MMR), and single-strand break repair (SSBR). These indels produce somewhat unpredictable mutations that need to be addressed for many applications of genome editing.
Existing CRISPR-Cas technologies tend to create indel mutations by NHEJ, MMR, SSBR, and related pathways, and there is little room to control this outcome without directly inhibiting these indel-producing pathways. There remains a need for a more precise editing mechanism, including an editing composition and system that can reliably function in dividing and non-dividing cells.
In one aspect, this disclosure is directed to a method of editing a genome in a cell. This method includes exposing the cell to an engineered Cas nuclease comprising one or more mutations within the DNA binding cleft of the Cas nuclease, wherein exposure to the engineered Cas nuclease decreases, inhibits, or prevents an indel-producing DNA repair pathway, related non-homologous DNA repair pathways, or other means of indel generation in the cell, and wherein exposure to the engineered Cas nuclease increases one or more precise editing repair pathways within the cell.
In one embodiment, the engineered Cas nuclease is an engineered Cas9 nuclease.
In another embodiment, the homology-driven repair pathway is homology directed repair (HDR), non-homologous end joining (NHEJ), or microhomology mediated end-joining (MMEJ).
In another embodiment, the precise editing repair pathway is a combination of micro-homology end joining (MMEJ) and homology directed repair (HDR).
In another embodiment, the Cas nuclease decreases indel production. In some embodiments, the Cas nuclease decreases indel production through a particular mechanism. In some embodiments, the Cas nuclease decreases indel production through multiple pathways. In some embodiments, the Cas nuclease decreases indel production generally.
In another embodiment, the level of NHEJ and the level of HDR are measured by sequencing.
In another embodiment, the ratio of NHEJ to HDR is decreased as compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the homology-driven repair pathway is microhomology mediated end-joining (MMEJ).
In another embodiment, the level of NHEJ and the level of MMEJ are measured by sequencing.
In another embodiment, the ratio of NHEJ to MMEJ is decreased as compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of NHEJ in the cell exposed to the engineered Cas nuclease is decreased by at least 10% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of NHEJ in the cell exposed to the engineered Cas nuclease is decreased by at least 25% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of NHEJ in the cell exposed to the engineered Cas nuclease is decreased by at least 40% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of NHEJ in the cell exposed to the engineered Cas nuclease is decreased by at least 50% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of HDR in the cell exposed to the engineered Cas nuclease is increased by at least 10% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of HDR in the cell exposed to the engineered Cas nuclease is increased by at least 25% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of HDR in the cell exposed to the engineered Cas nuclease is increased by at least 40% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of HDR in the cell exposed to the engineered Cas nuclease is increased by at least 50% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of MMEJ in the cell exposed to the engineered Cas nuclease is increased by at least 10% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment the level of MMEJ in the cell exposed to the engineered Cas nuclease is increased by at least 25% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of MMEJ in the cell exposed to the engineered Cas nuclease is increased by at least 40% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the level of MMEJ in the cell exposed to the engineered Cas nuclease is increased by at least 50% compared to that of a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In another embodiment, the genome is in a non-dividing cell.
In another embodiment, the non-dividing cell is a quiescent cell, a senescent cell, or a fully differentiated cell.
In another embodiment, the one or more mutations comprise mutations of an amino acid residue at a position corresponding to D54, S55, K848, R976, N980, H982, K1003, T1314, N1317, or A1322 of SEQ ID NO: 2.
In another embodiment, the one or more mutations comprise mutations of one or more amino acid residues that occupy the same position in the three-dimensional structure of the DNA binding cleft as amino acids S55, R976, K1003, or T1314 from a Streptococcus pyogenes Cas9 protein.
In another embodiment, the engineered Cas9 nuclease comprises one or more mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises a replacement of a sequence in the DNA binding cleft, wherein two or more sequential amino acids in the DNA binding cleft are replaced.
In another embodiment, the replacement sequence comprises the same number of amino acids, fewer amino acids, or more amino acids than the original sequence.
In another embodiment, the Cas9 nuclease further comprises a mutation outside of the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises one mutation in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises two mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises three mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises four mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises S55R, R976A, K1003A, and T1314A mutations.
In another embodiment, the engineered Cas9 nuclease comprises K848A and H982A mutations.
In some embodiments, the engineered Cas9 nuclease system is part of a prime editing system.
In some embodiments, the engineered Cas9 comprises amino acids R221 and N394.
In some embodiments, the engineered Cas9 comprises amino acids R221, N394, A848, and A982.
In another embodiment, wherein the engineered Cas nuclease decreases, inhibits, or prevents non-homologous end joining when compared to a reference Cas nuclease lacking said mutations in the DNA binding cleft.
In another embodiment, the Cas nuclease is a Cas9 nuclease.
In another embodiment, the reference Cas9 comprises mutations, insertions, or deletions of amino acids outside of the DNA binding cleft.
In another embodiment, the engineered Cas nuclease retains at least 85%, at least 95% or at least 99% of the activity of a reference Cas nuclease without the corresponding mutations in the DNA binding cleft.
In another embodiment, the engineered Cas nuclease has the same or greater activity than a reference Cas nuclease without the corresponding mutations in the DNA binding cleft.
In another embodiment, the Cas nuclease is a fusion protein.
In another embodiment, the fusion protein is a fusion of a Cas nuclease fused to a reverse transcriptase.
In another embodiments, the Cas nuclease is a fusion protein, which is further fused to a reverse transcriptase.
In another aspect, this disclosure is directed to a method of precisely editing the genome of a non-dividing cell, the method comprising administering to the cell an agent capable of inhibiting or preventing non-homologous end joining (NHEJ) and increasing homology-driven repair (HDR).
In another embodiment, the agent is a modified Cas nuclease.
In another embodiment, the agent is a modified Cas9 nuclease.
In another embodiment, the engineered Cas9 nuclease comprises one mutation in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises two mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises three mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises four mutations in the DNA binding cleft.
In another embodiment, the modified Cas9 nuclease comprises mutations at one or more amino acid residues in the DNA binding cleft.
In another aspect, this disclosure is directed to an engineered Cas nuclease variant comprising two or more amino acid substitutions, mutations, or deletions in the DNA binding cleft such that the engineered Cas nuclease variant predominantly engages a homology-driven DNA repair pathway.
In another embodiment, the Cas nuclease is a Cas9 nuclease.
In another embodiment, the engineered Cas9 nuclease inhibits or prevents non-homologous end joining.
In another embodiment, the inhibition or prevention of NHEJ is determined by sequencing.
In another embodiment, the homology-driven DNA repair pathway is homology directed repair (HDR).
In another embodiment, the homology-driven DNA repair pathway is micro-homology end joining (MMEJ).
In another embodiment, the homology-driven DNA repair pathway is a combination of micro-homology end joining (MMEJ) and homology directed repair (HDR).
In another embodiment, the engineered Cas9 nuclease decreases the number of semi-random insertion/deletion (indel) mutations when compared to a reference Cas9 nuclease in a non-dividing cell.
In another embodiment, the two or more substitutions, mutations, or deletions within the DNA binding cleft are located at an amino acid residue corresponding to D54, S55, R221, N394, K848, R976, N980, H982, K1003, T1314, N1317, or A1322 of SEQ ID NO: 2, or any combination thereof.
In another embodiment, the one or more mutations within the DNA binding cleft comprise mutations S55R, R976A, K1003A, and T1314A corresponding to SEQ ID NO: 2.
In another embodiment, the one or more mutations within the DNA binding cleft comprise mutations at amino acid residues R221, N394, K848 and H982 corresponding to SEQ ID NO: 2.
In another aspect, this disclosure is directed to a method of switching a cell from a predominantly non-homologous DNA repair pathway to a homology-driven DNA repair pathway, the method comprising exposing the cell to a modified or engineered Cas nuclease.
In another aspect, this disclosure is directed to a method of decreasing or preventing indel generation in a cell during DNA repair pathways, the method comprising exposing the cell to a modified or engineered Cas nuclease.
In another embodiment, the Cas nuclease is a Cas9 nuclease.
In another embodiment, the modified or engineered Cas9 nuclease comprises one or mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises one mutation in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises two mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises three mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises four mutations in the DNA binding cleft.
In another embodiment, the modified or engineered Cas9 nuclease comprises one or more mutations located at an amino acid residue corresponding to D54, S55, R976, N980, K1003, T1314, N1317, or A1322 of SEQ ID NO: 2, or any combination thereof.
In another embodiment, the modified or engineered Cas 9 nucleases comprise the amino acids R221, N394, A848, and A982.
In another aspect, this disclosure is directed to a method of editing a genome in a cell. This method includes exposing the cell to an engineered Cas nuclease comprising one or more mutations within the DNA binding cleft of the Cas nuclease, wherein the engineered Cas nuclease is fused or otherwise associated with a polymerase to form a prime editor, wherein exposure to the engineered Cas nuclease increases precise genome editing initiated by action of the associated polymerase, and wherein exposure to the engineered Cas nuclease decreases, inhibits, or prevents byproduct indel formation in the cell.
In one embodiment, the engineered Cas nuclease is an engineered Cas9 nuclease within a prime editing system.
In another embodiment, the ratio of byproduct indels to precise genome edits is decreased compared to that of a cell exposed to a reference Cas nuclease within a prime editing system lacking the same mutations in the DNA binding cleft.
In another embodiment, the engineered Cas9 nuclease comprises mutations at one or more amino acid residues in the DNA binding cleft.
In another embodiment, the one or more mutations comprise mutations of an amino acid residue at a position corresponding to R780, K810, K848, K855, R976, H982, or T1314 of SEQ ID NO: 2.
In another embodiment, the one or more mutations comprise mutations of one or more amino acid residues that occupy the same position in the three-dimensional structure of the DNA binding cleft as amino acids R780, K810, K848, K855, R976, H982, or T1314 from a Streptococcus pyogenes Cas9 protein.
In another embodiment, the engineered Cas nuclease comprises the amino acids R221, N394, or both R221 and N394.
In another embodiment, the engineered Cas nuclease is vPE.
In another aspect, this disclosure is directed to an engineered Cas nuclease variant comprising one or more amino acid substitutions, mutations, or deletions in the DNA binding cleft, wherein the engineered Cas nuclease is fused or otherwise associated with a polymerase to form a prime editor, such that the engineered Cas nuclease variant suppresses indel formation.
Aspects, features, benefits, and advantages of the embodiments described herein will be apparent with regard to the following description, appended claims, and accompanying drawings.
It will be appreciated that for clarity, the following discussion will describe various aspects of embodiments of the applicants teachings. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).
As used herein, the singular forms “a”, “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.
As used herein, the term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
As used here, the terms “about” or “approximately” when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/−10% or less, +/−5% or less, +/−1% or less, +/−0.5% or less, and +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically disclosed.
As used herein, the term “engineered” refers to molecules or cells that have an alteration from their natural state.
As used herein, the term “DNA” means a deoxyribonucleic acid comprising two polynucleotide chains that coil around each other to form a double helix carrying genetic instructions.
As used herein, the term “PAM” means a protospacer adjacent motif. A PAM is a region of between two and six nucleotides adjacent to, but not part of, a target sequence that is recognized by a Cas nuclease to identify a sequence of DNA for cleavage. The commonly accepted PAM sequence for Cas nucleases derived from Streptococcus pyogenes is nGG, wherein n is any nucleotide selected from cytosine, adenine, guanine, and thymine. However, the engineered Cas nucleases disclosed herein are not limited to identifying PAM sequences of the formula nGG.
As used herein, the term “guide RNA sequence,” or “gRNA sequence,” refers to a sequence of ribonucleic acid capable of targeting a specific complementary sequence. The present disclosure contemplates guide RNA sequences which target a complementary sequence within a genome. The present disclosure also contemplates guide RNA sequences which target a complementary sequence within a non-genomic vector. The present disclosure also contemplates guide RNA sequences which target a complementary sequence ex vivo.
As used herein, the term “non-homologous end joining,” or “NHEJ,” refers to the process of repairing double-strand breaks in DNA by directly joining both ends of the break.
As used herein, the term “microhomology-mediated end joining,” or “MMEJ,” refers to the process of repairing double-strand breaks in DNA by using a homologous sequences flanking the double-strand break.
As used herein, the terms “homology directed repair,” or “HDR,” refers to the process of repairing double-strand breaks in DNA by using a homologous DNA repair template.
As used herein, the terms “prime editing system” and “prime editors” refer to an editing system that is not classified as HDR or NHEJ. Primer editors use a fused polymerase, generally reverse transcriptase, to write new sequences directly into a nicked genomic DNA strand in a cell. They use a pegRNA as a template for writing these new sequences in. They also result in indels when that direct writing process fails. The precise edits are therefore homology-dependent, and the indels are non-homologous, meaning they are considered neither HDR or NHEJ, but nor are they non-homologous.
As used herein, the term “polypeptide” and the likes refer to an amino acid sequence including a plurality of consecutive polymerized amino acid residues (e.g., at least about 2 consecutive polymerized amino acid residues). “Polypeptide” refers to an amino acid sequence, oligopeptide, peptide, protein, enzyme, nuclease, or portions thereof, and the terms “polypeptide,” “oligopeptide,” “peptide,” “protein,” “enzyme,” and “nuclease,” are used interchangeably.
Polypeptides as described herein also include polypeptides having various amino acid additions, deletions, or substitutions relative to the native amino acid sequence of a polypeptide. In some embodiments, polypeptides that are homologs of a polypeptide contain non-conservative changes of certain amino acids relative to the native sequence of a polypeptide. In some embodiments, polypeptides that are homologs of a polypeptide contain conservative changes of certain amino acids relative to the native sequence of a polypeptide, and thus may be referred to as conservatively modified variants. A conservatively modified variant may include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well-known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles.
As used herein, the term “fusion protein” refers to at least a first polypeptide sequence linked either directly or indirectly to at least a second polypeptide sequence with which it is not normally directly linked in nature.
As used herein, the term “DNA binding cleft” may be used to refer to the amino acid residues of a Cas protein or variant, or amino acid residues in a region of a Cas protein or variant, which come into contact with DNA, either stably or transiently. “DNA binding cleft” may also be used to refer to residues that may act to stabilize these residues and/or regions.
As used herein, the term “indel” refers to a molecular edit to a nucleic acid sequence, including but not limited to genomic sequences, that can be either an insertion or a deletion of a number of nucleotides within a nucleic acid sequence. The present disclosure contemplates both small and large indels.
As used herein, the terms “mutation,” “variant,” and the like refer to a polypeptide or nucleotide sequence that differs from a given polypeptide or nucleotide sequence in amino acid or nucleic acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids or nucleotides, but that retains some or all the biological activity of the given polypeptide (e.g., a variant nucleic acid could still encode the same or a similar amino acid sequence). A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol., 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. The present disclosure provides amino acids having hydropathic indexes of ±2 that can be substituted. The hydrophilicity of amino acids also can be used to reveal substitutions that would result in proteins retaining some or all biological functions. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101). The term “variant” also can be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains some or all its biological and/or antigen reactivities. Use of “variant” herein is intended to encompass fragments of a variant unless otherwise contradicted by context.
Alternatively, or additionally, a “variant” is to be understood as a polynucleotide or protein which differs in comparison to the polynucleotide or protein from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a protein variant is derived is also known as the parent polypeptide or polynucleotide. The term “variant” comprises “fragments” or “derivatives” of the parent molecule. Typically, “fragments” are smaller in length or size than the parent molecule, whilst “derivatives” exhibit one or more differences in their sequence in comparison to the parent molecule. Also encompassed modified molecules such as but not limited to post-translationally modified proteins (e.g. glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins). Also, mixtures of different molecules such as but not limited to RNA-DNA hybrids, are encompassed by the term “variant.” Typically, a variant is constructed artificially by gene-technological means whilst the parent polypeptide is a wild-type protein. However, naturally occurring variants are to be understood to be encompassed by the term “variant” as used herein. Further, the variants usable in the present disclosure may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant exhibits at least one or more biological activity of the parent molecule, i.e. is functionally active.
Alternatively, or additionally, a “variant” as used herein can be characterized by a certain degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. A protein variant in the context of the present disclosure can exhibit at least 90% sequence identity to its parent polypeptide. A polynucleotide variant in the context of the present disclosure can exhibit at least 80% sequence identity to its parent polynucleotide. A polynucleotide variant in the context of the present disclosure can exhibit at least 70% sequence identity to its parent polynucleotide. A polynucleotide variant in the context of the present disclosure can exhibit at least 60% sequence identity to its parent polynucleotide. A polynucleotide variant in the context of the present disclosure can exhibit at least 50% sequence identity to its parent polynucleotide. The term “at least 50% sequence identity” refers to a sequence identity of at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide.
The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on http://www.ebi.ac.uk/Tools/clustalw/or on http://www.ebi.ac.uk/Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html. Parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/or http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.
Some embodiments disclosed herein are directed to non-naturally occurring or engineered systems, methods, and compositions for the repair of DNA targets. DNA repair pathways are discussed in more detail below.
Cells have developed a number of pathways for repairing single (SSB) and double-strand breaks (DSB) in DNA. One pathway normally active in non-dividing cells is non-homologous end joining (NHEJ). NHEJ repairs double stranded DNA breaks by directly ligating the two ends of nucleic acid together. The result of NHEJ is a semi-random insertion or deletion of a DNA sequence, called an indel, that are generally small. NHEJ does not employ a homologous sequence as a template for these indels and may result in losing sequence information in the process. In other words, this repair mechanism can be mutagenic. NHEJ relies on chance pairings, or microhomologies, between the single-stranded tails of the two DNA fragments to be joined.
Another DSB repair pathway is microhomology-mediated end-joining (MMEJ). MMEJ process generally involves the following steps: resection of the DSB ends, annealing of microhomologous region, removal of heterologous flaps, fill-in synthesis and ligation. A certain degree of end resection may also be needed for MMEJ. Following end resection, the exposed microhomologous sequence is annealed to form an intermediate with 3′-flap and gaps on both sides of the DSB. The microhomologous sequences then move closer and perform annealing, which may start in a thermodynamically-driven fashion and be regulated with proteins factors or enzymes. After microhomologous annealing, the non-homologous 3′ tail (3′-heterologous flaps) is removed to allow DNA polymerase to fill-in the gap and stabilize the annealed intermediate. This step can be executed by substrate structure specific endonuclease, such as XPF/ERCC1 in mammals. The final step of MMEJ is DNA ligase mediated break end ligation.
Another DSB repair pathway is homology-directed repair (HDR). HDR process is one of the most accurate for DSB repair due to the requirement of higher sequence homology between the damaged and intact donor strands of DNA. The process can be error-free as long as the DNA template used for repair is identical to the original DNA sequence at the DSB. If not, specific mutations could be introduced into the damaged DNA.
The HDR process generally involves the following steps: the 5′-ended DNA strand is resected at the break to create a 3′ overhang. This can serve as both a substrate for proteins required for strand invasion and a primer for DNA repair synthesis. The invasive strand can then displace one strand of the homologous DNA duplex and pair with the other, which results in the formation of the hybrid DNA (a displacement loop or D loop). The recombination intermediates can then be resolved to complete the DNA repair process.
Prime editors are Cas9 nickases (Cas9n) paired with extended gRNAs featuring homologous templates (pegRNAs) whose sequences can be copied into nicked DNA ends by a fused reverse transcriptase (RT). Unlike the DSB pathways described above, nicked DNA is a single strand break.
Another DNA repair pathway is mismatch repair (MMR). MMR repairs mismatched DNA sequences between complementary strands. The MMR process generally involves the following steps: the mismatch is identified by the bulged DNA structure it creates. One strand of mismatched DNA, generally the newly synthesized and nicked strand, is partially excised. The other strand is used as a template to resynthesize the excised strand. The resynthesized strand is then ligated to complete the repair process.
An additional DNA repair pathway is single-strand break repair (SSBR). SSBR is comprised of several related pathways that repair single-strand breaks, or nicks. The nicks can arise through many processes. The SSBR process generally involves the following steps: the single-strand break is identified. The DNA ends at the break are processed, which may lead to large or small gaps in the nicked strand. The other strand is used as a template to resynthesize the DNA and fill in the gap. The resynthesized strand is then ligated to complete the repair process.
Some embodiments disclosed herein are directed to non-naturally occurring or engineered systems, methods, and compositions comprising a Cas protein. Cas proteins are discussed in more details below.
In the conflict between bacterial hosts and their associated viruses, CRISPR-Cas systems provide an adaptive defense mechanism that utilizes programmed immune memory. CRISPR-Cas systems provide their defense through three stages: adaptation, the integration of short nucleic acid sequences into the CRISPR array that serves as memory of past infections; expression, the transcription of the CRISPR array into a pre-crRNA (CRISPR RNA) transcript and processing of the pre-crRNA into functional crRNA species targeting foreign nucleic acids; and interference, the programming of CRISPR effectors by crRNA to cleave nucleic acid of foreign threats. Across all CRISPR-Cas systems, these fundamental stages display enormous variation, including the identity of the target nucleic acid (either RNA, DNA, or both) and the diverse domains and proteins involved in the effector ribonucleoprotein complex of the system.
CRISPR-Cas systems can be broadly split into two classes based on the architecture of the effector modules involved in pre-crRNA processing and interference. Class 1 systems have multi-subunit effector complexes composed of many proteins, whereas Class 2 systems rely on single-effector proteins with multi-domain capabilities for crRNA binding and interference; Class 2 effectors often provide pre-crRNA processing activity as well. Class 1 systems contain 3 types (type I, III, and IV) and 33 subtypes, including the RNA and DNA targeting type III-systems. Class 2 CRISPR families encompass 3 types (type II, V, and VI) and 17 subtypes of systems, including the RNA-guided DNases Cas9 and Cas12 and the RNA-guided RNase Cas13. Continual sequencing of novel bacterial genomes and metagenomes uncovers new diversity of CRISPR-Cas systems and their evolutionary relationships, necessitating experimental work that reveals the function of these systems and develops them into new tools.
Some embodiments disclosed herein are directed to non-naturally occurring or engineered systems, methods, and compositions comprising a Cas protein variant fused to a polymerase in a prime editing system. The Cas variants contemplated herein include variants of Cas9, Cas12, or Cas13. The polymerases contemplated herein include variants of DNA polymerases including reverse transcriptases. Prime editing systems are discussed in more detail below.
Prime editing systems are made of a Cas nuclease fused to a DNA polymerase, or ‘prime editor,’ coupled with extended guide RNAs which include a DNA synthesis template, or ‘pegRNA.’ The Cas enzyme in the fusion protein may produce a DSB, or may be engineered to instead produce a single-strand break, or ‘nick.’ The polymerase may be a reverse transcriptase or other DNA polymerase that can utilize the DNA synthesis template in the pegRNA as a primer for incorporating genome edits into a target DNA sequence.
The prime editing system components work in concert to introduce a programmed genome edit in the target DNA. In this process, the Cas enzyme in the prime editor is guided to a target DNA sequence, such as a genomic DNA sequence, using the guide RNA sequence in the pegRNA. The Cas enzyme makes a DNA break, such as a DSB or nick, in the target DNA sequence. The DNA synthesis template extension of the pegRNA then anneals to complementary sequences in the cut target DNA. The polymerase in the prime editor then extends the cut target DNA, copying the DNA synthesis template extension sequence in the pegRNA. The newly extended target DNA strand now contains a replacement sequence for the original target DNA sequence that includes the programmed genome edit. Through DNA repair, such as HDR, MMR, SSBR, and related mechanisms, the endogenous sequence is replaced by the newly synthesized target DNA strand extension. Thus, the prime editing system directly writes replacement DNA sequences into target DNA and utilizes DNA repair to resolve the resulting mismatches between the original and replacement sequences.
Prime editing systems may fail to incorporate the programmed genome edit, and may instead introduce a byproduct mutation referred to as an indel as described above. These indels may result from failure of the cut target DNA to bind, failure of the polymerase to extend the target DNA sequence with the template extension sequence, failure of DNA repair mechanisms in replacing the endogenous target DNA sequence with the replacement sequence, errors in DNA repair, or other processes. The DNA repair mechanisms introducing indels may be NHEJ, MMEJ, MMR, SSBR, and related mechanisms.
Some embodiments disclosed herein are directed to non-naturally occurring or engineered systems, methods, and compositions comprising a Cas protein variant. The Cas variants contemplated herein include variants of Cas9, Cas12, or Cas13. Cas protein variants are discussed in more detail below.
The Cas protein variants of the disclosure can be variants of a Cas nuclease, such as a Cas9 nuclease variant. The Cas protein of the disclosure can be a fusion protein. The Cas protein variant of the disclosure can be an engineered Cas nuclease, such as an engineered Cas9 nuclease. In one embodiment, the fusion protein can be Cas9 fused to Reverse Transcriptase. Such engineered Cas9-nickase fusions may be referred to as prime editors. Further disclosed herein are Cas protein variants linked either directly or indirectly to a Cas nickase, which is a Cas nuclease with one or more mutations (including, but not limited to, modifications to amino acids D10, D54, S55, E762 H840, K848, D839, N863, R976, N980, H982, H983, D986 K1003, T1314, N1317, or A1322 of SEQ ID NO: 2, that inactive one nuclease domain.
The engineered Cas nuclease variant can comprise one or more mutations at one or more amino acids residues, such as one or more mutations at one or more amino acid residues of a DNA binding cleft of the nuclease, such that the engineered Cas nuclease variant engages a different repair pathway as compared to the natural nuclease under normal circumstances. The DNA-binding cleft includes those amino acids in contact with DNA, either stably (e.g., in a particular conformation) or transiently (e.g., during conformational shifts) and can also include amino acid residues in positions that could possibly stabilize these regions. In one embodiment, the engineered Cas nuclease variant predominantly engages a homology-driven DNA repair pathway. In another embodiment, the engineered Cas9 nuclease variant inhibits or prevents non-homologous end joining (NHEJ). The inhibition or prevention of NHEJ can be determined by sequencing. In another embodiment, the homology-driven DNA repair pathway is homology directed repair (HDR). In another embodiment, the homology-driven DNA repair pathway is micro-homology end joining (MMEJ). In another embodiment, the homology-driven DNA repair pathway is a combination of micro-homology end joining (MMEJ) and homology directed repair (HDR). In another embodiment, the engineered Cas9 nuclease variant decreases the number of semi-random insertion/deletion (indel) mutations when compared to a reference Cas9 nuclease in a non-dividing cell.
The engineered Cas nuclease can comprise the same number of amino acids, fewer amino acids, or more amino acids than the original sequence. In some embodiments, the amino acid sequence of the engineered Cas nuclease has at least about 1 mutation, about 2 mutations, about 3 mutations, about 4 mutations, about 5 mutations, about 6 mutations, about 7 mutations, about 8 mutations, about 9 mutations, about 10 mutations, about 11 mutations, about 12 mutations, about 13 mutations, about 14 mutations, about 15 mutations, about 16 mutations, about 17 mutations, about 18 mutations, about 19 mutations, about 20 mutations, about 21 mutations, about 22 mutations, about 23 mutations, about 24 mutations, about 25 mutations, or any ranges that are made of any two or more points in the above list.
The engineered Cas nuclease can comprise one or more mutations of an amino acid residue inside, outside, or a mixture of both inside and outside of the DNA binding cleft of the nuclease. In another embodiment, the mutations correspond to one or more of to D54, S55, R976, N980, K1003, T1314, N1317, or A1322 of SEQ ID NO: 2. The engineered Cas nuclease can comprise one or more mutations of one or more amino acid residues that occupy the same position in the three-dimensional structure of the DNA binding cleft as amino acids S55, R976, K1003, or T1314 from a Streptococcus pyogenes Cas9 protein. The engineered Cas nuclease can comprise one or more of the D54, S55, R976, N980, K1003, T1314, N1317, or T1314 mutations of SEQ ID NO: 2. The engineered Cas nuclease can comprise all of the S55R, R976A, K1003A, and T1314A mutations of SEQ ID NO: 2.
The engineered Cas nuclease of the disclosure can comprise one or more replacements of a sequence, such as one or more replacement of a sequence in a DNA binding cleft of the nuclease and/or one or more replacement of a sequence outside a DNA binding cleft of the nuclease. The engineered Cas nuclease can have at least about 1 non-sequential or sequential amino acids, about 2 non-sequential or sequential amino acids, about 3 non-sequential or sequential amino acids, about 4 non-sequential or sequential amino acids, about 5 non-sequential or sequential amino acids, about 6 non-sequential or sequential amino acids, about 7 non-sequential or sequential amino acids, about 8 non-sequential or sequential amino acids, about 9 non-sequential or sequential amino acids, or about 10 non-sequential or sequential amino acids that are replaced in the DNA binding cleft.
The engineered Cas nuclease of the disclosure can retain between about 10% and 100% nuclease activity when compared to a reference Cas nuclease without the corresponding one or more mutations as measured by sequencing. In some embodiments, nuclease activity can be measured by next generation sequencing. In some embodiments, nuclease activity can be measured by Sanger sequencing. In some embodiments, nuclease activity can be measured by long-read sequencing.
The engineered Cas nuclease of the disclosure can retain about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24% about 25%, about 26%, about 27%, about 28%, about 29%, about 30% about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the nuclease activity of a reference Cas nuclease without the corresponding one or more mutations. The engineered Cas nuclease disclosed herein can have the same or greater activity than a reference Cas nuclease without mutation.
The engineered Cas nuclease disclosed herein can have the same or greater activity than a reference Cas nuclease without mutation. The engineered Cas nuclease can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-13. For example, the engineered Cas nuclease can comprise an amino acid sequence at least about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-13.
The engineered Cas nuclease can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-13. For example, the engineered Cas nuclease can comprise an amino acid sequence at least about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-13.
The engineered Cas nuclease of the disclosure can be used in a method of editing a genome in a cell. The method can comprise exposing the cell to an engineered Cas nuclease comprising one or more mutations within the DNA binding cleft of the Cas nuclease. The exposure to the engineered Cas nuclease can decrease, inhibit, or prevent non-homologous end joining (NHEJ) in the cell, and can increase one or more homology-driven repair pathways within the cell. The engineered Cas nuclease can be an engineered Cas9 nuclease. The homology-driven repair pathway can be homology directed repair (HDR) or microhomology mediated end-joining (MMEJ). The level of NHEJ, HDR and MMEJ can be measured by sequencing.
The ratio of NHEJ to HDR is decreased in some embodiments. In some embodiments, the level of NHEJ in the cell exposed to the engineered Cas nuclease is decreased by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In some embodiments, the level of HDR in the cell exposed to the engineered Cas nuclease is increased by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft.
In some embodiments, the homology-driven repair pathway is microhomology mediated end-joining (MMEJ). The level of NHEJ and the level of MMEJ are measured by sequencing. In some embodiments, the ratio of NHEJ to MMEJ is decreased. In some embodiments, the level of MMEJ in the cell exposed to the engineered Cas nuclease is increased by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to a cell exposed to a reference Cas nuclease lacking the same mutations in the DNA binding cleft. The genome can be in a non-dividing cell, such as a quiescent cell, a senescent cell, or a fully differentiated cell, or a dividing cell. In another embodiment, the method comprises precisely editing the genome of a non-dividing cell by administering to the cell an agent capable of inhibiting or preventing non-homologous end joining (NHEJ) and increasing homology-driven repair. In another embodiment, the method includes switching a cell from a predominantly non-homologous DNA repair pathway to a homology-driven DNA repair pathway by exposing the cell to a modified or engineered Cas nuclease.
In any of the methods described herein, the engineered Cas nuclease can contain any or all of the modifications and/or sequences described above.
The Cas protein of the disclosure can be a fusion protein. Fusion proteins may include fusions with heterologous domains or functional domains (e.g., localization signals, catalytic domains, etc.). In certain embodiments, various modifications may be combined (e.g., a mutated nuclease which is catalytically inactive, and which further is fused to a functional domain. As used herein, “altered functionality” includes without limitation an altered specificity (e.g., altered target recognition, increased (e.g., “enhanced” Cas proteins) or decreased specificity, or altered PAM recognition), altered activity (e.g., increased or decreased catalytic activity, including catalytically inactive), and/or altered stability (e.g., fusions with destabilization domains). Suitable heterologous domains include without limitation a nuclease, a ligase, a repair protein, a methyltransferase, (viral) integrase, a recombinase, a transposase, an argonaute, a cytidine deaminase, a retron, a group II intron, a phosphatase, a phosphorylase, a sulfurylase, a kinase, a polymerase, an exonuclease, etc. Examples of all these modifications are known in the art. It will be understood that a “modified” nuclease as referred to herein, and in particular a “modified” Cas or “modified” Cas system or complex has the capacity to interact with or bind to the polynucleic acid (e.g., in complex with the guide molecule).
In some embodiments, the nuclease is introduced into a cell as a nucleic acid encoding each protein. The nucleic acid introduced into the eukaryotic cell is a plasmid DNA or viral vector. In some embodiments, the target specific nuclease and blunting enzyme are introduced into a cell via a ribonucleoprotein (RNP).
Delivery is in the form of a vector which may be a viral vector, such as a lenti- or baculo- or adeno-viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided. The viral vector may be selected from a variety of families/genera of viruses, including, but not limited to Myoviridae, Siphoviridae, Podoviridae, Corticoviridae, Lipothrixviridae, Poxviridae, Iridoviridae, Adenoviridae, Polyomaviridae, Papillomaviridae, Mimiviridae, Pandoravirusa, Salterprovirusa, Inoviridae, Microviridae, Parvoviridae, Circoviridae, Hepadnaviridae, Caulimoviridae, Retroviridae, Cystoviridae, Reoviridae, Birnaviridae, Totiviridae, Partitiviridae, Filoviridae, Orthomyxoviridae, Deltavirusa, Leviviridae, Picornaviridae, Marnaviridae, Secoviridae, Potyviridae, Caliciviridae, Hepeviridae, Astroviridae, Nodaviridae, Tetraviridae, Luteoviridae, Tombusviridae, Coronaviridae, Arteriviridae, Flaviviridae, Togaviridae, Virgaviridae, Bromoviridae, Tymoviridae, Alphaflexiviridae, Sobemovirusa, or Idaeovirusa.
A vector may mean not only a viral or yeast system (for instance, where the nucleic acids of interest may be operably linked to and under the control of (in terms of expression, such as to ultimately provide a processed RNA) a promoter), but also direct delivery of nucleic acids into a host cell. For example, baculoviruses may be used for expression in insect cells. These insect cells may, in turn be useful for producing large quantities of further vectors, such as AAV or lentivirus adapted for delivery of the present engineered nuclease. Also envisaged is a method of delivering the target specific nuclease and blunting enzyme comprising delivering to a cell mRNAs encoding each.
In some embodiments, expression of a nucleic acid sequence encoding the nuclease may be driven by a promoter. In some embodiments, the nuclease is a Cas. In some embodiments, the nuclease is a Cas9. In some embodiments, the nuclease is a mutant or variant Cas9. In some embodiments, a single promoter drives expression of a nucleic acid sequence encoding a Cas. In some embodiments, the Cas is operably linked to and expressed from the same promoter. In some embodiments, the CRISPR enzyme is expressed from different promoters. For example, the promoter(s) can be, but are not limited to, a UBC promoter, a PGK promoter, an EF1A promoter, a CMV promoter, an EFS promoter, a SV40 promoter, and a TRE promoter. The promoter may be a weak or a strong promoter. The promoter may be a constitutive promoter or an inducible promoter. In some embodiments, the promoter can also be an AAV ITR, and can be advantageous for eliminating the need for an additional promoter element, which can take up space in the vector. The additional space freed up by use of an AAV ITR can be used to drive the expression of additional elements, such as guide sequences. In some embodiments, the promoter may be a tissue specific promoter.
In some embodiments, an enzyme coding sequence encoding a nuclease is codon-optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cas protein correspond to the most frequently used codon for a particular amino acid.
In some embodiments, a vector encodes a nuclease comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the Cas protein comprises about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known. In some embodiments, the NLS is between two domains, for example between the Cas protein and the viral protein. The NLS may also be between two functional domains separated or flanked by a glycine-serine linker.
In general, the one or more NLSs are of sufficient strength to drive accumulation of the nuclease in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the nuclease, the particular NLS used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the nuclease, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus such as DAPI). Examples of detectable markers include fluorescent proteins (such as green fluorescent proteins, or GFP; RFP; CFP), and epitope tags (HA tag, FLAG tag, SNAP tag). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.
In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a Cas protein optionally in combination with (and optionally complexed) with a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding a target specific nuclease and/or a blunting enzyme to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, nucleic acid complexed with a delivery vehicle, such as a liposome, and ribonucleoprotein. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-8313 (1992); Navel and Felgner, TIBTECH 11:211-217 (1993); Mitani and Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
The nuclease can be delivered using adeno-associated virus (AAV), lentivirus, adenovirus, or other viral vector types, or combinations thereof. In some embodiments, Cas protein(s) can be packaged into one or more viral vectors. In some embodiments, the targeted trans-splicing system is delivered via AAV as a split intein system, similar to Levy et al. (Nature Biomedical Engineering, 2020, DOI: https://doi.org/10.1038/s41551-019-0501-5). In other embodiments, the target specific nuclease and/or the blunting enzyme can be delivered via AAV as a trans-splicing system, similar to Lai et al. (Nature Biotechnology, 2005, DOI: 10.1038/nbt1153). In some embodiments, the viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, intrathecal, intracranial or other delivery methods. Such delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector chosen, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
The use of RNA or DNA viral based systems for the delivery of nucleic acids takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
In certain embodiments, delivery of the nuclease and to a cell is non-viral. In certain embodiments, the non-viral delivery system is selected from a ribonucleoprotein, cationic lipid vehicle, electroporation, nucleofection, calcium phosphate transfection, transfection through membrane disruption using mechanical shear forces, mechanical transfection, and nanoparticle delivery.
In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.
The present disclosure provides kits for carrying out a method. The present disclosure provides kits containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the kit comprises a vector system comprising regulatory elements and polynucleotides encoding the target specific nuclease and/or the blunting enzyme. In some embodiments, the kit comprises a viral delivery system of the target specific nuclease and/or the blunting enzyme. In some embodiments, the kit comprises a non-viral delivery system of the target specific nuclease and/or the blunting enzyme. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instruction in one or more languages, for examples, in more than one language.
In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.
pyogenes
pyogenes
T1314R (vCas9)
While several experimental Examples are contemplated, these Examples are intended to be non-limiting.
To examine the balance between precise homology related repair and semi-random indel repair, indel size and frequency were examined in HEK293T cells that were exposed to a Cas9 from Streptococcus pyogenes (SEQ ID NO: 1) in the presence or absence of a single-stranded oligodeoxynucleotide (ssODN) for use in HDR. gRNAs in the presence or absence of ssODN HDR templates were targeted to the EMX1 locus (
The specific repair pathway engaged in the presence or absence of repair template was also examined. In HEK293T cells in which no repair template was supplied, there was no precise editing by HDR. In three of four loci examined without repair template, the majority of DNA repair was the result of NHEJ. The remainder is the result of MMEJ of varying lengths of microhomology (
MMEJ repair was competitively inhibited to a greater degree than NHEJ when HDR was engaged using a template. It was hypothesized that if MMEJ was promoted over NHEJ, a greater proportion of HDR template would be utilized in DNA repair. Although no known Cas9 variants alter the frequency of DNA repair pathways, different Cas9 structures are known to produce different double strand break structures upon cutting of the DNA (
To further examine these Cas9 variants in their editing capability, a GFP transgene was inserted into the genomes of HEK293T cells. An HDR ssODN template was supplied to the cells that, if inserted, would convert the GFP transgene to a blue fluorescent protein (BFP) (
The R976A mutation produced the greatest change in repair pathway frequency along with the largest drop in total editing activity and sits at one end of the nontarget cleft near where the substrate DNA strands separate (
These triple, quadruple, and quintuple mutants were analyzed in HEK293T cells with gRNAs and HDR templates (
vCas9 activity across numerous loci was compared to Cas9 from Streptococcus pyogenes (SEQ ID NO: 1). Whereas Cas9 demonstrated precise editing frequencies of 9.9-37.5% (mean 24.1%), vCas9 increased these to 43.3-73.7% (mean 58.3%), corresponding to a 1.4- to 2.8-fold (mean 1.9-fold) suppression of indel frequency. Further highlighting the unique nature of locus-specific activity issues, the editing percentage varied greatly across the tested loci. Compared to Cas9, vCas9 exhibited and increased fraction of editing attributed to precise HDR editing, and a decreased percentage of indels (
When vCas9 and Cas9 from Streptococcus pyogenes (SEQ ID NO: 1) were tested for their ability to precisely edit a GFP transgene to BFP (as described above), vCas9 exhibited a significant increase in the level of blue fluorescence observed by flow cytometry, indicating a significant increase in precise HDR editing (
We also examined the cell-type dependence of precise editing of vCas9 compared to unmutated Cas9. We applied similar HDR templates to produce small edits at several loci in HeLa, A549, and Panc1 cells. In each cell model and locus, vCas9 consistently altered precise editing and indel frequencies (
Finally, we also compared other engineered Cas9 variants and fusions and found that repair outcomes for vCas9 are far more biased toward precise editing (
We first studied the effect of Cas variants on DNA break structure and the downstream effect of regulation of repair pathway outcomes. Break structures in cells were assessed by creating two concurrent DSBs and analyzing junctions of the DNA ends for sequences resulting from blunt versus staggered cutting (
Editing outcomes for each engineered Cas9 variant from the Alanine substitution screen were studied with a gRNA and a precise HDR editing template. Methods were the same as described in Example 3. There was a strong correlation between precise editing frequency and mean indel size (
When both Cas9 from Streptococcus pyogenes (SEQ ID NO: 1) and vCas9 were introduced into HEK293T cells in the presence of a gRNA and an ssODN HDR template, vCas9 consistently made HDR dominant, with the remaining minor repair outcomes comprised mostly of NHEJ insertions and MMEJ deletions (
Engagement of repair pathways was also tested in the presence of NHEJ and MMEJ inhibitors. Cas9 from Streptococcus pyogenes (SEQ ID NO: 1) and vCas9 along with a gRNA and ssODN HDR template were introduced into HEK293T cells that were also treated with either an inhibitor of MMEJ (Rucaparib) or an inhibitor of NHEJ (NU7026). Cas9 from Streptococcus pyogenes (SEQ ID NO: 1) combined with the NHEJ inhibitor NU7026 led to editing patterns that resembled vCas9 at both the EMX1 locus (
Comparing repair frequencies in HEK293T cells exposed to a gRNA with or without an HDR template, vCas9 exhibited an increased degree to which precise editing outcompeted any size indel (
A major limitation of CRISPR technology for precise editing to generate genetic models or treat certain diseases is the lack of HDR in non-dividing cells. The major pathways engaged in DNA repair in non-dividing cells are MMEJ and NHEJ. As such, the ability to precisely edit the genome of non-dividing cells is a critical and undermet need in the gene editing field. To determine if vCas9 might be reliably engage precise HDR repair in non-dividing cells, an MMEJ-driven template strategy was developed. This hybrid strategy was termed “microhomology-directed recombination” (MDR). It utilizes a partially double-stranded DNA templates with single-stranded microhomology arms complementary to sequences distal to the DSB ends (
When Cas9 from Streptococcus pyogenes (SEQ ID NO: 1) and vCas9 was paired with an MDR template with a 5 nt 5′ homology arm and a 20 nt 3′ homology arm, cells transfected with vCas9 exhibited significantly increased precise editing as measured by blue fluorescence, as well as a significant decrease in non-precise editing as measured by the presence of indels (
To examine engagement of precise HDR/MDR editing pathways in both dividing and non-dividing cells, MDR templates from several loci (
We next set out to determine if altered Cas9 variants could be employed in other genome editing systems to similarly suppress indels and favor precise gene editing. We first examined the use of these Cas9 variants in a prime editing system. Prime editing systems employ Cas9 nickases fused to a reverse transcriptase, which is then paired with an engineered guide RNA which contains a sequence complementary to target DNA, as well as an extension region which encodes the desired change (pegRNA). Although prime editors favor precise editing and decrease indels relative to Cas9 nuclease editing by HDR, they do not completely eliminate indel production.
We determined whether mutations incorporated into a Cas9 nickase established within a prime editing system could alter the frequency of repair outcomes (
Since the PE prime editor system has slightly reduced indel frequency relative to PEmax, we used PE as a basis for further engineering of Cas9 within prime editor systems. We next introduced the 14 mutations that we previously introduced into Cas9 (R780A, R783A, K810A, R832A, K848A, K855A, R859A, S964A, K968A, R976A, H982A, K1003A, K1047A, and R1060A) into the Cas9 nickase within the PE prime editor system. To measure precise editing and indel frequencies for these mutants, we combined them with a pegRNA and nicking gRNA (double-nicking prime editing, or PE3) that produces a small sequence replacement at the KRAS locus in HEK293T cells by prime editing and analyzed alterations using deep sequencing of the amplified locus. Several PE single-mutant variants demonstrated reduced indel frequency and increased precise editing frequency (
We further created and analyzed several dual-mutation variants of the Cas9 nickase within the PE prime editor system (R780A-K810A; R780A-K848A; R780A-H982A; K810A-K848A; K810A-H982A; K848A-H982A). We tested these engineered variants by applying prime editing with a pegRNA and nicking gRNA to convert a GFP transgene to BFP, similar to the HDR assay. One variant, K848A-H982A, nearly eliminated indels while promoting precise editing (
We next explored mechanisms and robustness for these effects on prime editor repair outcomes. We compared the precise editing frequencies of each PE single-mutant with the break structure alterations observed for corresponding Cas9 single-mutants. We again found a strong correlation between precise editing frequency and altered break structure for these PE variants (
We next examined whether vPE produces precise editing by again applying prime editing to convert a GFP transgene to BFP. Here, vPE resulted in efficient precise gene conversion with limited indels (
Considering this broad suppression of all indel types, we also studied whether vPE reduced indel frequency at several loci in HEK293T cells using pegRNAs without nicking gRNAs (single-nicking prime editing, or PE2). Intriguingly, though PE resulted in fairly low indel frequencies at all loci, vPE produced lower indel frequencies (
Except as otherwise stated in the above Examples, the following general methods were employed throughout.
All mammalian cell cultures were maintained in a 37° C. incubator at 5% CO2. HEK293T human embryonic kidney, HeLa human cervical cancer, A549 human lung cancer, and Panc1 human pancreatic cancer cells were maintained in Dulbecco's Modified Eagle's Medium with high glucose, sodium pyruvate, and GlutaMAX (DMEM; ThermoFisher, 10569) supplemented with 10% Fetal Bovine Serum (FBS; ThermoFisher, 10438), and 100 U/mL Penicillin-Streptomycin (ThermoFisher, 15140). For inhibitor studies, cell media was supplemented with 20 μM Rucaparib (MilliporeSigma, PZ0036) or NU7026 (MilliporeSigma, N1537) dissolved in DMSO (MilliporeSigma, D8418).
Wild-type Cas9 was obtained from pSpCas9 (pX165) and a cloning backbone for gRNA expression was obtained from pX330-U6-Chimeric BB-CBh-hSpCas9 (pX330). Cas9 mutagenesis was performed using PCR-driven splicing by overlap extension using primers listed in Supplementary Table 1. Briefly, one fragment was amplified by PCR from pX165 using the cas9-mut-FWD or cas9-mid-FWD and mutant-BOT primers and a second fragment was amplified using the mutant-TOP and cas9-mid-REV or cas9-mut-REV primers for each mutant. Each pair of fragments was then spliced by overlap extension PCR using the cas9-mut-FWD and cas9-mid-REV or cas9-mid-FWD and cas9-mut-REV primers to create a Cas9 gene fragment with a single residue mutation. These Cas9 gene fragments were then each cloned back into pX165 using unique BshTI, ApaI, and EcoRI restriction sites to replace the wild-type sequence with the mutant sequence. Additional mutants (double-, triple-, and quadruple-mutants) were made iteratively starting from these single-mutant plasmids. A custom gRNA cloning backbone vector was created by PCR amplification from pX330 using the gRNA-scaffold-NheI-FWD and gRNA-scaffold-EcoRI-REV primers and restriction cloning into pUC19 (ThermoFisher) using NheI and EcoRI digestion. The gRNA spacer sequence oligos, listed in Supplementary Table 2, were phosphorylated with T4 polynucleotide kinase (NEB) and cloned into gRNA cloning backbone by Golden Gate cloning with BpiI digestion.
PE2 and PEmax prime editors were obtained from pCMV-PE2 and pCMV-PEmax, and a cloning backbone for pegRNA expression was obtained from pU6-pegRNA-GG-acceptor. PE was created by restriction cloning of Cas9n (H840A) from pCMV-PE2 into pCMV-PEmax using NotI and SacI digestion. PE mutagenesis was performed using PCR-driven splicing by overlap extension using primers listed in Supplementary Table 1. Briefly, one fragment was amplified by PCR from PE using the pe-FWD or pe-mid-FWD and mutant-BOT primers and a second fragment was amplified using the mutant-TOP and pe-mid-REV or pe-rt-REV primers for each mutant. Each pair of fragments was then spliced by overlap extension PCR using the pe-FWD and pe-mid-REV or pe-mid-FWD and pe-rt-REV primers to create a PE gene fragment with a single residue mutation. These PE gene fragments were then each cloned back into PE using unique NotI, SacI, and BamHI restriction sites to replace the PE sequence with the mutant sequence. Additional mutants (double- and triple-mutants) were made iteratively starting from these single-mutant plasmids. The pegRNA oligos, listed in Supplementary Table 3, were phosphorylated with T4 polynucleotide kinase (NEB) and cloned into pU6-pegRNA-GG-acceptor by Golden Gate cloning with Eco31I digestion. Primers were synthesized by IDT. Restriction enzymes were obtained from ThermoFisher. T7 DNA ligase was obtained from NEB. Plasmids were transformed into competent Stbl3 chemically competent E. coli (ThermoFisher). Sequences for the wild-type Cas9, vCas9, gRNA cloning backbone, PE, and vPE vectors are presented in the Sequences section.
High-fidelity Cas9 variants were obtained from pX165-Cas9-HF1, pX165-eSpCas9, and pX165-HypaCas9. Cas9 fusions Cas9-CtIP and Cas9-dn53bp1 were created by restriction cloning of custom geneblocks synthesized by IDT into pX165 at unique KflI and EcoRI restriction sites.
Crystal structures of Cas9 with substrate DNA bound (5F9R) or without substrate DNA bound (4ZTO) were analyzed using PyMol (Schrödinger).
Cells were seeded in the maintenance medium without Pen-Strep into 24-well plates at 100,000 cells/well or 48-well plates at 50,000 cells/well. Transfections of HEK293T without repair templates were carried out 24 hrs after seeding using 400 ng Cas9 expression vector and 144 ng gRNA expression vector formulated with 1.36 μL Lipofectamine 2000 (ThermoFisher) at a total volume of 54.4 μL in OptiMEM I (ThermoFisher) per well for 24-well plates, or half these volumes for 48-well plates. Transfections of HEK293T, HeLa, A549, and Panc1 with HDR templates were carried out 24 hrs after seeding using 400 ng Cas9 expression vector, 144 ng gRNA expression vector, and 400 ng ssODN HDR template formulated with 2.11 μL Lipofectamine 2000 at a total volume of 84.4 μL in OptiMEM I per well for 24-well plates, or half these volumes for 48-well plates. Transfections of HEK293T with dual gRNAs were carried out 24 hrs after seeding using 400 ng Cas9 expression vector and 144 ng of each gRNA expression vector formulated with 1.72 μL Lipofectamine 2000 (ThermoFisher) at a total volume of 68.8 μL in OptiMEM I (ThermoFisher) per well for 24-well plates, or half these volumes for 48-well plates. Transfections of HEK293T with prime editing vectors were carried out 24 hrs after seeding using 475 ng PE expression vector, 114 ng pegRNA expression vector, and 144 ng nicking gRNA expression vector (for PE3) formulated with 1.47-1.83 μL (equal volume/DNA) Lipofectamine 2000 at a total volume of 58.9-73.3 μL (equal DNA concentration) in OptiMEM I (ThermoFisher) per well for 24-well plates, or half these volumes for 48-well plates. For sequencing assays, genomic DNA was extracted 72 hrs after transfection using QuickExtract (Epicentre). For flow cytometry assays, cells were transferred to 6-well plates 72 hrs after transfection, split 7 days after transfection, and harvested 10 days after transfection in PBS with 5% FBS (ThermoFisher). Repair templates, listed in Supplementary Table 4, were synthesized by IDT.
The targeted loci were amplified from extracted genomic DNA by PCR using Herculase II polymerase (Agilent). The PCR primers included Illumina sequencing handles as well as replicate-specific barcodes. These PCR products were then tagged with sample-specific barcodes and sequenced on an Illumina MiSeq. Primers, listed in Supplementary Table 5, were synthesized by IDT.
The targeted loci were amplified from extracted genomic DNA by PCR using Herculase II polymerase (Agilent). PCR amplicons were sequenced using primers ˜200 bp from the expected cut site. To measure editing frequencies, the sequencing traces were analyzed using TIDE 24. Primers, listed in Supplementary Table 6, were synthesized by IDT.
Flow cytometry analysis was performed on an LSR Fortessa analyzer and data was collected using FACSDiva (BD Biosciences). Cells were first gated comparing SSC-A and FSC-A, then SSC-H and SSC-W, then FSC-H and FSC-W parameters to select for single cells. To assess editing frequencies, cells were gated for GFP (488 nm laser excitation, 530/30 nm filter detection) and BFP (405 nm laser excitation, 450/50 nm filter detection). To profile cell cycle stage, cells were gated for propidium iodide (561 nm laser excitation, 610/20 nm filter detection) and Alexa Fluor 647 (640 nm laser excitation, 670/30 nm filter detection). Flow cytometry data were analyzed using FlowJo (FlowJo).
To measure editing outcomes, the high-throughput sequencing data were analyzed using CRISPResso2 25. Total editing rates were quantified as the fraction of edited reads out of total sequencing reads. Indel rates were quantified as the fraction of reads containing indels out of total sequencing reads. Precise editing rates were quantified as the fraction of reads containing a perfect match to the expected edit out of total sequencing reads. Frequencies of specific indel sizes were quantified as the fraction of reads containing these sizes out of all edited reads. Depletion of specific indel sizes by templated repair was quantified as the fractional reduction in the frequency of that indel size, comparing frequencies for when a template was present versus absent. Mean indel sizes were calculated as the mean of the absolute values of indel sizes weighted by their indel fractions.
To measure DNA break structures for Cas9 variants, editing outcomes for dual-gRNA cutting of genomic DNA were analyzed as previously described 13. HEK293T cells were edited with pairs of gRNAs targeting the EMX1 (EMX1 gRNA 1 and gRNA 2) or CXCR4 (CXCR4 gRNA 1 and gRNA 2) loci. The gRNA pairs were complementary to the same strand at each locus and were expected to make cuts 84 bp apart, resulting in large precise deletions. The loci were amplified and sequenced by high-throughput sequencing. The high-throughput sequencing data were analyzed using CRISPResso2 25, using the expected 84 bp deletion junction as a reference sequence. To assess DNA break structure, sequencing reads aligned to the deletion junction reference were analyzed for insertion sequences perfectly matching the sequences flanking the expected gRNA cut sites. The positions of these matching sequences at the two gRNA sites were used to determine cut positions leading to each read. Frequencies of these cut positions were quantified as the fraction of reads resulting from these specific cut positions out of all reads containing the deletion junctions with or without insertions.
For repair pathway analysis, next-generation sequencing reads were classified using annotated repair mechanisms determined by Indelphi 4. The high-throughput sequencing data for each editing experiment were analyzed using CRISPResso2 25. The same gRNA and locus sequences were also analyzed using Indelphi to identify whether each predicted indel was associated with a microhomology (MMEJ) or not (NHEJ), along with microhomology sizes. These repair pathway labels for each edited sequence from Indelphi analysis were then applied to the matching sequencing reads for the editing experiment. Frequencies of NHEJ, MMEJ, and precise editing were quantified as the fraction of reads containing these types of edits out of all edited reads.
To assess off-target cutting activity, indel rates were analyzed at known off-target sites previously reported for two gRNAs (EMX1 gRNA 2 and VEGFA gRNA 1) 19. Indel rates were determined by analysis of Sanger sequencing traces at these on-target and off-target loci using TIDE 24.
Specific statistical comparisons are indicated in the figure legends. Error bars indicate the standard error for three independent replicates. In most comparisons, significance was assessed using unpaired, two-tailed Student's t-tests. For correlations, significance was assessed using Pearson's tests. For linear regressions, significance was assessed using ANCOVA tests.
One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
This application claims the benefit of U.S. Provisional Patent Application No. 63/268,340, filed on Feb. 22, 2022, the entire disclosure of which is hereby incorporated herein by reference.
This invention was made with government support under R01 CA208205 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63268340 | Feb 2022 | US |
