WO2022152746A1 - Variants de cas9 k526d et applications associées - Google Patents

Variants de cas9 k526d et applications associées Download PDF

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WO2022152746A1
WO2022152746A1 PCT/EP2022/050535 EP2022050535W WO2022152746A1 WO 2022152746 A1 WO2022152746 A1 WO 2022152746A1 EP 2022050535 W EP2022050535 W EP 2022050535W WO 2022152746 A1 WO2022152746 A1 WO 2022152746A1
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mutations
mutation
cell
cas9 protein
modified cas9
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PCT/EP2022/050535
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Antonio CASINI
Anna CERESETO
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Alia Therapeutics Srl
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • RNP direct ribonucleoprotein delivery
  • Cas9 protein delivery allows for potentially favorable off-target profiles due to the transient nature of the nuclease activity in target cells which is rapidly cleared by proteolytic digestion. Nevertheless, for particular targets even the potential for increased specificity profile granted by Cas9 RNP delivery may not be sufficient to avoid off-target cleavages in the cellular genome.
  • SpCas9 variants characterized by a lower propensity to cleave off- target sites has been reported.
  • SpCas9 mutants with both high efficiency and specificity were reported in Slaymaker et al., 2016, Science 351 (6268):84-8, Kleinstiver et al., 2016, Nature 529(7587):490-495, Casini, et al., 2018, Nat. BiotechnoI 36:265-271 , and WO 2018/149888.
  • WO 2018/149888 describes several high fidelity SpCas9 variants, including the quadruple mutants M495V/Y515N/K526E/R661Q (referred to therein as evoCas9), and M495V/Y515N/K526E/R661S (referred to therein as evoCas9 II).
  • This disclosure is based, in part, on the discovery that a SpCas9 protein having a K526E mutation exhibited lower than desired activity when delivered to cells as a ribonucleoprotein (RNP) complex. Unexpectedly, it was found that a SpCas9 protein having a K526D mutation exhibited high fidelity and suitability for delivery to cells as a RNP complex, for example to cells in which alteration of a gene is desirable.
  • RNP ribonucleoprotein
  • the disclosure provides modified Cas9 proteins (e.g., Streptococcus pyogenes Cas9 (SpCas9) or a SpCas9 orthologue) having a K526D mutation alone or in combination with one or more other modifications, for example, one or more additional mutations described in WO 2018/149888, WO 2019/040650, or WO 2019/051419.
  • modified Cas9 proteins e.g., Streptococcus pyogenes Cas9 (SpCas9) or a SpCas9 orthologue
  • the disclosure provides fusion proteins comprising a modified Cas9 protein of the disclosure fused to a second amino acid sequence, for example a tag, a nuclear localization signal, a transcriptional activator, a transcriptional repressor, a histone-modifying protein, an integrase, or a recombinase.
  • a modified Cas9 protein of the disclosure fused to a second amino acid sequence, for example a tag, a nuclear localization signal, a transcriptional activator, a transcriptional repressor, a histone-modifying protein, an integrase, or a recombinase.
  • the disclosure provides nucleic acids and pluralities of nucleic acids encoding a modified Cas9 protein of the disclosure and, optionally, a guide RNA, for example a sgRNA.
  • a guide RNA for example a sgRNA.
  • the disclosure provides systems comprising the modified Cas9 proteins and one or more gRNAs, e.g., sgRNAs.
  • a system can comprise a ribonucleoprotein (RNPs) comprising a modified Cas9 protein complexed with a gRNA, e.g., an sgRNA or separate crRNA and tracrRNA.
  • RNPs ribonucleoprotein
  • Exemplary features of systems are described in Section 6.5 and specific embodiments 66 to 86, infra.
  • the disclosure provides particles comprising the modified proteins, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.5 and specific embodiments 87 to 95, infra.
  • the disclosure provides cells and populations of cells containing or contacted with a modified Cas9 protein, nucleic acid, plurality of nucleic acids, system, or particle of the disclosure. Exemplary features of such cells and cell populations are described in Section 6.5 and specific embodiments 97 to 104 and 148, infra.
  • compositions comprising a modified Cas9 protein, nucleic acid, plurality of nucleic acids, system, particle, cell, or population of cells together with one or more excipients.
  • exemplary features of pharmaceutical compositions are described in Section 6.6 and specific embodiment 96, infra.
  • the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the modified Cas9 proteins, fusion proteins, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure.
  • Cells altered according to the methods of the disclosure can be used, for example, to treat subjects having a disease or disorder.
  • some embodiments of the disclosure relate to editing a hemoglobin subunit gamma (HBG) gene in a cell to disrupt a binding site for the transcription repressor leukemia/lymphoma-related factor (LRF) in the HBG1 or HBG2 promoter in order to increase expression of fetal hemoglobin.
  • HBG hemoglobin subunit gamma
  • LRF transcription repressor leukemia/lymphoma-related factor
  • Such altered cells can be used to treat subjects suffering from a hemoglobinopathy, for example sickle cell disease or p-thalassemia.
  • exemplary methods of altering cells are described in Section 6.7 and specific embodiments 105 to
  • FIGS. 1A-1B shows that previously identified high-fidelity SpCas9 variants have suboptimal activity when delivered as RNPs.
  • FIG. 1A the activity and specificity of high-fidelity SpCas9 variants was evaluated by targeting EGFP in 293multiGFP cells through RNP lipofection. The on-target activity of each SpCas9 variant was measured using a perfectly matching gRNA (GFPon), while the specificity was assayed by targeting the same EGFP locus using two different gRN As having mismatches in position 12 (GFPoff12) and 18 (GFPoff18), counting from the PAM-proximal side of the spacer (see Table 4).
  • FIG. 1 B the on-target activity of the two best performing high-fidelity variants in FIG. 1A (containing the mutation K526E and K526E+R661S) was evaluated in comparison with wild-type (wt) SpCas9 on a panel of endogenous genomic loci by RNP nucleofection in U2OS cells.
  • FIGS 2A-2B show editing efficiency and on/off target ratios for various K526 mutants.
  • FIG. 2A the editing efficiency and the specificity of different SpCas9 variants with amino acid substitutions in position K526 were evaluated by measuring EGFP downregulation after transient transfection of 293multiEGFP cells with each indicated variant in combination with either a fully matching sgRNA (sgGFPon) or a sgRNA containing mismatches with the intended target (sgGFPoff1819).
  • FIG. 2B on-Zoff-target ratios calculated from the data shown in FIG. 2A.
  • FIGS. 3A-B show the on-target activity of the K526D high-fidelity variant.
  • FIG. 3A SpCas9 K526D cleavage activity is shown in comparison with wt SpCas9 and the K526E variant on a panel of endogenous genomic loci by RNP nucleofection in U2OS cells.
  • FIG. 3B SpCas9 K526D cleavage activity is shown in comparison with wt SpCas9 on a panel of endogenous genomic loci by RNP nucleofection in U2OS cells. Data presented as mean ⁇ SEM for n 2 independent runs.
  • FIGS. 4A-4B show SpCas9 K526D specificity by RNP nucleofection.
  • FIG. 4A indel formation evaluated at the HBB, CCR5 and EMX1 genomic loci and previously validated off- target sites (one for each locus: HBB OT, CCR5 OT and EMX1 OT) after RNP nucleofection of wt SpCas9 or the K526D mutant in U2OS cells.
  • FIG. 4B on-/off-target ratios calculated from the data shown in FIG. 4A. Data presented as mean SEM for n 2 independent runs.
  • FIGS. 5A-5B show SpCas9 K526D specificity by transient plasmid transfection.
  • FIG. 5A indel formation evaluated at the CCR5, EMX1 and ZSCAN2 genomic loci and previously validated off-target sites (one for each locus: CCR5 OT, EMX1 OT and ZSCAN2 OT) after transient plasmid transfection of wt SpCas9 or the K526D mutant together with the indicated sgRNAs in HEK293T cells.
  • FIG. 5B on-/off-target ratios calculated from the data shown in FIG. 5A.
  • FIGS. 6A-6B show SpCas9 K526D editing of the HBG therapeutic locus.
  • FIG. 6A indel formation evaluated at the HBG therapeutic target locus and at a previously validated off-target site associated with HBG cleavage (HBG OT) after RNP nucleofection of wt SpCas9 or the K526D mutant in U2OS cells.
  • FIG. 7 shows indel formation at a target locus (Sillele AQ 3 days after transient transfection of HEK293T/17 cells with wt SpCas9 or high-fidelity SpCas9 variants together with either a sgRNA targeting allele A (on-target) or targeting allele G (off-target), as indicated. Data are presented as mean SEM for at least 2 independent runs.
  • the disclosure provides modified Cas9 proteins (e.g., Streptococcus pyogenes Cas9 (SpCas9) or a SpCas9 orthologue) having a K526D mutation alone or in combination with one or more other amino acid substitutions.
  • modified Cas9 proteins e.g., Streptococcus pyogenes Cas9 (SpCas9) or a SpCas9 orthologue
  • Exemplary features of Cas9 proteins of the disclosure are described in Section 6.2.
  • the disclosure provides fusion proteins comprising a modified Cas9 protein of the disclosure fused to a second amino acid sequence. Exemplary features of fusion proteins of the disclosure are described in Section 6.3. Unless required otherwise by context, disclosures relating to Cas9 proteins are also disclosures relating to fusion proteins.
  • the disclosure provides nucleic acids encoding a modified Cas9 protein of the disclosure. Exemplary features of nucleic of the disclosure are described in Section 6.3.
  • the disclosure provides nucleic acids and pluralities of encoding a modified Cas9 protein of the disclosure. Exemplary features of nucleic acids of the disclosure are described in Section 6.4.
  • the disclosure provides systems such as RNPs comprising modified Cas9 proteins and gRNAs, and particles comprising the modified proteins, nucleic acids, and systems of the disclosure. Exemplary features of systems and particles of the disclosure are described in Section 6.5.
  • the disclosure provides cells and populations of cells comprising or contacted with a modified Cas9 protein, nucleic acid, plurality of nucleic acids, system, or particle of the disclosure. Exemplary features of such cells and cell populations are described in Section 6.5.
  • compositions comprising a modified Cas9 protein, nucleic acid, plurality of nucleic acids, system, particle, cell, or population of cells the disclosure together with one or more excipients.
  • exemplary features of pharmaceutical compositions are described in Section 6.6.
  • the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the modified Cas9 proteins, fusion proteins, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure.
  • methods of altering cells e.g., editing the genome of a cell
  • the modified Cas9 proteins, fusion proteins, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure are described in Section 6.7.
  • an agent includes a plurality of agents, including mixtures thereof.
  • a Cas9 protein refers to a wild-type or engineered Cas9 protein.
  • Engineered Cas9 proteins can also be referred to as Cas9 variants.
  • any disclosure pertaining to a Cas9 or Cas9 protein pertains to wild-type Cas9 proteins and Cas9 variants, unless the context dictates otherwise.
  • Identical or percent identity in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like).
  • This definition also refers to, or may be applied to, the complement of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 10 amino acids or 15 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN- 2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • sequence comparisons typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al., (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 .
  • Guide RNA molecule refers to an RNA capable of forming a complex with a Cas9 protein and which can direct the Cas9 protein to a target DNA.
  • gRNAs typically comprise a spacer of 15 to 30 nucleotides in length in length.
  • gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically comprise the spacer at the 5 end of the molecule and a 3 sgRNA segment.
  • sgRNAs single guide RNAs
  • 3 sgRNA segments are known in the art.
  • An sgRNA can in some embodiments comprise no uracil base at the 3 end of the sgRNA sequence.
  • a sgRNA can comprise one or more uracil bases at the 3 end of the sgRNA sequence.
  • a sgRNA can comprise 1 uracil (U) at the 3Dend of the sgRNA sequence, 2 uracil (UU) at the 3 end of the sgRNA sequence, 3 uracil (UUU) at the 3 end of the sgRNA sequence, 4 uracil (ULIUU) at the 3 end of the sgRNA sequence, 5 uracil (UUUUU) at the 3 end of the sgRNA sequence, 6 uracil (UUUUU) at the 3 end of the sgRNA sequence, 7 uracil (UUUUUU) at the 3 end of the sgRNA sequence, or 8 uracil (UUUUUUUU) at the 3 end of the sgRNA sequence.
  • 3 sgRNA sequences set forth in Table 1 can be modified by adding or removing one or more uracils at the end of the sequence.
  • Peptide, protein, and polypeptide are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
  • the amino acids may be natural or synthetic, and can contain chemical modifications such as disulfide bridges, substitution of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide range of other modifications.
  • a polypeptide may be attached to other molecules, for instance molecules required for function.
  • polypeptides examples include, without limitation, cofactors, polynucleotides, lipids, metal ions, phosphate, etc.
  • polypeptides include peptide fragments, denatured/unstructured polypeptides, polypeptides having quaternary or aggregated structures, etc. There is expressly no requirement that a polypeptide must contain an intended function; a polypeptide can be functional, non-functional, function for unexpected/unintended purposes, or have unknown function.
  • a polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids which are not members of the standard twenty amino acids may also be used.
  • the standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr,
  • Polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxy ribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers and gRNAs.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • Tiucleotide sequence the alphabetical representation of a polynucleotide molecule.
  • Spacer refers to a region of a gRNA molecule which is partially or fully complementary to a target sequence found in the + or - strand of genomic DNA.
  • the gRNA directs the Cas9 to the target sequence in the genomic DNA.
  • a spacer of a Cas9 gRNA is typically 15 to 30 nucleotides in length (e.g., 20-25 nucleotides).
  • the nucleotide sequence of a spacer can be, but is not necessarily, fully complementary to the target sequence.
  • a spacer can contain one or more mismatches with a target sequence, e.g., the spacer can comprise one, two, or three mismatches with the target sequence.
  • Wild-type in reference to a genomic DNA sequence, refers to a genomic DNA sequence that predominates in a species, e.g., Homo sapiens.
  • Cas9 proteins of the disclosure comprise a K526D mutation.
  • the position of the K526D mutation is identified by reference to the amino acid numbering in an unmodified mature Streptococcus pyogenes Cas9 (SpCas9) as set forth in SEQ ID NO: 1.
  • SpCas9 protein having the amino acid sequence of SEQ ID NO:1 is sometimes referred to herein as wild-type (wt) SpCas9.
  • the modified Cas9 proteins of the disclosure can have the K526D mutation alone, or can comprise one or more additional mutations.
  • a mutation X1nnnX2 means that at position nnn the amino acid X2 is present in place of the amino acid X1 which is present in the wild-type polypeptide; so, for example, K526D means that the amino acid at position 526 corresponds to an Aspartic acid (Asp or D), in place of the amino acid lysine (Lys or K) which is present in the wild-type polypeptide.
  • Exemplary additional mutations that can be included in a modified Cas9 protein of the disclosure are described in Section 6.2.1.
  • a modified Cas9 protein of the disclosure can be a S. pyogenes Cas9 proteins or an SpCas9 orthologue (e.g., S. thermophilus, S. aureus, or N. meningitides). SpCas9 and Cas9 orthologues are discussed in more detail in Section 6.2.2.
  • the modified Cas9 proteins of the disclosure can comprise one or more mutations in addition to K526D.
  • the K526D mutation can be combined with one or more mutations disclosed in WO 2018/149888, WO 2019/040650, or WO 2019/051419, the contents of each of which are incorporated herein by reference in their entirety.
  • the modified Cas9 proteins of the disclosure comprise a K526D mutation and one or more additional mutations described in WO 2018/149888.
  • the numbering of amino acid positions set forth herein corresponds to the amino acid numbering of an unmodified mature SpCas9 set forth in SEQ ID NO: 1.
  • the modified Cas9 comprises at least one mutation located at one or more of the following positions: K377, E387, D397, R400, D406, A421 , L423, R424, Q426, Y430, K442, P449, V452, A456, R457, W464, M465, K468, E470, T474, P475, W476, F478, K484, S487, A488, T496, F498, L502, N504, K506, P509, F518, N522, E523, K526, L540, S541 , I548, D550, F553, V561 , K562, E573, A589, L598, D605, L607, N609, N612, E617, D618, D628, R629, R635, K637, L651 , K652, R654, T657, G658, L666, K673, S675C, I679V, L680,
  • a modified Cas9 having a K526D mutation can comprise one or more further mutations (e.g., 1-9, 1-8, 1-5, 1-4, 4-8, 2-6, 1 , 2, 3, 4, 5, 6, 7, or 8), for example located at one or more of the following amino acid residue positions:
  • the one or more further mutations comprise one or more of the following:
  • the Cas9 protein comprises a K526D mutation and one or more further mutations at one or more of positions Y450, M495, Y515, R661 , N690, R691 , Q695, and H698, for example M495, Y515, R661 , and H698.
  • a Cas9 variant of the disclosure comprises one or more of the following mutations: Y450S, M495V, Y515N, R661X, N690I, R691Q, Q695H, and H698Q, where X is L, Q or S, preferably where X is Q or S.
  • the one or more additional mutations are one or more of M495V, Y515N, R661X, and H698Q, where X is L, Q or S, preferably where X is Q or S.
  • the Cas9 protein comprises a K526D mutation and a R661S, R661Q, R661L, R661 D, R661 E, R661 F, R661 M, R661W, R661Y, or R661A mutation, and optionally further comprises a M495V mutation and/or a H698Q mutation.
  • a Cas9 protein of the disclosure comprises a double mutation, for example a double mutant which is K526D+Y450S, K526D+M495V, K526D+Y515N, K526D+R661X, K526D+N690I, K526D+R691Q, K526D+Q695H or K526D+H698Q; wherein X is L, Q or S, preferably where X is Q or S.
  • a Cas9 protein comprises a triple mutation, for example, a triple mutation which is M495V+K526D+R661X, Y515N+K526D+R661X, K526D+R661X+H698Q and M495V+Y515N+K526D, where X is L, Q or S, preferably where X is Q or S.
  • a Cas9 protein comprises K526D, R661W, and Y515N mutations.
  • a Cas9 protein comprises K526D, R661 D, and Y515N mutations.
  • a Cas9 protein comprises K526D, R661 E, and Y515N mutations. In some embodiments, a Cas9 protein comprises K526D, R661 F, and Y515N mutations. In some embodiments, a Cas9 protein comprises K526D, R661M, and Y515N mutations. In some embodiments, a Cas9 protein comprises K526D, R661Y, and Y515N mutations.
  • a Cas9 protein comprises a quadruple mutation, for example a quadruple mutation which is M495V+Y515N+K526D+R661X or M495V+K526D+R661X+H698Q, where X is L, Q or S, preferably where X is Q or S.
  • a Cas9 protein comprises the mutations M495V+Y515N+K526D+R661Q.
  • the Cas9 variant comprises the mutations M495V+Y515N+K526D+R661S.
  • the Cas9 variant comprises the mutations M495V+Y515N+K526D+R661A.
  • a Cas9 protein comprises at least one of the following mutations in addition to K526D: K377E, E387V, D397E, R400H, Q402R, R403H, F405L, D406Y, D406V, N407P, N407H, A421V, L423P, R424G, Q426R, Y430C, K442N, P449S, Y450S, Y450H, Y450N, V452I, A456T, R457P, R457Q, W464L, M465R, K468N, E470D, T472A, I473F, I473V, T474A, P475H, W476R, F478Y, F478V, K484M, S487Y, A488V, M495V, M495T, T496A, F498I, F498Y,
  • a Cas9 protein comprises a K526D mutation and a D406Y, W464L, T474A, N612K, or L683P mutation.
  • a Cas9 protein comprises a K526D mutation and at least two additional mutations, for example R400H+Y450S, D406V+E523K, A421V+R661W, R424G+Q739P, W476R+L738P, P449S+F704S, N522K+G658E, E523D+E617K, L540Q+L607P, W659R+R661W, S675C+Q695L or I679V+H723L.
  • additional mutations for example R400H+Y450S, D406V+E523K, A421V+R661W, R424G+Q739P, W476R+L738P, P449S+F704S, N522K+G658E, E523D+E617K, L540Q+L607P, W659R+R661W, S675C+Q695L
  • a Cas9 protein comprises a K526D mutation and at least three additional mutations, for exampleK377E+L598P+L651 H, D397E+Y430C+L666P, Q402R+V561M+Q695L, N407P+F498I+P509L, N407H+K637N+N690I, Y450H+F553L+Q716H, Y450N+H698P+Q739K, T472A+P475H+A488V, I473F+D550N+Q739E, F478Y+N522I+L727H, K484M+Q695H+Q712R, S487Y+N504S+E573D, T496A+N609D+A728G, R654H+R691Q+H698Q or R691L+H721 R+I733V
  • a Cas9 protein comprises a K526D mutation and at least four additional mutations, for example F405L+F518L+L651 P+I724V, L423P+M465R+Y515N+K673M, R457P+K468N+R661W+G715S, E470D+I548V+A589T+Q695H, A488V+D605V+R629G+T657A or M495V+K526N+S541 P+K562E.
  • additional mutations for example F405L+F518L+L651 P+I724V, L423P+M465R+Y515N+K673M, R457P+K468N+R661W+G715S, E470D+I548V+A589T+Q695H, A488V+D605V+R629G+T657A or M495V+
  • a Cas9 protein comprises a K526D mutation and at least five additional mutations, for example R403H+N612Y+L651 P+K652E+G715S.
  • a Cas9 variant comprises a K526D mutation and at least six additional mutations, for example E387V+V561A+D618N+D628G+L680P+S701 F, R403H+M495T+N612Y+L651 P+K652E+G715S, R403H+L502P+N612Y+L651 P+K652E+G715S, R403H+K506N+N612Y+L651 P+K652E+G715S, or
  • a Cas9 protein comprises a K526D mutation and at least seven additional mutations, for example R403H+A456T+N612Y+L651 P+K652E+G715S+G728T, R403H+F498Y+N612Y+L651 P+K652E+R661 L+G715S, or R403H+Q426R+F478V+N612Y+L651 P+K652E+G715S.
  • a Cas9 protein comprises a K526D mutation and at least eight additional mutations, for example
  • a Cas9 protein comprises a K526D mutation and at least nine additional mutations, for example R403H+R457Q+F518I+N612Y+R635G+L651 P+K652E+F693Y+G715S.
  • a Cas9 protein comprises a K526D mutation and at least one additional mutation, for example Y450S, M495V, Y515N, R661X, N690I, R691Q, Q695H, or H698Q, where X is L, Q or S, preferably where X is Q or S.
  • a Cas9 variant comprises a K526D mutation and the additional mutations N692A, M694A, Q695A, and H698A (see Ikeda et al., 2019, Commun Biol 2, 371 , describing a Cas9 variant with N692A, M694A, Q695A, and H698A mutations identified as HypaCas9)
  • a Cas9 variant comprises a K526D mutation and the additional mutations K848A, K1003A, and R1060A (see Slaymaker et al., 2016, Science, 351(6268):84O 88, describing a Cas9 variant with K848A, K1003A, and R1060A mutations identified as eSPCas9(1.1)).
  • a Cas9 variant comprises a K526D mutation and the additional mutations F539S, M763I, and K890N (see Lee et al.., 2018, Nat Commun. 9:3048, describing a Cas9 variant with F539S, M763I, and K890N mutations identified as Sniper-Cas).
  • a Cas9 variant comprises a K526D mutation and the additional mutations N497A, R661A, Q695A, and Q926A (see Kleinstiver et al. 2016, Nature, 529:4900 495, describing a Cas9 variant with N497A, R661A, Q695A, and Q926A mutations identified as SpCas9-HF1 ).
  • a Cas9 variant comprises a K526D mutation and a R691A mutation (see Vakulskas et al., 2018, Nat Med 24:121601224, describing a Cas9 variant with a R691A mutation identified as HiFi Cas9).
  • Cas9 proteins described herein can further comprise one or more additional mutations, for example one or more of L169A, K810A, K848A, Q926A, R1003A, R1060A, and D1135E.
  • Cas9 variants having mutations described above can have improved specificity compared to wild-type Cas9 and other reported Cas9 variants.
  • the mutations identified above for Cas9 are suitable to improve the specificity of a Cas9 nickase, dCas9-Fokl or dCas9.
  • Cas9 proteins of the disclosure can further comprise at least one additional mutation at a residue selected from D10, E762, D839, H840, N863, H983 and D986 to decrease nuclease activity.
  • additional mutations are D10A, or D10N and H840A, H840N or H840Y.
  • said mutations result in a Cas9 nickase or in a catalytically inactive Cas9 (Ran F et al., 2013, Cell, 154(6):1380-1389; Maeder M et al., Nature Methods., 2013, 10(10):977-979).
  • a Cas9 protein can have improved specificity for recognizing alternative PAM sequences. Therefore, optionally Cas9 proteins of the disclosure can further comprise one or more additional mutations at residues D1135V/R1335Q/T1337R (QVR variant), D1135E/R1335Q/T1337R (EVR variant), D1135V/G1218R/R1335Q/T1337R (VRQR variant), D1135V/G1218R/R1335E/T1337R (VRER variant), as described in US US2016/0319260, the contents of which are incorporated by reference in their entirety.
  • a modified form of a Cas9 protein can comprise a mutation such that it can induce a SSB on a target nucleic acid (e.g., by cutting only one of the sugar-phosphate backbones of a double-strand target nucleic acid).
  • the mutation can result in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity in one or more of the plurality of nucleic acid-cleaving domains of the wild-type site directed polypeptide (e.g., Cas9 from S. pyogenes, supra).
  • the mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid, but reducing its ability to cleave the non-complementary strand of the target nucleic acid.
  • the mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid, but reducing its ability to cleave the complementary strand of the target nucleic acid. For example, residues in the wild-type exemplary S.
  • pyogenes Cas9 polypeptide such as Asp 10, His840, Asn854 and Asn856, can be mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g., nuclease domains).
  • the residues to be mutated can correspond to residues Asp 10, His840, Asn854 and Asn856 in the wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., as determined by sequence and/or structural alignment).
  • Non-limiting examples of mutations include D10A, H840A, N854A or N856A. Mutations other than alanine substitutions can be suitable.
  • a D10A mutation can be combined with one or more of H840A, N854A, or N856A mutations to produce a DNA endonuclease substantially lacking DNA cleavage activity.
  • a H840A mutation can be combined with one or more of D10A, N854A, or N856A mutations to produce a DNA endonuclease substantially lacking DNA cleavage activity.
  • a N854A mutation can be combined with one or more of H840A, D10A, or N856A mutations to produce a DNA endonuclease substantially lacking DNA cleavage activity.
  • a N856A mutation can be combined with one or more of H840A, N854A, or D10A mutations to produce a DNA endonuclease substantially lacking DNA cleavage activity.
  • DNA endonucleases that comprise one substantially inactive nuclease domain are referred to as BiickasesD
  • nickase variants of Cas9 each only cut one strand, in order to create a double-strand break it is necessary for a pair of nickases to bind in close proximity and on opposite strands of the target nucleic acid, thereby creating a pair of nicks, which is the equivalent of a double-strand break.
  • nickases can also be used to promote HDR versus NHEJ.
  • HDR can be used to introduce selected changes into target sites in the genome through the use of specific donor sequences that effectively mediate the desired changes.
  • the modified Cas9 protein can be a S. pyogenes Cas9 or an SpCas9 orthologue (e.g., S. thermophilus, S. aureus, or N. meningitides).
  • the Cas9 orthologue has at least 10% or 25% amino acid identity to the Red -II domain of SpCas9 and complete amino acid identity of any percentage between 10% or 25% and 100% to SpCas9.
  • Those skilled in the art can determine the appropriate homologous residues to be modified by sequence and/or structural alignments.
  • Identified amino acids can be modified conservatively with substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.
  • Non-limiting examples of Cas9 orthologues from other bacterial strains include, but are not limited to, Cas proteins identified in Acaryochloris marina MBIC11017; Acetohalobium arabaticum DSM 5501 ; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1 ; Alicyclobacillus acidocaldarius subsp.
  • PCC 8005 Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS10; Burkholderiales bacterium 1 1 47; Caldicrudosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrolhermahs_ 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree Clostridium botulinum Ba4 str. 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501 ; Cyanothece sp.
  • Lactobacillus salivarius ATCC 11741 Listeria innocua ; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMMO 1 ;
  • the modified Cas9 proteins of the disclosure are modified SpCas9 proteins.
  • the amino sequence of S. pyogenes Cas9 has the reference NP_269215 (NCBI) and is as follows:
  • the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence which is at least 90% identical to SEQ ID NO:1. In other embodiments, the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence which is at least 95% identical to SEQ ID NO:1. In other embodiments, the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence which is at least 96% identical to SEQ ID NO:1. In other embodiments, the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence which is at least 97% identical to SEQ ID NO:1.
  • the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence which is at least 98% identical to SEQ ID NO:1. In other embodiments, the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence which is at least 99% identical to SEQ ID NO:1. In other embodiments, the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence which is identical to SEQ ID NO:1 other than the K526D mutation and any additional mutations, e.g., one or more additional mutations described in Section 6.2.1 .
  • the amino acid sequence of the modified Cas9 protein of the disclosure comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAED AKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS
  • SEQ ID N0:2 corresponds to the sequence of the SpCas9 shown in SEQ ID NO:1 , but with a K526D mutation.
  • a modified Cas9 protein of the disclosure comprises a K526D mutation and comprises an amino acid sequence which is at least 90% identical to SEQ ID NO:2.
  • a modified Cas9 protein of the disclosure comprises a K526D mutation and comprises an amino acid sequence which is at least 95% identical to SEQ ID NO:2.
  • a modified Cas9 protein of the disclosure comprises a K526D mutation and comprises an amino acid sequence which is at least 96% identical to SEQ ID NO:2.
  • a modified Cas9 protein of the disclosure comprises a K526D mutation and comprises an amino acid sequence which is at least 97% identical to SEQ ID NO:2. In other embodiments, a modified Cas9 protein of the disclosure comprises a K526D mutation and comprises an amino acid sequence which is at least 98% identical to SEQ ID NO:2. In other embodiments, a modified Cas9 protein of the disclosure comprises a K526D mutation and comprises an amino acid sequence which is at least 99% identical to SEQ ID NO:2. In other embodiments, a modified Cas9 protein of the disclosure comprises a K526D mutation and comprises an amino acid sequence which is identical to SEQ ID NO:2.
  • the modified Cas9 proteins of the disclosure are modified S. thermophilus Cas9 proteins.
  • the amino sequence of S. thermophilus Cas9 has UniProt ID G3ECR1 and is as follows:
  • the disclosure provides Cas9 proteins in the form of a fusion protein with a second amino acid sequence, such as a nuclear localization signal, non-native tag, a transcriptional activator, a transcriptional repressor, a histone-modifying protein, an integrase, or a recombinase.
  • a second amino acid sequence such as a nuclear localization signal, non-native tag, a transcriptional activator, a transcriptional repressor, a histone-modifying protein, an integrase, or a recombinase.
  • Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 31), PKKKRRV (SEQ ID NO: 32), KRPAATKKAGQAKKKK (SEQ ID NO: 33), YGRKKRRQRRR (SEQ ID NO: 34), RKKRRQRRR (SEQ ID NO: 35), PAAKRVKLD (SEQ ID NO: 36), RQRRNELKRSP (SEQ ID NO: 37), VSRKRPRP (SEQ ID NO: 38), PPKKARED (SEQ ID NO: 39), PQPKKKPL (SEQ ID NO: 40), SALIKKKKKMAP (SEQ ID NO: 41 ), PKQKKRK (SEQ ID NO: 42), RKLKKKIKKL (SEQ ID NO: 43), REKKKFLKRR (SEQ ID NO: 44), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 45), RKCLQAGMNLEARKTKK (SEQ ID NO: 46), NQSSNFGPMKGGN
  • Exemplary second amino acid sequences include protein tags (e.g., V5-tag, FLAG-tag, myc-tag, HA-tag, GST-tag, polyHis-tag, MBP-tag), protein domains, transcription modulators, enzymes acting on small molecule substrates, DNA, RNA and protein modification enzymes (e.g., adenosine deaminase, cytidine deaminase, guanosyl transferase, DNA methyltransferase, RNA methyltransferases, DNA demethylases, RNA demethylases, dioxygenases, polyadenylate polymerases, pseudouridine synthases, acetyltransferases, deacetylase, ubiquitin-ligases, deubiquitinases, kinases, phosphatases, NEDD8-ligases, de- NEDDylases, SUMO-ligases, deSUMOylases, histone deacet
  • the disclosure provides nucleic acids (e.g., DNA or RNA) encoding modified Cas9 proteins (e.g., a Cas9 protein which is in the form of a fusion protein or a Cas9 protein which is not in the form of a fusion protein) and pluralities of nucleic acids, for example comprising a nucleic acid encoding the modified Cas9 protein and a gRNA.
  • modified Cas9 proteins e.g., DNA or RNA
  • nucleic acids e.g., DNA or RNA
  • modified Cas9 proteins e.g., a Cas9 protein which is in the form of a fusion protein or a Cas9 protein which is not in the form of a fusion protein
  • pluralities of nucleic acids for example comprising a nucleic acid encoding the modified Cas9 protein and a gRNA.
  • a nucleic acid encoding the Cas9 protein and/or gRNA can be, for example, a plasmid or a viral genome (e.g., a lentivirus, retrovirus, adenovirus, or adeno-associated virus genome).
  • Plasmids can be, for example, plasmids for producing virus particles, e.g., lentivirus particles, or plasmids for propagating the Cas9 and gRNA coding sequences in bacterial (e.g., E. coll) or eukaryotic (e.g., yeast) cells.
  • a nucleic acid encoding a Cas9 protein can, in some embodiments, further encode a gRNA.
  • a gRNA can be encoded by a separate nucleic acid (e.g., DNA or mRNA).
  • Nucleic acids encoding a Cas9 protein can be codon optimized, e.g., where at least one non-common codon or less-common codon has been replaced by a codon that is common in a host cell.
  • a codon optimized nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system.
  • a human codon-optimized polynucleotide encoding Cas9 can be used for producing a Cas9 polypeptide.
  • Nucleic acids of the disclosure can comprise one or more regulatory elements such as promoters, enhancers, and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • regulatory elements e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences.
  • Such regulatory elements are described, for example, in Goeddel, 1990, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • a tissue-specific promoter may direct expression primarily in a desired tissue of interest or in particular cell types. Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
  • a nucleic acid of the disclosure comprises one or more pol III promoter (e.g., 1 , 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1 , 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1 , 2, 3, 4, 5, or more pol I promoters), or combinations thereof, e.g., to express a Cas9 protein and a gRNA separately.
  • pol III promoters include, but are not limited to, U6 and H1 promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous Sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, 1985, Cell 41 :521-530), the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 > promoter.
  • exemplary enhancer elements include WPRE; CMV enhancers; the R-U5 sgment in LTR of HTLV-I;
  • SV40 enhancer and the intron sequence between exons 2 and 3 of rabbit -globin. It will be appreciated by those skilled in the art that the design of an expression vector can depend on such factors as the choice of the host cell, the level of expression desired, etc.
  • vector refers to a polynucleotide molecule capable of transporting another nucleic acid to which it has been linked.
  • polynucleotide vector includes a "plasmid”, which refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated.
  • plasmid refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated.
  • viral vector Another type of polynucleotide vector; wherein additional nucleic acid segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors can be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors can be referred to herein as “recombinant expression vectors", or more simply “expression vectors”, which serve equivalent functions.
  • operably linked means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence.
  • regulatory sequence is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.
  • Vectors can include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus (e.g., AAV2, AAV5, AAV7m8, AAV8), SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors.
  • retrovirus e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a
  • vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTI, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-l, pCTx-2, and pCTx-3. Other vectors can be used so long as they are compatible with the host cell.
  • a vector can comprise one or more transcription and/or translation control elements.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector.
  • the vector can be a self-inactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements.
  • Non-limiting examples of suitable eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 promoter (EF1), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphogly cerate kinase-1 locus promoter (PGK), and mouse metallothionein-l.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • LTRs long terminal repeats
  • EF1 human elongation factor-1 promoter
  • CAG chicken beta-actin promoter
  • MSCV murine stem cell virus promoter
  • PGK phosphogly cerate kinase-1 locus promoter
  • An expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector can also comprise appropriate sequences for amplifying expression.
  • the expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.
  • a promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor- regulated promoter, etc.).
  • the promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter).
  • the promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.).
  • the disclosure further provides gRNAs targeting a target sequence disclosed herein, e.g., disclosed in the Examples.
  • the gRNAs can comprise a spacer sequence, e.g., as disclosed in the Examples and 3 sgRNA sequence, for example, as described in Table 1.
  • the disclosure provides systems comprising a modified Cas9 protein of the disclosure (including fusion proteins) and a gRNA (e.g., a gRNA having a spacer sequence described in the Examples).
  • the systems can comprise a ribonucleoprotein particle (RNP) in which the Cas9 protein as described herein is complexed with a gRNA, for example a sgRNA or separate crRNA and tracrRNA.
  • RNP ribonucleoprotein particle
  • Systems of the disclosure can in some embodiments further comprise genomic DNA complexed with the Cas9 protein and the gRNA. Accordingly, the disclosure provides systems comprising a modified Cas9 protein of the disclosure, a genomic DNA, and gRNA, all complexed with one another.
  • the systems of the disclosure can exist within a cell (whether the cell is in vivo, ex vivo, or in vitro) or outside a cell (e.g., in a particle our outside of a particle).
  • the disclosure further provides particles comprising modified Cas9 proteins of the disclosure (e.g., a Cas9 protein which is not in the form of a fusion protein or a Cas9 protein which is in the form of a fusion protein) and provides particles comprising a nucleic acid encoding a modified Cas9 protein of the disclosure.
  • the particles can further comprise a gRNA, or a nucleic acid encoding the gRNA (e.g., DNA or mRNA).
  • the particles can comprise a RNP of the disclosure.
  • Exemplary particles include lipid nanoparticles, vesicles, and gold nanoparticles.
  • WO 2020/012335 the contents of which are incorporated herein by reference in their entireties, which describes vesicles that can be used to deliver gRNA molecules and Cas9 proteins to cells (e.g., complexed together as a RNP).
  • the disclosure further provides particles (e.g., virus particles) comprising a nucleic acid encoding a Cas9 protein of the disclosure.
  • the particles can further comprise a nucleic acid encoding a gRNA.
  • the disclosure further provides pluralities of particles (e.g., pluralities of virus particles).
  • Such pluralities can include a particle encoding a Cas9 protein and a different particle encoding a gRNA.
  • a plurality of particles can comprise a virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO virus particle) encoding a Cas9 protein and a second virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO virus particle) encoding a gRNA.
  • virus particle e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO virus particle
  • a second virus particle e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8
  • the disclosure further provides cells and populations of cells that can comprise a Cas9 protein (e.g., introduced to the cell as a RNP) or a nucleic acid encoding the Cas9 protein (e.g., DNA or mRNA).
  • the cells and populations of cells can be, for example, human cells such as a stem cell, e.g., a hematopoietic stem cell (HSC), a pluripotent stem cell, an induced pluripotent stem cell (iPS), or an embryonic stem cell.
  • HSC hematopoietic stem cell
  • iPS induced pluripotent stem cell
  • Methods for introducing proteins and nucleic acids to cells are known in the art.
  • a RNP can be produced by mixing a Cas9 protein and one or more guide RNAs in an appropriate buffer.
  • An RNP can be introduced to a cell, for example, via electroporation and other methods known in the art.
  • the cell populations of the disclosure can be cells in which gene editing by the systems of the disclosure has taken place, or cells in which the components of a system of the disclosure have been introduced or expressed but gene editing has not taken place, or a combination thereof.
  • a cell population can comprise, for example, a population in which at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells have undergone gene editing by a system of the disclosure.
  • Suitable excipients include, but are not limited to, salts, diluents, (e.g., Tris-HCI, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, sorbents, solvents, pH modifying agents, antioxidants, antinfective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and other components and combinations thereof.
  • salts e.g., Tris-HCI, acetate, phosphate
  • preservatives e.g., Thimerosal, benzyl alcohol, parabens
  • binders fillers, solubilizers, disintegrants, sorbents, solvents, pH modifying agents, antioxidants, antinfective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents,
  • Suitable pharmaceutically acceptable excipients can be selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. Suitable excipients and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable dosage forms for administration, e.g., parenteral administration, include solutions, suspensions, and emulsions.
  • the components of the pharmaceutical formulation can be dissolved or suspended in a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride.
  • a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride.
  • the formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1 ,3-butanedioL
  • formulations can include one or more tonicity agents to adjust the isotonic range of the formulation.
  • Suitable tonicity agents are well known in the art and include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.
  • the formulations can be buffered with an effective amount of buffer necessary to maintain a pH suitable for parenteral administration.
  • Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.
  • the formulation can be distributed or packaged in a liquid form, or alternatively, as a solid, obtained, for example by lyophilization of a suitable liquid formulation, which can be reconstituted with an appropriate carrier or diluent prior to administration.
  • the formulations can comprise a guide RNA and a Cas9 protein in a pharmaceutically effective amount sufficient to edit a gene in a cell.
  • the pharmaceutical compositions can be formulated for medical and/or veterinary use.
  • a method of altering a cell comprises contacting a eukaryotic cell (e.g., a human cell) with a nucleic acid, particle, system or pharmaceutical composition described herein.
  • a eukaryotic cell e.g., a human cell
  • Contacting a cell with a disclosed nucleic acid, particle, system or pharmaceutical composition can be achieved by any method known in the art and can be performed in vivo, ex vivo, or in vitro.
  • the methods can include obtaining one or more cells from a subject prior to contacting the cell(s) with a herein disclosed nucleic acid, particle, system or pharmaceutical composition.
  • the methods can further comprise returning or implanting the contacted cell or a progeny thereof to the subject.
  • Cas9 and gRNA as well as nucleic acids encoding Cas9 and gRNAs can be delivered to a cell by any means known in the art, for example, by viral or non-viral delivery vehicles, electroporation or lipid nanoparticles.
  • a polynucleotide encoding Cas9 and a gRNA can be delivered to a cell (ex vivo or in vivo) by a lipid nanoparticle (LNP).
  • LNPs can have, for example, a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
  • a nanoparticle can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.
  • LNPs can be made from cationic, anionic, neutral lipids, and combinations thereof.
  • Neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as 'helper lipids' to enhance transfection activity and nanoparticle stability.
  • LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Lipids and combinations of lipids that are known in the art can be used to produce a LNP. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC- cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE- polyethylene glycol (PEG).
  • DOTMA DOSPA
  • DOTAP DOTAP
  • DMRIE DC- cholesterol
  • DOTAP-cholesterol DOTAP-cholesterol
  • GAP-DMORIE-DPyPE GAP-DMORIE-DPyPE
  • PEG polyethylene glycol
  • Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2- DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 , and 7C1.
  • Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM.
  • Examples of PEG-modified lipids are: PEG-DMG, PEG- CerCI4, and PEG-CerC20.
  • Lipids can be combined in any number of molar ratios to produce a LNP.
  • the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.
  • Cas9 and/or gRNAs can be delivered to a cell via an adeno-associated viral vector (e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO serotype), or by another viral vector.
  • adeno-associated viral vector e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO serotype
  • Other viral vectors include, but are not limited to lentivirus, adenovirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplex virus.
  • a Cas9 mRNA is formulated in a lipid nanoparticle, while a sgRNA is delivered to a cell in an AAV or other viral vector.
  • one or more AAV vectors e.g., one or more AAV2, AAV5, AAV7m8, or AAV8 viral vectors
  • a Cas9 and a sgRNA are delivered using separate vectors (e.g., when the Cas9 is spCas9).
  • a Cas9 and a sgRNA are delivered using a single vector (e.g., when the Cas9 is Nme2Cas9).
  • Nme2Cas9 with its relatively small size, can be delivered with a gRNA (e.g., sgRNA) using a single AAV vector.
  • compositions and methods for delivering Cas9 and gRNAs to a cell and/or subject are further described in PCT Patent Application Publications WO 2019/102381 , WO 2020/012335, and WO 2020/053224, each of which is incorporated by reference herein in its entirety.
  • DNA cleavage can result in a single-strand break (SSB) or double-strand break (DSB) at particular locations within the DNA molecule.
  • SSB single-strand break
  • DSB double-strand break
  • Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-dependent repair (HDR) and non- homologous end-joining (NHEJ).
  • HDR homology-dependent repair
  • NHEJ non- homologous end-joining
  • These repair processes can edit the targeted polynucleotide by introducing a mutation, thereby resulting in a polynucleotide having a sequence which differs from the polynucleotides sequence prior to cleavage by a Cas9.
  • NHEJ and HDR DNA repair processes consist of a family of alternative pathways.
  • Non- homologous end-joining refers to the natural, cellular process in which a doublestranded DNA-break is repaired by the direct joining of two non-homologous DNA segments. See, e.g. Cahill et al., 2006, Front. Biosci. 11 :1958-1976.
  • DNA repair by non-homologous endjoining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair.
  • NHEJ repair mechanisms can introduce mutations into the coding sequence which can disrupt gene function.
  • NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with a modification of the polynucleotide sequence such as a loss of or addition of nucleotides in the polynucleotide sequence.
  • the modification of the polynucleotide sequence can disrupt (or perhaps enhance) gene expression.
  • Homology-dependent repair utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point.
  • the homologous sequence can be in the endogenous genome, such as a sister chromatid.
  • the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double- stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus.
  • a third repair mechanism includes microhomology-mediated end joining (MMEJ), also referred to as Alternative NHEJ (ANHEJ), in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site.
  • MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome. In some instances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.
  • Modifications of a cleaved polynucleotide by HDR, NHEJ, and/or ANHEJ can result in, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and/or gene mutation.
  • the aforementioned process outcomes are examples of editing a polynucleotide.
  • Advantages of ex vivo cell therapy approaches include the ability to conduct a comprehensive analysis of the therapeutic prior to administration.
  • Nuclease-based therapeutics can have some level of off-target effects.
  • Performing gene correction ex vivo allows a method user to characterize the corrected cell population prior to implantation, including identifying any undesirable off-target effects. Where undesirable effects are observed, a method user may opt not to implant the cells or cell progeny, may further edit the cells, or may select new cells for editing and analysis.
  • Other advantages include ease of genetic correction in iPSCs compared to other primary cell sources. iPSCs are prolific, making it easy to obtain the large number of cells that will be required for a cell-based therapy. Furthermore, iPSCs are an ideal cell type for performing clonal isolations. This allows screening for the correct genomic correction, without risking a decrease in viability.
  • Additional promoters are inducible, and therefore can be temporally controlled if the nuclease is delivered as a plasmid.
  • the amount of time that delivered protein and RNA remain in the cell can also be adjusted using treatments or domains added to change the half-life.
  • In vivo treatment would eliminate a number of treatment steps, but a lower rate of delivery can require higher rates of editing.
  • In vivo treatment can eliminate problems and losses from ex vivo treatment and engraftment.
  • An advantage of in vivo gene therapy can be the ease of therapeutic production and administration.
  • the same therapeutic approach and therapy has the potential to be used to treat more than one patient, for example a number of patients who share the same or similar genotype or allele.
  • ex vivo cell therapy typically requires using a subjects own cells, which are isolated, manipulated and returned to the same patient.
  • Progenitor cells are capable of both proliferation and giving rise to more progenitor cells, which in turn have the ability to generate a large number of cells that can in turn give rise to differentiated or differentiable daughter cells.
  • the daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
  • stem cell refers then to a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating.
  • progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues.
  • Cellular differentiation is a complex process typically occurring through many cell divisions.
  • a differentiated cell can derive from a multipotent cell that itself is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered stem cells, the range of cell types that each can give rise to can vary considerably.
  • Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity can be natural or can be induced artificially upon treatment with various factors.
  • stem cells can also be "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required.
  • Human cells described herein can be induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • An advantage of using iPSCs in the methods of the disclosure is that the cells can be derived from the same subject to which the progenitor cells are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then differentiated into a progenitor cell to be administered to the subject (e.g., an autologous cell). Because progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells from another subject or group of subjects. In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one aspect, the stem cells used in the disclosed methods are not embryonic stem cells.
  • Methods are known in the art that can be used to generate pluripotent stem cells from somatic cells. Pluripotent stem cells generated by such methods can be used in the method of the disclosure.
  • Reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described. Mouse somatic cells can be converted to ES cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc; see, e.g., Takahashi and Yamanaka, 2006, Cell 126(4): 663-76.
  • iPSCs resemble ES cells, as they restore the pluripotency-associated transcriptional circuitry and much of the epigenetic landscape.
  • mouse iPSCs satisfy all the standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission (see, e.g., Maherali and Hochedlinger, 2008, Cell Stem Cell. 3(6):595-605), and tetrapioid complementation.
  • iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency; see, e.g., 2014, Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57; Barrett et al, 2014, Stem Cells Trans Med 3: 1-6 sctm.2014-0121 ; Focosi et al, 2014, Blood Cancer Journal 4: e211.
  • the production of iPSCs can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, historically using viral vectors.
  • iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell.
  • reprogramming can be induced by the non-viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., 2010, Cell Stem Cell, 7(5):6I8- 30.
  • Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct- 3/4 or Pouf5l), Soxl, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klfl, Klf2, Klf4, Klf5, NR5A2, c-Myc, 1- Myc, n-Myc, Rem2, Tert, and LIN28.
  • Reprogramming using the methods and compositions described herein can further comprise introducing one or more of Oct- 3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell.
  • the methods and compositions described herein can further comprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming.
  • the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein.
  • the reprogramming is not affected by a method that alters the genome.
  • reprogramming can be achieved, e.g., without the use of viral or plasmid vectors.
  • Efficiency of reprogramming (the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various agents, e.g., small molecules, as shown by Shi et al., 2008, Cell-Stem Cell 2:525-528; Huangfu et al., 2008, Nature Biotechnology 26(7):795-797; and Marson et al., 2008, Cell-Stem Cell 3: 132-135.
  • an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patient-specific or disease-specific iPSCs.
  • agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HD AC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.
  • reprogramming enhancing agents include: Suberoylanilide Hydroxamic Acid (SAHA (e.g ., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC Toxin, Nullscript (4-(l,3-Dioxo-IH,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A) and other short chain fatty acids), Scriptaid, Suramin Sodium, Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate, pi valoyloxy methyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228 or
  • reprogramming enhancing agents include, for example, dominant negative forms of the HDACs (e.g, catalytically inactive forms), siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs.
  • HDACs e.g., catalytically inactive forms
  • siRNA inhibitors of the HDACs e.g., antibodies that specifically bind to the HDACs.
  • Such inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.
  • isolated clones can be tested for the expression of a stem cell marker.
  • a stem cell marker can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdfi, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl.
  • a cell that expresses Oct4 or Nanog is identified as pluripotent.
  • Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. Detection can involve not only RT-PCR, but also detection of protein markers. Intracellular markers can be best identified via RT-PCR, or protein detection methods such as immunocytochemistry, while cell surface markers are readily identified, e.g., by immunocytochemistry.
  • Pluripotency of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate into cells of each of the three germ layers.
  • teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones.
  • the cells can be introduced into nude mice and histology and/or immunohistochemistry can be performed on a tumor arising from the cells.
  • the growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.
  • Patient-specific iPS cells or cell line can be created.
  • the creating step can comprise: a) isolating a somatic cell, such as a skin cell or fibroblast, from the patient; and b) introducing a set of pluripotency-associated genes into the somatic cell in order to induce the cell to become a pluripotent stem cell.
  • the set of pluripotency-associated genes can be one or more of the genes selected from the group consisting of OCT4, SOX1 , SOX2, SOX3, SOX15, SOX18, NANOG, KLF1 , KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.
  • a biopsy or aspirate of a subjects bone marrow can be performed.
  • a biopsy or aspirate is a sample of tissue or fluid taken from the body.
  • biopsies or aspirates There are many different kinds of biopsies or aspirates. Nearly all of them involve using a sharp tool to remove a small amount of tissue. If the biopsy will be on the skin or other sensitive area, numbing medicine can be applied first.
  • a biopsy or aspirate can be performed according to any of the known methods in the art. For example, in a bone marrow aspirate, a large needle is used to enter the pelvis bone to collect bone marrow.
  • a mesenchymal stem cell can be isolated from a subject.
  • Mesenchymal stem cells can be isolated according to any method known in the art, such as from a subjects bone marrow or peripheral blood. For example, marrow aspirate can be collected into a syringe with heparin. Cells can be washed and centrifuged on a Percoll’ density gradient. Cells, such as blood cells, liver cells, interstitial cells, macrophages, mast cells, and thymocytes, can be separated using density gradient centrifugation media, Percoll’ .
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • the modified Cas9 proteins of the disclosure can be used to alter various genomic targets, for example to treat a genetic disease associated with the genomic target.
  • the methods of altering a cell are methods for altering a hemoglobin subunit gamma (HBG) gene, beta-2 microglobulin (B2M) gene, chemokine receptor 5 (CCR5) gene, empty spriacles homeobox 1 (EMX1 ) gene, FANCF2 gene, hemoglobin subunit beta (HBB), PD1 gene, hypoxanthine phosphoribosyltransferase (HPRT) gene, T Cell Receptor Alpha Constant (TRAC) or IL2RG gene.
  • HBG hemoglobin subunit gamma
  • B2M beta-2 microglobulin
  • CCR5 chemokine receptor 5
  • EX1 empty spriacles homeobox 1
  • FANCF2 gene gene
  • HBB hemoglobin subunit beta
  • PD1 hypoxanthine phosphoribo
  • the methods of altering a cell are methods for altering a breakpoint cluster region protein (BCR) gene, a Calcium Voltage-Gated Channel Auxiliary Subunit Alpha 2/Delta 4 (CACNA2D4) gene, a T Cell Receptor Beta Constant (TRBC) gene, or a Zinc Finger And SCAN Domain Containing 2 (ZSCAN2) gene.
  • BCR breakpoint cluster region protein
  • CACNA2D4 Calcium Voltage-Gated Channel Auxiliary Subunit Alpha 2/Delta 4
  • TRBC T Cell Receptor Beta Constant
  • ZSCAN2 Zinc Finger And SCAN Domain Containing 2
  • the methods of altering a cell are methods for altering a hemoglobin subunit gamma (HBG) gene, e.g., HBG1 or HBG2.
  • HBG hemoglobin subunit gamma
  • the gene identifier numbers from the Ensembl Genome Browser database for HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565, respectively, -hemoglobinopathies such as -thalassemia and Sickle Cell Disease (SCD) are caused by mutations affecting quantitatively or qualitatively the production of the adult hemoglobin (Hb) -globin chain encoded by the HBB gene.
  • SCD Sickle Cell Disease
  • the clinical severity of -hemoglobinopathies is alleviated by the co-inheritance of genetic mutations causing a sustained fetal -globin chain production at adult age, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Elevated fetal -globin levels reduces globin chain imbalance in -thalassemias and exert a potent anti-sickling effect in SCD. Disruption of the binding site for the transcriptional repressor Leukemia/lymphoma-related factor (LRF) in the HBG1 and HBG2 promoters has been identified as therapeutic target for the treatment of - hemoglobinopathies.
  • LRF Leukemia/lymphoma-related factor
  • methods of the disclosure comprise contacting a cell with a system of the disclosure (e.g., a RNP) comprising a gRNA targeting the HBG1 or HBG2 promoter, for example with a gRNA, e.g., sgRNA, having a spacer sequence or full gRNA sequence as described in WO 2020/053224.
  • a system of the disclosure e.g., a RNP
  • a gRNA e.g., sgRNA, having a spacer sequence or full gRNA sequence as described in WO 2020/053224.
  • the gRNA comprises a spacer sequence CAUUGAGAUAGUGUGGGGAA (SEQ ID NO: 50).
  • the cell is a cell from a subject having a -hemoglobinopathy such as - thalassemia or SCD, or a progeny thereof.
  • a method of the disclosure can comprise editing a HBG1 or HBG2 gene of a cell or population of cells, for example a stem cell, and transplanting the cell, population of cells, or progeny thereof to a subject having a hemoglobinopathy such as -thalassemia or SCD.
  • the cell or population of cells is autologous the subject.
  • the methods of altering a cell are methods for altering a hemoglobin subunit beta (HBB) gene. HBB mutations are associated with -thalassemia and SCD. Dever et al., 2016 Nature 539(7629):384-389.
  • the methods of altering a cell are methods for altering a CCR5 gene.
  • CCR5 has demonstrated involvement in several different disease states including, but not limited to, human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS).
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • WO 2018/119359 describes CCR5 editing by CRISPR-Cas to make loss of function CCR5 in order to provide protection against HIV infection, decrease one or more symptoms of HIV infection, halt or delay progression of HIV to AIDS, and/or decrease one or more symptoms of AIDS.
  • the methods of altering a cell are methods for altering a PD1 , B2M gene, TRAC gene, or a combination thereof.
  • CAR-T cells having PD1 , B2M and TRAC genes disrupted by CRISPR-Cas9 have demonstrated enhanced activity in preclinical glioma models. Choi et al., 2019, Journal for ImmunoTherapy of Cancer 7:309.
  • the methods of altering a cell are methods for altering a TRAC gene.
  • the methods of altering a cell are methods for altering a TRBC gene. TRAC and TRBC genes can be targeted for the generation of CAR-T cells. See, e.g., Li et al., 2020, Brief Funct Genomics 19(3): 175-182.
  • the methods of altering a cell are methods for altering a CACNA2D4 gene. Mutations in CACNA2D4 are associated with autosomal recessive cone dystrophy. Wycisk et al., 2006, Am J Hum Genet. 79(5):973-7. Thus, for example, cells with an altered CACNA2D4 gene can be used to treat subjects having autosomal recessive cone dystrophy, e.g., retinal cone dystrophy type 4.
  • Example 1 Identification of a Cas9 variant, K526D, suitable for RNP delivery
  • This Example describes studies performed to identify a Cas9 variant having both high specificity and high activity when delivered to target cells as a RNP.
  • a derivative of PX330 (Addgene #42230), where the sgRNA cassette has been removed by Ndel digestion (pX-Cas9), was used to express SpCas9 in mammalian cells.
  • pX- Cas9 plasmids encoding different mutants generated in the study were obtained by site- directed mutagenesis of the wild-type pX-Cas9 plasmid using the oligonucleotides reported in
  • sgRNAs were expressed in cells from a pUC19 plasmid containing a U6-driven sgRNA expression cassette. Desired spacer sequences were cloned as annealed oligonucleotides (see Table 3) into a double Bbsl site immediately upstream of the sgRNA scaffold, according to previously published cloning strategies.
  • U2OS and HEK293T cells were obtained from ATCC (HTB-96).
  • HEK293 cells stably expressing multiple copies of EGFP (293multiEGFP cells) have been previously described (Casini, et al., 2018, Nat. BiotechnoI 36:265 271 ).
  • Cells were cultured in DMEM (Gibco) supplemented with fetal bovine serum (FBS, 10%, Gibco), glutamine (Gibco) and penicillin/streptomycin (Gibco) and maintained at 37°C in 5% CO2 humidified atmosphere.
  • 293multiEGFP culture medium was additionally supplemented with 1 g/ml puromycin, which was removed during transfection studies.
  • Plasmid transfections were performed by seeding 10 5 HEK293T or 293multiEGFP cells in a 24 well plate the day before transfection. 400 ng of each pX-Cas9 plasmid (wild-type and mutants) together with 200 ng of pUC19-sgRNA plasmids were transfected using the T ransIT- LT 1 reagent (Mirus Bio) according to the manufacturer's protocol. Cells were then collected 7 days post transfection and analyzed by flow cytometry using a FACSCanto (BD Biosciences) to evaluate EGFP KO.
  • Mass cultures were grown at 37°C until OD600 reached 0.6, they were then transferred at 18°C and after 30 minutes IPTG (final concentration 400 mM) was added to the medium to induce SpCas9 expression. Cultures were left shaking at 18°C overnight. Bacterial pellets were then resuspended in lysis buffer (20mM Tris pH 8, 500 mM NaCI and 5 mM imidazole) supplemented with lysozyme (0.5 mg/ml), incubated for 20 minutes at 4°C shaking and lysed by sonication.
  • fractions containing SpCas9 were pooled, concentrated using a centrifugal concentrator (50000 MWCO) to reach at least 10 mg/ml and stored in aliquots at -80°C until use.
  • the purity of prepared batches was verified through SDS-PAGE and Coomassie staining.
  • Synthetic guide RNAs were obtained as separate crRNAs and tracrRNA (IDT). Spacer and target sequences are shown in Table 4.
  • RNP complexes were assembled in OptiMEM (Gibco) by incubating 6 pmol of each crRNA:tracrRNA duplex and 3 pmol of each SpCas9 recombinant protein (ratio 2:1 ) in a final volume of 50 > for 10 minutes at room temperature (about 20 °C). 1 I of TranslT-X2 (Mirus) was then added to the mix which was further incubated for 15 minutes at room temperature (about 20 °C) before dropwise addition to cell cultures (1.5x10 5 293multiGFP cells per well plated the day before transfection in a 24-well plate). Cells were then collected 7 days post- transfection for FACS analysis.
  • RNP complexes were assembled by incubating 120 pmol of each crRNA:tracrRNA duplex and 100 pmol of each SpCas9 recombinant protein (ratio 1 :1 .2) in a final volume of 5 l for 15 minutes at room temperature (about 20 °C).
  • 2x10 5 U2OS cells were electroporated using the Amaxa Nucleofector 4D device (Lonza) and the SE cell line kit (Lonza) according to the manufactures instructions. Briefly, before electroporation cells were washed once in PBS, resuspended in the appropriate amount of SE nucleofection solution (20 I per sample) and mixed with the previously assembled RNP complexes of interest.
  • a total of 25 I of cell suspension was transferred to a well of a 16-well Nucleocuvette Strip and electroporated using the DN-100 program. After the pulse, cells were left in the cuvette for 10 minutes at room temperature (about 20 °C) before being transferred to a pre-warmed 12-well plate with complete culture medium.
  • amplicon pools were Sanger sequenced (Microsynth) and the indel levels were evaluated using the TIDE webtool (shinyapps.datacurators.nl/tide/).
  • the single K526E substitution was also included because previous studies (Casini, et al., 2018, Nat. BiotechnoL 36:2650271) demonstrated its ability to particularly boost SpCas9 specificity.
  • FIG. 1A despite recovering part of the cleavage activity, all triple and the majority of double mutants still demonstrated reduced on-target editing compared to wt SpCas9.
  • the K526E variant and the K526E+R661S double mutant showed on-target editing levels comparable to those of wt SpCas9 while presenting an overall more favorable specificity profile: for this reason, these two variants were selected for further characterization.
  • K526D variant was then evaluated to verify its ability to restore the loss of SpCas9 cleavage activity observed with the K526E substitution after RNP delivery. Indel formation by the K526D mutant was thus evaluated on a benchmark set of genomic target sites previously used to characterize the K526E variant. As shown in FIGS. 3A-3B, K526D is characterized by a more favorable on-target profile compared to K526E, having increased cleavage activity on many of the sites that K526E failed to cleave (compare also with FIG. 1B).
  • the specificity profile of SpCas9 K526D was characterized by measuring editing levels at previously validated off-target sites generated by specific gRNAs after RNP delivery. Given the intrinsic increase in specificity of direct protein delivery, off-target (OT) sites which are cleaved above background levels by wt SpCas9 after RNP electroporation into cells were selected. As shown in FIG. 4A, the K526D mutant produced less off-target editing at each of the tested sites (HBB OT, CCR5 OT and EMX1 OT). Further confirmation of the increased editing specificity of the K526D mutant was obtained by calculating the on -Zoff-target ratio for the tested sites (FIG. 4B) which shows that the K526D variant has an overall more favorable editing profile than wt SpCas9.
  • Reactivation of gamma-globin expression can be achieved by altering or removing the binding sites for transcriptional repressors such as LRF or BCL11 A from the HBG promoter, to mimic the situation found in people naturally affected by hereditary persistence of fetal hemoglobin.
  • the K526D variant was evaluated in combination with this HBG-targeting gRNA by measuring both on-target cleavage and editing at the known off-target sites.
  • FIG. 6A while SpCas9 K526D was showing lower editing activity towards the HBG locus than wt SpCas9, it was also able to completely abolish off-target cleavage that could be readily detected when the gRNA was used in combination with wt SpCas9.
  • the superior editing profile of SpCas9 K526D becomes even more evident (FIG. 6B).
  • the mutations included in each of the high- fidelity SpCas9 variants evaluated are reported in Table 6.
  • each SpCas9 variant was evaluated for indel formation at an endogenous locus in HEK293T cells in combination with a guide RNA perfectly matching the sequence present in the HEK293T genome (corresponding to an A allele present in the human population) and a guide RNA perfectly matching a G allele which is present in the human population but absent in the HEK293T genome.
  • a guide RNA perfectly matching the sequence present in the HEK293T genome corresponding to an A allele present in the human population
  • a guide RNA perfectly matching a G allele which is present in the human population but absent in the HEK293T genome As shown in FIG. 7, many of the evaluated variants were able to efficiently cleave the on-target site (corresponding to the A allele), up to levels comparable to wt SpCas9. Consistently, while being able to edit the target site at good levels, the high-fidelity variants characterized by a higher number of mutations showed a slight decrease in editing activity compared to
  • the different variants When evaluated for their ability to discriminate the two alleles using the surrogate off- target model described above, the different variants showed varying levels of allele-specificity, with triple and quadruple mutants being more proficient in reducing off-target cleavage (FIG. 7). Among all the candidates, the quadruple mutants evoCas9 and DQNV were able to reduce unwanted cleavages close to the limit of detection of the assay used.
  • a modified Cas9 protein comprising a K526D mutation, wherein the position of the mutation is identified by reference to the amino acid numbering in an unmodified mature Streptococcus pyogenes Cas9 (SpCas9) as set forth in SEQ ID NO: 1.
  • modified Cas9 protein of embodiment 1 further comprising one or more additional mutations relative to an unmodified mature Streptococcus pyogenes Cas9 (SpCas9) as set forth in SEQ ID NO: 1.
  • the modified Cas9 protein of embodiment 3 or embodiment 4 which comprises a K526D mutation and one or more mutations at one or more positions selected from Y450, M495, Y515, R661 , N690, R691 , Q695, and H698.
  • M495V, R661X, and H698Q mutations n. M495V, Y515N, and R661X mutations; or o. R403H, N612Y, L651 P, K652E, and G715S mutations; wherein X is L, Q or S.
  • R661 E mutation 25.
  • the modified Cas9 protein of any one of 16 to 31 which comprises a H698Q mutation.
  • the modified Cas9 protein of embodiment 16 which comprises Y515N, K526D, and R661 L mutations.
  • the modified Cas9 protein of any one of embodiments 1 to 36 which is a modified S. pyogenes Cas9. 38.
  • modified Cas9 protein of embodiment 1 wherein the amino acid sequence of the modified Cas9 protein comprises an amino acid sequence which is 100% identical to SEQ ID NO:2.
  • the modified Cas9 protein of any one of embodiments 1 to 36 which is a modified S. pyogenes Cas9 orthologue.
  • a fusion protein comprising the modified Cas9 protein of any one of embodiments 1 to 46 fused to a second amino acid sequence.
  • the fusion protein of embodiment 47 wherein the second amino acid sequence comprises a non-native tag.
  • the second amino acid sequence comprises a transcriptional activator, a transcriptional repressor, a histone-modifying protein, an integrase, or a recombinase.
  • nucleic acid of embodiment 51 which is a plasmid.
  • nucleic acid of embodiment 51 which is a viral genome.
  • nucleic acid of embodiment 53, wherein the viral genome is an adeno- associated virus (AAV) genome.
  • AAV adeno-associated virus
  • AAV genome is an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhW genome.
  • nucleic acid of embodiment 55, wherein the AAV genome is an AAV5 genome.
  • nucleic acid of embodiment 55, wherein the AAV genome is an AAV8 genome.
  • nucleic acid of embodiment 55, wherein the AAV genome is an AAV9 genome.
  • nucleic acid of embodiment 55, wherein the AAV genome is an AAVrh8r genome.
  • nucleic acid of embodiment 54 wherein the AAV genome is an AAV2, AAV5, AAV7m8, or AAV8 genome.
  • nucleic acid of any one of embodiments 51 to 63 further encoding a gRNA.
  • a plurality of nucleic acids comprising (a) the nucleic acid of any one of embodiments 51 to 63, and (b) a nucleic acid encoding a gRNA.
  • a system comprising the modified Cas9 protein of any one of embodiments 1 to 46 or the fusion protein of any one of embodiments 47 to 50 and a gRNA.
  • RNP ribonucleoprotein
  • gRNA is a gRNA for editing a human hemoglobin subunit gamma (HBG) gene, a HBB gene, B2M gene, chemokine receptor 5 (CCR5) gene, EMX1 gene, FANCF2 gene, HPRT gene, PD1 gene or IL2RG gene.
  • HBG human hemoglobin subunit gamma
  • B2M B2M gene
  • CCR5 chemokine receptor 5
  • EMX1 gene FANCF2 gene
  • HPRT gene HPRT gene
  • PD1 gene or IL2RG gene IL2RG gene
  • gRNA is a gRNA for editing a human BCR gene, CACNA2D4 gene, TRAC gene, TRBC gene, or ZSCAN2 gene.
  • nucleic acid comprising a spacer comprising the sequence CAUUGAGAUAGUGUGGGGAA (SEQ ID NO: 50).
  • gRNA is a gRNA for editing a human B2M gene.
  • gRNA is a gRNA for editing a human chemokine receptor 5 (CCR5) gene.
  • nucleic acid a gRNA for editing a human BCR gene.
  • nucleic acid, plurality of nucleic acids, or system of embodiment 71 wherein the gRNA is a gRNA for editing a human CACNA2D4 gene.
  • nucleic acid, plurality of nucleic acids, or system of embodiment 71 wherein the gRNA is a gRNA for editing a human TRBC gene.
  • nucleic acid a gRNA for editing a human ZSCAN2 gene.
  • a particle comprising the modified Cas9 protein of any one of embodiments 1 to 46, the fusion protein of any one of embodiments 47 to 50, the nucleic acid of any one of embodiments 51 to 64 and 68 to 86, the plurality of nucleic acids of any one of embodiments 65 and 68 to 86, or the system of any one of embodiments 66 to 86.
  • the particle of embodiment 87 which is a lipid nanoparticle, a vesicle, a gold nanoparticle, or a viral particle.
  • invention 88 which is a lipid nanoparticle.
  • the particle of embodiment 88 which is a vesicle. 91 .
  • the particle of embodiment 88 which is a gold nanoparticle.
  • invention 88 which is a viral particle.
  • invention 92 which is an adeno-associated virus (AAV) particle.
  • AAV adeno-associated virus
  • AAV particle is an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO particle.
  • AAV particle is an AAV2, AAV5, AAV7m8, or AAV8 particle.
  • a pharmaceutical composition comprising the modified Cas9 protein of any one of embodiments 1 to 46, the fusion protein of any one of embodiments 47 to 50, the nucleic acid of any one of embodiments 51 to 64 and 68 to 86, the plurality of nucleic acids of any one of embodiments 65 and 68 to 86, the system of any one of embodiments 66 to 86, or the particle of any one of embodiments 87 to 95 and at least one pharmaceutically acceptable excipient.
  • a cell comprising the modified Cas9 protein of any one of embodiments 1 to 46, the fusion protein of any one of embodiments 47 to 50, the nucleic acid of any one of embodiments 51 to 64 and 68 to 86, the plurality of nucleic acids of any one of embodiments 65 and 68 to 74, the system of any one of embodiments 66 to 86, or the particle of any one of embodiments 87 to 95.
  • invention 98 The cell of embodiment 97, which is a human cell.
  • stem cell is a hematopoietic stem cell (HSC), a pluripotent stem cell, or an induced pluripotent stem cell (iPS).
  • HSC hematopoietic stem cell
  • iPS induced pluripotent stem cell
  • stem cell is an embryonic stem cell.
  • 105. A method for altering a cell, the method comprising contacting the cell with the modified Cas9 protein of any one of embodiments 1 to 46, the fusion protein of any one of embodiments 47 to 50, the nucleic acid of any one of embodiments 51 to 64 and 68 to 86, the plurality of nucleic acids of any one of embodiments 65 and 68 to 86, the system of any one of embodiments 66 to 86, the particle of any one of embodiments 876 to 95 or the pharmaceutical composition of embodiment 96.
  • invention 106 which comprises lipid-mediated delivery of the system to the cell, optionally wherein the lipid-mediated delivery is cationic lipid-mediated delivery.
  • invention 106 which comprises delivery of the system to the cell by nucleofection.
  • embodiment 105 which comprises contacting the cell with the modified Cas9 protein of any one of embodiments 1 to 46 or the fusion protein of any one of embodiments 47 to 50.
  • stem cell is a hematopoietic stem cell (HSC), a pluripotent stem cell, or an induced pluripotent stem cell (iPS).
  • HSC hematopoietic stem cell
  • iPS induced pluripotent stem cell
  • stem cell is an embryonic stem cell.
  • hemoglobinopathy has sickle cell disease or -thalassemia.
  • hemoglobinopathy is sickle cell disease.
  • hemoglobinopathy is -thalassemia.

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Abstract

L'invention concerne des protéines Cas9 modifiées ayant une mutation K526D, des acides nucléiques codant pour les protéines Cas9 modifiées, des ribonucléoprotéines (RNP) comprenant les protéines Cas9 modifiées transformées en complexe afin d'obtenir des ARNg, des systèmes comprenant les protéines Cas9 modifiées, des particules comprenant ce qui précède et des utilisations de ce qui précède, par exemple pour modifier l'ADN génomique d'une cellule.
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