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WO2025059585A1 - Nucléases ingénierisées et chimériques - Google Patents

Nucléases ingénierisées et chimériques Download PDF

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Publication number
WO2025059585A1
WO2025059585A1 PCT/US2024/046782 US2024046782W WO2025059585A1 WO 2025059585 A1 WO2025059585 A1 WO 2025059585A1 US 2024046782 W US2024046782 W US 2024046782W WO 2025059585 A1 WO2025059585 A1 WO 2025059585A1
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sequence
engineered
seq
nos
guide polynucleotide
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Inventor
Brian C. Thomas
Lisa ALEXANDER
Alan Brooks
Christopher Brown
Cristina Noel BUTTERFIELD
Daniela S.A. Goltsman
Jyun-Liang LIN
Isabel NOCEDAL
Kartik RALLAPALLI
Morayma TEMOCHE-DIAZ
Sarah Laperriere
Leanna MONTELEONE
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Metagenomi Inc
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Metagenomi Inc
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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 [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • Cas enzymes along with their associated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (RNAs) appear to be a pervasive (-45% of bacteria, -84% of archaea) component of prokaryotic immune systems, serving to protect such microorganisms against non-self-nucleic acids, such as infectious viruses and plasmids by CRISPR-RNA guided nucleic acid cleavage. While the deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse, containing a wide variety of nucleic acid-interacting domains.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR DNA elements have been observed as early as 1987, the programmable endonuclease cleavage ability of CRISPR/Cas complexes has only been recognized relatively recently, leading to the use of recombinant CRISPR/Cas systems in diverse DNA manipulation and gene editing applications.
  • engineered nuclease systems comprising: a) an engineered endonuclease comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence.
  • the engineered guide polynucleotide is a single guide nucleic acid.
  • the engineered guide polynucleotide is a dual guide nucleic acid.
  • the engineered guide polynucleotide is RNA. In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3954-4050. In some embodiments, the engineered endonuclease binds non- covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is fused to the engineered guide polynucleotide.
  • fusion endonucleases comprising: a) an N-terminal sequence comprising at least part of a RuvC domain, a REC domain, or an HNH domain of an endonuclease and having at least 60% sequence identity to any one of SEQ ID NOs: 4222-4224 and 4227; and b) a C-terminal sequence comprising WED, TOPO, or CTD domains of an endonuclease and having at least 60% sequence identity to any one of SEQ ID NOs: 4173-4221, 4225-4226, 4228-4238, wherein the N-terminal sequence and the C- terminal sequence do not naturally occur together in a same reading frame.
  • engineered nuclease systems comprising: a) an engineered endonuclease comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4051-4078; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence.
  • the engineered guide polynucleotide is a single guide nucleic acid.
  • the engineered guide polynucleotide is a dual guide nucleic acid.
  • the engineered guide polynucleotide is RNA. In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 4094- 4105. In some embodiments, the engineered endonuclease binds non-covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is fused to the engineered guide polynucleotide.
  • fusion endonucleases comprising: a) an N-terminal sequence comprising at least part of a RuvC domain, a REC domain, or an HNH domain of an endonuclease and having at least 60% sequence identity to any one of SEQ ID NOs: 4239-4252 and 4255; and b) a C-terminal sequence comprising WED, TOPO, or CTD domains of an endonuclease and having at least 60% sequence identity to any one of SEQ ID NOs: 4253-4254, wherein the N-terminal sequence and the C-terminal sequence do not naturally occur together in a same reading frame.
  • engineered nuclease systems comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3749-3787 and 3825-3861.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3749-3787 and 3825-3861.
  • the engineered guide polynucleotide is a single guide nucleic acid.
  • the engineered guide polynucleotide is a dual guide nucleic acid.
  • the engineered guide polynucleotide is RNA.
  • the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.
  • the endonuclease is covalently linked to the engineered guide polynucleotide.
  • the endonuclease is fused to the engineered guide polynucleotide.
  • engineered nuclease systems comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3710-3748 and 3788-3824.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3710-3748 and 3788-3824.
  • the engineered guide polynucleotide is a single guide nucleic acid.
  • the engineered guide polynucleotide is a dual guide nucleic acid.
  • the engineered guide polynucleotide is RNA.
  • the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.
  • the endonuclease is covalently linked to the engineered guide polynucleotide.
  • the endonuclease is fused to the engineered guide polynucleotide.
  • engineered nuclease systems comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAV S 1 gene or within an intron of the AAV S 1 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4106-4122.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 4106-4122. In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the engineered endonuclease binds non- covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is fused to the engineered guide polynucleotide.
  • engineered nuclease systems comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4123-4172.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 4123-4172.
  • the engineered guide polynucleotide is a single guide nucleic acid.
  • the engineered guide polynucleotide is a dual guide nucleic acid.
  • the engineered guide polynucleotide is RNA.
  • the engineered endonuclease binds non- covalently to the engineered guide polynucleotide.
  • the endonuclease is covalently linked to the engineered guide polynucleotide.
  • the endonuclease is fused to the engineered guide polynucleotide.
  • modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.
  • the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the modification is in vitro.
  • the modification is in vivo. In some embodiments, the modification is ex vivo.
  • the gRNA is encoded by a sequence having any one of SEQ ID NOs: 251-260, 271-274, and 279-290.
  • the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 261-270, 275-278, and 291-302.
  • Described herein, in certain embodiments, are methods of modifying an albumin gene comprising contacting the albumin gene using an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3749-3787 and 3825-3861.
  • Described herein, in certain embodiments, are methods of modifying a HAO 1 gene comprising contacting the HA01 gene using an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3710-3748 and 3788-3824.
  • Described herein, in certain embodiments, are methods of modifying an AAVS1 gene comprising contacting the AAVS1 gene using an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the AAVS1 gene or within an intron of the AAVS1 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4106-4122.
  • Described herein, in certain embodiments, are methods of modifying a TRAC gene comprising contacting the TRAC gene using an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4123-4172.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is an immortalized cell.
  • the cell is an insect cell.
  • the cell is a yeast cell.
  • the cell is a plant cell.
  • the cell is a fungal cell.
  • the cell is a prokaryotic cell.
  • the cell is an A549, HEK- 293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.
  • the cell is an engineered cell.
  • the cell is a stable cell.
  • FIG. 1 depicts a three-dimensional structure prediction of the MG71-2 effector.
  • the MG71-2 chassis is shown in light grey, while the PAM-interacting and WED domains are shown in dark grey.
  • the star represents the breakpoint for swapping the PID and WED domains.
  • FIG. 2 depicts PCR results showing the presence of amplified cleavage bands.
  • Nuclease MG71-2 was used as a backbone for chimeragenesis. Three breakpoints were selected, which retain different amounts of the parent nuclease sequence. Two homologous nucleases, MG71-1 and MG18-1, were selected for domains swaps. The chimeras were tested with three sgRNA (MG71-2, MG71-1, and MG18-1). The presence of amplified cleavage bands indicates successful in vitro cleavage activity.
  • FIGs. 3A-3D depict bar plots showing editing activity (Indel (%)) for the chimeric nucleases.
  • the chimeric nucleases were screened in K562 cells via nucleofection of mRNA (500 ng) and single guide (150 pmol). Editing activity was detected viaNGS. Error bars are s.d. of two replicates.
  • FIG. 4 depicts a bar plot showing editing activity (Indel (%)) of MG3-6 3-8 150-1.
  • MG3-6_3-8_150-l was screened in K562 cells via nucleofection of mRNA (500 ng) and guide (150 pmol). Editing activity was detected via NGS. Error bars are s.d. of two replicates.
  • FIG. 5 depicts the predicted structure of an MG3-6/3-4 single-guide RNA (SEQ ID NO: 35) at a temperature of 37 °C.
  • FIG. 6 depicts a graph showing gene editing outcomes (% indel) at the DNA level for mouse HA01 guide 7 and mouse ALB guide 34 - round 1 engineering.
  • FIG. 7 depicts a graph showing the correlation between relative indel % between different engineered versions of MG3-6/3-4 mouse HA01 guide 7 and mouse ALB guide 34
  • FIG. 8 depicts a graph showing gene editing outcomes (% indel) at the DNA level for mouse HA01 guide 7 and mouse ALB guide 34 - round 2 engineering.
  • FIG. 9 depicts a graph showing the correlation between relative indel % between different engineered versions of MG3-6/3-4 mouse HA01 guide 7 and mouse ALB guide 34
  • FIGs. 10A-10B depict design of MG71 chimeras.
  • FIG. 10A shows a three- dimensional cartoon structure of the MG71-2. The MG71-2 chassis is shown in light grey, while the WED PAM-interacting domains are shown in dark grey. The residues used as breakpoints for chimeragenesis are depicted using the space filling representation (N1106, SI 133, and A1095).
  • FIG. 10B shows multiple sequence alignment of MG71-2 with its close homologs used for determining the breakpoints in homologs.
  • FIG. 11 depicts in vitro PAMs of MG71-2 chimeras.
  • FIG. 12 depicts in vitro PAMs of MG71-2 chimeras.
  • the chimeras were expressed in an in vitro transcription/translation reaction, then incubated with guide and 8N plasmid library. Cut plasmids were then sequenced by NGS and aligned to generate the PAM. Most PAMs match the parent PID domain, sometimes with additional minor base preferences.
  • FIG. 12 depicts in vitro PAMs of MG71-2 chimeras.
  • the chimeras were expressed in an in vitro transcription/translation reaction, then incubated with guide and 8N plasmid library. Cut plasmids were then sequenced by NGS and aligned to generate the PAM. Most PAMs match the parent PID domain, sometimes with additional minor base preferences.
  • FIG. 13 depicts in vitro PAMs of MG71-2 ASR chimeras.
  • the zeroth nucleotide “T” is masked as it represents the constant flanking base adjacent to the spacer in the 5N PAM enrichment library (TNNNNN).
  • FIG. 14 depicts in vitro PAMs of MG71-2 chimeras.
  • the zeroth nucleotide “T” is masked as it represents the constant flanking base adjacent to the spacer in the 5N PAM enrichment library (TNNNNN).
  • FIG. 15 depicts pooled screening of MG71 chimeras at PAH p.R408W SNV in engineered immortalized K562 cells.
  • Each nuclease was delivered as codon-optimized mRNA along with 10 guide RNAs that target the SNV of interest. Guides are offset from each other by 1 nt. Each guide’s cut site lies within 10 nt of the desired edit site. Editing efficiency was evaluated by NGS.
  • FIG. 16 depicts a nuclease activity of MG71 chimeras at safe harbor AAVS1 locus in the immortalized engineered K562 cells.
  • Guide RNAs Al, Bl, Cl, DI, El, Gl, A2, C2, and D2 were designed to target different regions of AAVS1 and were tested with selected MG71-54, MG71-55, and MG71-56.
  • Spacers targeting Cl and C2 region of AAVS1 resulted in the editing levels of about 5% whereas spacers at Al, DI, and D2 reached >80% InDeis.
  • MG71-56 showed slightly improved levels of editing compared to MG71-54 and MG71-55.
  • FIG. 17 depicts targeting therapeutically relevant sites with MG71 chimeras in the immortalized engineered K562 and Hepal-6 cell lines.
  • MG71-54, MG71-55, and MG71-56 show editing at both PAH c. 1315+1G>A and PAH p.R408W position in human (K562 engineered cell line) and mouse (Hepal-6) cell lines.
  • active chimeras include MG71-58 which showed activity at PAH c.1315+1G>A and PAH p.R408W position in human (K562 engineered cell line) while MG71-59 showed activity at in K562 engineered cells at PAH ampli.R408W target and in Hepal-6 cells showed activity at PAH c.1066-11G>A and PAH
  • FIGs. 18A-18B depict design of MG87 chimeras.
  • FIG. 18A shows a three- dimensional cartoon structure of the MG87-21. The predicted structure is aligned with the R- loop taken from the closest reported nuclease in literature. The MG87-21 chassis is shown in light grey, while the WED PAM-interacting domains are shown in black. The residues used as breakpoints for chimeragenesis are highlighted (D962 in MG87-21 and D972 in MG87- 70).
  • FIG. 18B shows multiple sequence alignment of MG87s used for determining the breakpoints in MG87-21 and MG87-70 homologs.
  • FIG. 19 depicts in vitro PAMs of MG87-21 “reverse” chimeras.
  • the chimeras were expressed in an in vitro transcription/translation reaction, then incubated with guide and 8N plasmid library. Cut plasmids were then sequenced by NGS and aligned to generate the PAM. Most PAMs match the parent PID domain, sometimes with additional minor base preferences.
  • FIG. 20 depicts in vitro PAMs of MG87-70 “reverse” chimeras.
  • the chimeras were expressed in an in vitro transcription/translation reaction, then incubated with a guide and 8N plasmid library. Cut plasmids were sequenced by NGS and aligned to generate the PAM. Most PAMs match the parent PID domain, sometimes with additional minor base preferences.
  • FIG. 21 depicts MG87-21 “reverse” chimeras outperformed parent enzyme in the mammalian cells. Upon testing the MG87-21 chimeras at TRAC and AAVS1 loci in the immortalized K562 cells, MG87-107 and MG87-110 demonstrated higher indel activity than the parent MG87-21 at most tested guides.
  • FIGs. 22A-22B depicts MG3 chimeras are active gene editors in the mammalian cells. 500 ng of codon-optimized mRNA and 150 pmol of chemically synthesized guide RNA was delivered to 100k K562 cells via nucleofection. Editing was assessed via NGS.
  • FIG. 22A shows MG3-6_3-8_3-78 is robustly active at the AAVS1 locus, showing editing >95%.
  • FIG. 22B shows MG3-6_3-8_3-90 is active at the TRAC locus.
  • FIG. 23 depicts MG3-6_3-8_3-96 chimera is an active gene editor in the mammalian cells. 500 ng of codon-optimized mRNA and 150 pmol of chemically synthesized guide RNA was delivered to 100k K562 cells via nucleofection. Editing was assessed via NGS.
  • FIGs. 24A-24C depict MG3 chimeras display high specificity in the mammalian cells.
  • Cells were assessed for double strand break (DSB) formation by co-nucleofection mRNA, gRNA, and annealed dsODN.
  • the dsODN can be incorporated into DSBs and used as a priming site for NGS library prep, allowing for unbiased sampling of all DSBs created in the cell.
  • FIG. 24A shows the percent of dsODN reads coming from the desired target site is mapped for up to 3 replicates for nuclease MG3-6_3-8_3-78. For 6/7 guides, all the reads passing analysis criteria come from the on-target site.
  • FIG. 24B shows the percent of dsODN reads coming from the desired target site is mapped for up to 3 replicates for nuclease MG3- 6 3-8 3-38. For 6/7 guides, all the reads passing analysis criteria come from the on-target site. Guide P1F4 has lower reads from the on-target for the one replicate that passed sequencing QC.
  • FIG. 24C shows the identified off-target sites (SEQ ID NOs: 4472 and 4472-4475, respectively, in order of appearance) for guide P1F4 are shown relative to the on- target sequence (top, NNNMWYY represents the PAM). Dots denote matching position. The boxed bases represent changes from the on target. 75% of reads come from the on-target site and 3 off-targets are seen.
  • SEQ ID NOs: 1-27 and 771-862 show the full-length peptide sequences of MG3-6 chimeric nucleases.
  • SEQ ID NO: 108 shows the nucleotide sequence of an MG3-6/3-4 nuclease containing 5' UTR, NLS, CDS, NLS, 3' UTR, and polyA tail.
  • SEQ ID NO: 722 shows the nucleotide sequence of a MG3-6/3-4 guide sgRNA scaffold.
  • SEQ ID Nos: 28-45, 605-610, 646-695, 863, 1789-1826, and 4380-4425 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6 chimeric nuclease.
  • SEQ ID NO: 603 shows the DNA coding sequence for MG3-6/3-4.
  • SEQ ID NO: 604 shows the protein sequence of the MG3-6/3-4 cassette coding sequence.
  • SEQ ID NOs: 109-110 and 2842-2854 show the full-length peptide sequences of MG29-1 chimeric nucleases.
  • SEQ ID NOs: 111-113 show the nucleotide sequences of sgRNAs engineered to function with an MG29-1 chimeric nuclease.
  • SEQ ID NO: 696 shows a N-terminal peptide sequence (1-742) of MG3-6.
  • SEQ ID NOs: 697-721 show C-terminal peptide sequences.
  • SEQ ID NOs: 4374-4379 and 3862-3915 show the full-length peptide sequences of MG71-2 chimeric nucleases.
  • SEQ ID NOs: 3954-4050 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 chimeric nuclease.
  • SEQ ID NOs: 4051-4078 show the full-length peptide sequences ofMG87 reverse chimeric nucleases.
  • SEQ ID NOs: 4094-4105 show the nucleotide sequences of sgRNAs engineered to function with a MG87 reverse chimeric nucleases.
  • SEQ ID NOs: 67-86, 3749-3787, and 3825-3861 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target albumin.
  • SEQ ID NOs: 119-138 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target TRAC.
  • SEQ ID NOs: 139-158 show the DNA sequences of TRAC target sites.
  • SEQ ID NOs: 922-924 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-3 nuclease in order to target TRAC.
  • SEQ ID NOs: 925-927 show the DNA sequences of TRAC target sites.
  • SEQ ID NOs: 972-991 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-7 nuclease in order to target TRAC.
  • SEQ ID Nos: 992-1011 show the DNA sequences of TRAC target sites.
  • SEQ ID NOs: 1088-1183, 1280-1320, 2390-2485, and 2582-2617 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-8 nuclease in order to target TRAC.
  • SEQ ID NOs: 4123-4172 the nucleotide sequences of sgRNAs engineered to function with MG3 chimeras.
  • SEQ ID NOs: 1184-1279, 1321-1361, 2486-2581, and 2618-2653 show the DNA sequences of TRAC target sites.
  • SEQ ID NOs: 159-184 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target B2M.
  • SEQ ID Nos: 185-210 show the DNA sequences of B2M target sites.
  • SEQ ID NOs: 211-251 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target TRBC1.
  • SEQ ID Nos: 252-292 show the DNA sequences of TRBC1 target sites.
  • SEQ ID NOs: 293-337 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target TRBC2.
  • SEQ ID Nos: 338-382 show the DNA sequences of TRBC2 target sites.
  • SEQ ID NOs: 383-477 and 1392-1489 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target ANGPTL3.
  • SEQ ID NOs: 478-572 and 1490-1587 show the DNA sequences of ANGPTL3 target sites.
  • SEQ ID NOs: 2120-2215 and 2312-2350 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-8 nuclease in order to target ANGPTL3.
  • SEQ ID Nos: 2216-2311 and 2351-2389 show the DNA sequences of ANGPTL3 target sites.
  • SEQ ID NOs: 573-587 and 1362-1376 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target PCSK9.
  • SEQ ID Nos: 588-602 and 1377-1391 show the DNA sequences of PCSK9 target sites.
  • SEQ ID NOs: 723-738 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-8 nuclease in order to target VCP R155.
  • SEQ ID NOs: 739-754 show the DNA sequences of VCPR155 target sites.
  • SEQ ID NOs: 755-762 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target VCP R155.
  • SEQ ID NOs: 763-770 show the DNA sequences of VCPR155 target sites.
  • SEQ ID NOs: 928-949 show the nucleotide sequences of sgRNAs engineered to function with an MG3 -6/3 -3 nuclease in order to target AAV S 1.
  • SEQ ID NOs: 950-971 show the DNA sequences of AAVS1 target sites.
  • SEQ ID NOs: 1012-1049 show the nucleotide sequences of sgRNAs engineered to function with an MG3 -6/3 -7 nuclease in order to target AAV S 1.
  • SEQ ID NOs: 1050-1087 show the DNA sequences of AAVS1 target sites.
  • SEQ ID NOs: 4106-4122 the nucleotide sequences of sgRNAs engineered to function with MG3 chimeras.
  • SEQ ID NOs: 1588-1656 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-7 nuclease in order to target M. musculus GPR146.
  • SEQ ID NOs: 1657-1725 show the DNA sequences of M. musculus GPR146 target sites.
  • SEQ ID NOs: 1726-1744 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target M. musculus APOA1.
  • SEQ ID NOs: 1745-1763 show the DNA sequences of M. musculus APOA1 target sites.
  • SEQ ID NOs: 1764-1774 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target H. sapiens APOA1.
  • SEQ ID NOs: 1775-1785 show the DNA sequences of H. sapiens APOA1 target sites.
  • SEQ ID NOs: 1866-1961 and 2058-2088 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-8 nuclease in order to target mouse APOA1.
  • SEQ ID NOs: 1962-2057 and 2089-2119 show the DNA sequences of mouse APOA1 target sites.
  • SEQ ID NOs: 611-633 and 1789-1826 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target M. musculus HA01.
  • SEQ ID NOs: 1827-1865, 3710-3748, and 3788-3824 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target human HAO I .
  • SEQ ID NOs: 87-102, 118, 634-645, and 1786-1787 show primer sequences [0101]
  • SEQ ID NOs: 103-107, 920-921, and 1788 show plasmid sequences.
  • nucleotide refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides.
  • Nucleotides are monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • the term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof.
  • ATP adenosine triphosphate
  • UDP uridine triphosphate
  • CTP cytosine triphosphate
  • GTP guanosine triphosphate
  • deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores) or quantum dots.
  • Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.
  • Fluorescent labels of nucleotides include but are not limited fluorescein, 5 -carboxyfluorescein (FAM), 2'7'- dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS).
  • FAM 5 -carboxyfluorescein
  • JE 2'7'- dimethoxy-4'5-dichloro-6-carboxyfluorescein
  • rhodamine
  • fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA] ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein- 15 -
  • nucleotide encompasses chemically modified nucleotides.
  • An exemplary chemically- modified nucleotide is biotin-dNTP.
  • biotinylated dNTPs include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin- 11-dUTP, biotin- 16-dUTP, biotin-20-dUTP).
  • polynucleotide oligonucleotide
  • nucleic acid a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form.
  • Contemplated polynucleotides include a gene or fragment thereof.
  • Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.
  • loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short
  • a T means U (Uracil) in RNA and T (Thymine) in DNA.
  • a polynucleotide can be exogenous or endogenous to a cell and/or exist in a cell-free environment.
  • the term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer.
  • Non-limiting examples of modifications include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholines, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • peptide refers to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary or tertiary structure (e.g, domains).
  • amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component.
  • amino acid and amino acids refer to natural and non-natural amino acids, including, but not limited to, modified amino acids.
  • Modified amino acids include amino acids that have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid.
  • amino acid includes both D-amino acids and L-amino acids.
  • non-native refers to a nucleic acid or polypeptide sequence that is non-naturally occurring.
  • Non-native refers to a non-naturally occurring nucleic acid or polypeptide sequence that comprises modifications such as mutations, insertions, or deletions.
  • the term non-native encompasses fusion nucleic acids or polypeptides that encodes or exhibits an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) of the nucleic acid or polypeptide sequence to which the non-native sequence is fused.
  • a non-native nucleic acid or polypeptide sequence includes those linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.
  • operably linked refers to an arrangement of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein an operation (e.g., movement or activation) of a first genetic element has some effect on the second genetic element.
  • the effect on the second genetic element can be, but need not be, of the same type as operation of the first genetic element.
  • two genetic elements are operably linked if movement of the first element causes an activation of the second element.
  • a regulatory element which may comprise promoter and/or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.
  • a “vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles.
  • the vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.
  • a “functional fragment” of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence.
  • a biological activity of a DNA sequence includes its ability to influence expression in a manner attributed to the full- length sequence.
  • engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to an object that has been modified by human intervention. For example, the terms refer to a polynucleotide or polypeptide that is non-naturally occurring.
  • An engineered peptide has, but does not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein.
  • low sequence identity e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity
  • VPR and VP64 domains are synthetic transactivation domains.
  • Non-limiting examples include the following: a nucleic acid modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid synthesized in vitro with a sequence that does not exist in nature; a protein modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein acquiring a new function or property.
  • An “engineered” system comprises at least one engineered component.
  • the term “tracrRNA” or “tracr sequence” means trans-activating CRISPR RNA.
  • tracrRNA encompasses a nucleic acid that can be at least about 60% identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from .S'. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides.
  • a wild type exemplary tracrRNA e.g., a tracrRNA from .S'. pyogenes, S. aureus, etc
  • a tracrRNA sequence has at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from .S'. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides.
  • Type II tracrRNA sequences can be predicted on a genome sequence by identifying regions with complementarity to part of the repeat sequence in an adjacent CRISPR array.
  • a “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid.
  • a guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA.
  • a guide nucleic acid can include a crRNA or a tracrRNA or a combination of both.
  • guide nucleic acid encompasses an engineered guide nucleic acid and a programmable guide nucleic acid to specifically bind to the target nucleic acid.
  • a portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid.
  • the strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid is the complementary strand.
  • the strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore is not complementary to the guide nucleic acid is called noncomplementary strand.
  • a guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.”
  • a guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.”
  • the term “guide nucleic acid” is inclusive, referring to both single guide nucleic acids and double guide nucleic acids.
  • a guide nucleic acid may comprise a segment referred to as a “nucleic acid-targeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.”
  • a nucleic acid-targeting segment can include a subsegment referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment.”
  • sequence identity or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm.
  • Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https://blast.ncbi.nlm.nih.gov); CLUSTALW with parameters of ; the Smith-Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MALLT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER hmmalign
  • RuvC III domain refers to a third discontinuous segment of a RuvC endonuclease domain (the RuvC nuclease domain being comprised of three discontiguous segments, RuvC I, RuvC II, and RuvC III).
  • a RuvC domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam HMM PL18541 for RuvC III).
  • HMMs Hidden Markov Models
  • WED domain refers to a domain (e.g., present in a Cas protein) interacting primarily with repeat: anti -repeat duplex of the sgRNA and PAM duplex.
  • a WED domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences.
  • HMMs Hidden Markov Models
  • PAM interacting domain refers to a domain interacting with the protospacer-adjacent motif (PAM) external to the seed sequence in a region targeted by a Cas protein.
  • PAM-interacting domains include, but are not limited to, Topoisomerase-homology (TOPO) domains and C-terminal domains (CTD) present in Cas proteins.
  • TOPO Topoisomerase-homology
  • CTD C-terminal domains
  • a PAM interacting domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences.
  • HMMs Hidden Markov Models
  • REC domain refers to a domain (e.g., present in a Cas protein) comprising at least one of two segments (REC 1 or REC2) that are alpha helical domains thought to contact the guide RNA.
  • a REC domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam PF19501 for domain RECI).
  • HMMs Hidden Markov Models
  • BH domain refers to a domain (e.g., present in a Cas protein) that is a bridge helix between NUC and REC lobes of a Type II Cas enzyme.
  • a BH domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam PF 16593 for domain BH).
  • HMMs Hidden Markov Models
  • HNH domain refers to an endonuclease domain having characteristic histidine and asparagine residues.
  • An HNH domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam HMM PF01844 for domain HNH).
  • HMMs Hidden Markov Models
  • variants of any of the enzymes described herein with one or more conservative amino acid substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three- dimensional structure or function of the polypeptide.
  • Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues without altering the basic functions of the encoded proteins.
  • Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity any one of the systems described herein.
  • such conservatively substituted variants are functional variants.
  • Such functional variants can encompass sequences with substitutions such that the activity of critical active site residues of the endonuclease is not disrupted.
  • a functional variant of any of the systems described herein lack substitution of at least one of the conserved or functional residues described herein.
  • a functional variant of any of the systems described herein lacks substitution of all of the conserved or functional residues described herein.
  • nucleotide symbols (irrespective of uppercase or lowercase letters) described herein correspond to the following:
  • Metagenomic sequencing from natural environmental niches that represent large numbers of microbial species may offer the potential to drastically increase the number of new CRISPR/Cas systems documented and speed the discovery of new oligonucleotide editing functionalities.
  • a recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX/CasY CRISPR systems from metagenomic analysis of natural microbial communities.
  • CRISPR/Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes.
  • CRISPR/Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally comprise two parts: (i) an array of short repetitive sequences (30- 40bp) separated by equally short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the Cas encoding the nuclease polypeptide directed by the RNA-based targeting element alongside accessory proteins/enzymes.
  • Class 1 CRISPR-Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.
  • Type IV CRISPR-Cas systems possess an effector complex that comprises a highly reduced large subunit nuclease (csfl), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.
  • csfl highly reduced large subunit nuclease
  • csf3 two genes for RAMP proteins of the Cas5
  • csf2 Cas7
  • Class 2 CRISPR-Cas systems generally have single -polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.
  • Type II CRISPR-Cas systems are considered the simplest in terms of components.
  • the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g., Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA.
  • Cas II nucleases are documented as DNA nucleases.
  • Type 2 effectors generally exhibit a structure comprising a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain.
  • the RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.
  • Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g., Casl2) structure similar to that of Type II effectors, comprising a RuvC-like domain.
  • Type V CRISPR systems Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs; however, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems are capable of using the effector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are again documented as DNA nucleases.
  • Type VI CRISPR-Cas systems have RNA-guided RNA endonucleases. Instead of RuvC-like domains, the single polypeptide effector of Type VI systems (e.g., Casl3) comprises two HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also appear to, in some embodiments, not require a tracrRNA for processing of pre-crRNA into crRNA. Similar to type V systems, however, some Type VI systems (e.g., C2C2) appear to possess robust single -stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.
  • Type VI systems e.g., C2C2C2
  • Class 2 CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease/genome editing applications.
  • One of the early adaptations of such a system for in vitro use involved (i) recombinantly-expressed, purified full-length Cas9 (e.g., a Class 2, Type II Cas enzyme) isolated from .S', pyogenes SF370, (ii) purified mature ⁇ 42 nt crRNA bearing a ⁇ 20 nt 5’ sequence complementary to the target DNA sequence desired to be cleaved followed by a 3’ tracr-binding sequence (the whole crRNA being in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg 2+ .
  • Cas9 e.g., a Class 2, Type II Cas enzyme
  • a later improved, engineered system involved the crRNA of (ii) joined to the 5’ end of (iii) by a linker (e.g., GAAA) to form a single fused synthetic guide RNA (sgRNA) capable of directing Cas9 to a target by itself.
  • a linker e.g., GAAA
  • sgRNA single fused synthetic guide RNA
  • Such engineered systems can be adapted for use in mammalian cells by providing DNA vectors encoding (i) an ORF encoding codon-optimized Cas9 (e.g., a Class 2, Type II Cas enzyme) under a suitable mammalian promoter with a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., TK pA signal); and (ii) an ORF encoding an sgRNA (having a 5 ’ sequence beginning with G followed by 20 nt of a complementary targeting nucleic acid sequence joined to a 3’ tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable Polymerase III promoter (e.g., the U6 promoter).
  • an ORF encoding codon-optimized Cas9 e.g., a Class 2, Type II Cas enzyme
  • a suitable mammalian promoter with a C
  • the engineered endonuclease is a chimera of two or more endonucleases. In some embodiments, the engineered endonuclease is a fusion of two or more endonucleases. [0140] In some embodiments, the engineered endonuclease comprises one or more fragments or domains of a nuclease, such as nucleic acid-guided nuclease.
  • the engineered endonuclease comprises one or more fragments or domains of a nuclease from orthologs of organisms, genus, species, or other phylogenetic groups described herein. In some embodiments, the engineered endonuclease comprises one or more fragments or domains from nuclease orthologs of different species.
  • the engineered endonuclease comprises one or more fragments or domains from at least two different nucleases. In some embodiments, the engineered endonuclease comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleases. In some embodiments, the engineered endonuclease comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleases from different species. In some embodiments, the engineered endonuclease comprises 2 fragments or domains, each from a different nuclease.
  • the engineered endonuclease comprises 3 fragments or domains, each from a different nuclease. In some embodiments, the engineered endonuclease comprises 4 fragments or domains, each from a different nuclease. In some embodiments, the engineered endonuclease comprises 5 fragments or domains, each from a different nuclease. In some embodiments, the engineered endonuclease comprises 3 fragments or domains, wherein at least one fragment or domain is from a different nuclease. In some embodiments, the engineered endonuclease comprises 4 fragments or domains, wherein at least one fragment or domain is from a different nuclease. In some embodiments, the engineered endonuclease comprises 5 fragments or domains, wherein at least one fragment or domain is from a different nuclease.
  • the engineered endonucleases are functional in prokaryotic or eukaryotic cells for in vitro, in vivo, or ex vivo applications.
  • the endonuclease is derived from an uncultivated microorganism.
  • the engineered endonuclease is not a Cas9 endonuclease, a Casl4 endonuclease, a Casl2a endonuclease, a Casl2b endonuclease, a Cas 12c endonuclease, a Casl2d endonuclease, a Casl2e endonuclease, a Casl3a endonuclease, a Casl3b endonuclease, a Casl3c endonuclease, or a Casl3d endonuclease.
  • the engineered endonuclease has less than 86% identity to a SpyCas9 endonuclease.
  • junctions between fragments or domains from different nucleases or species occur in stretches of unstructured regions.
  • Unstructured regions in polynucleotides include, for example, regions that have no predicted secondary structure elements such as alpha helices or beta strands.
  • Unstructured regions may include for example, regions which are exposed within a protein structure, loop regions, or regions that are not conserved within various protein orthologs as predicted by sequence or structural alignments.
  • engineered endonucleases e.g., chimeric endonucleases or fusion endonucleases
  • N-terminal portion comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least
  • the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 70% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 75% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 697-721.
  • the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 80% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 85% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 697-721.
  • the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 90% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 697- 721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 95% identity to SEQ ID NO: 696 and a C- terminal portion comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 697-721.
  • the engineered endonuclease comprises an N- terminal portion comprising a sequence having at least about 96% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 97% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 697-721.
  • the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 98% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 99% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 697-721.
  • the engineered endonuclease comprises an N-terminal portion comprising a sequence having 100% identity to SEQ ID NO: 696 and a C-terminal portion comprising a sequence having 100% identity to any one of SEQ ID NOs: 697-721.
  • engineered endonucleases e.g., chimeric endonucleases or fusion endonucleases
  • an N-terminal portion comprising RuvC, REC, or HNH domains of a Cas endonuclease and having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 696; and a C- terminal portion comprising WED, TOPO, or CTD domains of a Cas endonuclease and having at least 5
  • engineered endonucleases e.g., chimeric endonucleases or fusion endonucleases
  • an N-terminal sequence comprising at least part of a RuvC domain, a REC domain, or an HNH domain of an endonuclease and having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 4222-4224 and 4227; and a C-terminal sequence comprising WED, TOPO, or CTD domains of
  • the N-terminal sequence and the C- terminal sequence do not naturally occur together in a same reading frame.
  • engineered endonucleases comprising an N-terminal sequence comprising at least part of a RuvC domain, a REC domain, or an HNH domain of an endonuclease and having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 4239-4252 and 4255; and a C-terminal sequence comprising WED, TOPO, or CTD domains
  • the N-terminal portion of the engineered endonuclease comprises a RuvC domain, a REC domain, an HNH domain, a BH domain, or combinations thereof.
  • the C-terminal portion of the engineered endonuclease comprises a WED domain, TOPO domain, CTD domain, a PAM-interacting domain, or combinations thereof.
  • the N-terminal portion of the engineered endonuclease comprises RuvC, REC, and HNH domains.
  • the N- terminal portion of the engineered endonuclease comprises RuvC and HNH domains.
  • the N-terminal portion of the engineered endonuclease further comprises RuvC, REC, HNH, RuvC-I, BH, and RuvC-II domains.
  • the C- terminal portion of the engineered endonuclease comprises WED, TOPO, and CTD domains. In some embodiments, the C-terminal portion of the engineered endonuclease comprises a PAM-interacting domain.
  • the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease do not naturally occur together in a same reading frame. In some embodiments, the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are derived from different organisms.
  • the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are fused directly.
  • the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are joined by a linker.
  • the linker is a glycine and/or serine-rich linker, a large protein domain, a long helix structure, or a short helix structure.
  • the linker is (G4S)n (SEQ ID NO: 2950), and wherein n is an integer from 1 to 20.
  • the linker is GGGGS (SEQ ID NO: 2864).
  • the linker comprises a sequence selected from the group consisting of (GS)n (SEQ ID NO: 2951), (G2S)n (SEQ ID NO: 2952), (G3S)n (SEQ ID NO: 2953), (G4S)n (SEQ ID NO: 2950), and (G)n (SEQ ID NO: 2954), and wherein n is an integer from 1 to 20.
  • the one or more linkers comprises a sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 2955) or (GGSGGE)n (SEQ ID NO:
  • the one or more linkers comprises a sequence selected from the group consisting of (GGGSGGG)n (SEQ ID NO:
  • the one or more linkers comprises a sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8.
  • X is serine, aspartic acid, glutamic acid, threonine, or proline.
  • the engineered endonuclease (e.g., chimeric endonuclease or fusion endonuclease) comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity sequence identity to any one of SEQ ID NOs: 1-27 and 771- 862 (i.e., MG3-6 chimera endonucleases).
  • the engineered endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862.
  • the engineered endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862.
  • the engineered endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 1-27 and 771-862.
  • the engineered endonuclease (e.g., chimeric endonuclease or fusion endonuclease) comprises sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 109-110 and 2842-2854 (i.e., MG29-1 chimera endonucleases).
  • the engineered endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 109-110 and 2842-2854.
  • the engineered endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 109-110 and 2842- 2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 109-110 and 2842-2854.
  • the engineered endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 109-110 and 2842-2854.
  • the engineered endonuclease (e.g., chimeric endonuclease or fusion endonuclease) comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity sequence identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 (i.e., MG71-2 chimera endonucleases).
  • the engineered endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 4374- 4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915.
  • the engineered endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 4374- 4379 and 3862-3915.
  • the engineered endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915. In some embodiments, the engineered endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915.
  • the engineered endonuclease (e.g., chimeric endonuclease or fusion endonuclease) comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity sequence identity to any one of SEQ ID NOs: 4051-4078 (i.e., MG87 chimera endonucleases).
  • the engineered endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 4051- 4078. In some embodiments, the engineered endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 4051-4078. In some embodiments, the engineered endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 4051-4078. In some embodiments, the engineered endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 4051-4078.
  • the engineered endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 4051-4078. In some embodiments, the engineered endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 4051-4078. In some embodiments, the engineered endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 4051-4078. In some embodiments, the engineered endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 4051-4078.
  • the engineered endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 4051-4078. In some embodiments, the engineered endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 4051-4078. In some embodiments, the engineered endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 4051-4078.
  • the engineered endonuclease described herein have natural PAM specificities.
  • the present disclosure provides for the enablement of PAM specificity by protein engineering.
  • the enablement of PAM specificity is achieved by the domain swapping of RNA guided CRISPR-associated nucleases.
  • the optimal breakpoint is guided by the alignment of multiple sequences described herein.
  • the engineered endonuclease is configured to bind to a PAM that is not nnRGGnT. In some embodiments, the engineered endonuclease is configured to bind to a PAM in Table 1.
  • the engineered endonuclease is configured to bind to a PAM comprising a sequence of any one of nnRGGnT, nnnmtY, nnnCCCy, nnraww, nnRnYAY, nnnctC, nnRGGTY, nnRGGTY, nnnctC, nnRnYAY, nnnMWTY, nnrRTwy, nnRRTWY, nymwYY, nnrCyY, nnncMMc, nmY CC, nnncMMc, nnrRnCm, nnnYCMw, nnnmCmC, nnmynnn, nnnmCm, nnRnYCYr, nnRAYAC, nnnmCm, nnRMYT, nnTY Cm,
  • the engineered endonuclease is configured to bind to a PAM that is not nnRGGnT. In some embodiments, the engineered endonuclease is configured to bind to a PAM in Table 1. In some embodiments, the engineered endonuclease is configured to bind to a PAM that comprises any one of TTTn, TYYn, TTTn, nYTn, TTYn, nTTn, nYYn, and nYYn. In some embodiments, the engineered endonuclease is configured to bind to a PAM by recognizing the PAM sequence. In some embodiments, the engineered endonuclease recognizes and binds a PAM sequence.
  • the endonuclease or fusion endonuclease described herein comprises a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • the NLS is at an N- terminus of the endonuclease.
  • the NLS is at a C-terminus of the endonuclease.
  • the NLS is at an N-terminus and a C-terminus of the endonuclease.
  • the NLS comprises a sequence of any one of SEQ ID NOs: 4478-4521, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 4478- 4521.
  • the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 90% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 93% identity to SEQ ID NOs: 4478-4521.
  • the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having at least about 99% identity to SEQ ID NOs: 4478-4521. In some cases, the NLS comprises a sequence having 100% identity to SEQ ID NOs: 4478-4521.
  • engineered endonuclease and the NLS are fused directly.
  • engineered endonuclease and the NLS are joined by a linker.
  • the linker is a glycine and/or serine-rich linker, a large protein domain, a long helix structure, or a short helix structure.
  • the linker is (G4S)n (SEQ ID NO: 2950), and wherein n is an integer from 1 to 20.
  • the linker is GGGGS (SEQ ID NO: 2864).
  • the linker comprises a sequence selected from the group consisting of (GS)n (SEQ ID NO: 2951), (G2S)n (SEQ ID NO: 2952), (G3S)n (SEQ ID NO: 2953), (G4S)n (SEQ ID NO: 2950), and (G)n (SEQ ID NO: 2954), and wherein n is an integer from 1 to 20.
  • the one or more linkers comprises a sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 2955) or (GGSGGE)n (SEQ ID NO: 2956), and wherein n is an integer from 1 to 6.
  • the one or more linkers comprises a sequence selected from the group consisting of (GGGSGGG)n (SEQ ID NO: 2957), (GGGSGSGGGGS)n (SEQ ID NO: 2958) and (GGGGGPGGGGP)n (SEQ ID NO: 2959), and wherein n is an integer from 1 to 3.
  • the one or more linkers comprises a sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8.
  • X is serine, aspartic acid, glutamic acid, threonine, or proline.
  • Table 2 Example NLS Sequences that may be used with endonucleases according to the disclosure.
  • endonuclease systems comprising (a) an engineered nuclease disclosed herein, and (b) a guide polynucleotide e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • a guide polynucleotide e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • gRNA guide ribonucleic acid
  • a T means U (Uracil) in RNA and T (Thymine) in DNA.
  • the engineered guide polynucleotide is configured to form a complex with the engineered endonuclease.
  • the engineered guide polynucleotide comprises a spacer sequence.
  • the spacer sequence is configured to hybridize to a target nucleic acid sequence.
  • the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.
  • PAM protospacer adjacent motif
  • the guide polynucleotide targets a gene or locus in a cell.
  • the guide polynucleotide targets a gene or locus in a mammalian cell.
  • the mammalian cell is a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, or a human cell.
  • the target gene or target locus is albumin, TRAC, B2M, TRBC1, TRBC2, ANGPTL3, PCSK9, VCP R155, AAVS1, GPR146, AP0A1, or HA01.
  • the target gene is albumin.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the albumin gene or within an intron of the albumin gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 67- 86, 3749-3787, and 3825-3861.
  • the target gene is TRAC.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088- 1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1 19- 138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 119- 138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 119- 138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 119- 138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the TRAC gene or within an intron of the TRAC gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088- 1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence within the TRAC gene or within an intron of the TRAC gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the target gene is B2M.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 159-184 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the B2M gene or within an intron of the B2M gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 159-184 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 159-184. [0172] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the B2M gene or within an intron of the B2M gene.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence having at least about 99% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence having 100% identity to any one of SEQ ID NOs: 185-210.
  • the target gene is TRBC1.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 211-251 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 211 -251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 211 -251. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the TRBC1 gene or within an intron of the TRBC1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 211-251 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 211 -251.
  • the guide polynucleotide hybridizes or targets a sequence within the TRBC1 gene or within an intron of the TRBC1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 252-292 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 252-292.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 252-292.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 252-292.
  • the target gene is TRBC2.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 293-337 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the TRBC2 gene or within an intron of the TRBC2 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 293-337 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 293-337. [0178] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the TRBC2 gene or within an intron of the TRBC2 gene.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 338-382 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 338-382.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 338-382.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 338-382.
  • the target gene is ANGPTL3.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 383-477, 1392- 1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 383-477, 1392-1489, 2120- 2215, and 2312-2350 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide hybridizes or targets a sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351- 2389.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216- 2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.
  • the target gene is PCSK9.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 573-587 and 1362-1376 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 573- 587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 573-587 and 1362- 1376.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 573- 587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 573-587 and 1362- 1376. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the PCSK9 gene or within an intron of the PCSK9 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 573-587 and 1362-1376 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 573-587 and 1362- 1376.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 573-587 and 1362- 1376. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide hybridizes or targets a sequence within the PCSK9 gene or within an intron of the PCSK9 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 588-602 and 1377-1391 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 588-602 and 1377-1391.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 588-602 and 1377-1391.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 588-602 and 1377- 1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 588-602 and 1377-1391.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 588-602 and 1377-1391.
  • the target gene is VCP (e.g., VCP R155).
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 723-738 and 755-762 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 723- 738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 723-738 and 755- 762. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the guide polynucleotide hybridizes or targets a sequence within the VCP gene or within an intron of the VCP gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 739- 754 and 763-770 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 739-754 and 763-770.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 739-754 and 763- 770.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 739-754 and 763-770.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 739-754 and 763-770. [0188] In some embodiments, the target locus is AAVS1.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the AAVS1 locus. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 928-949, 1012- 1049, and 4106-4122.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106- 4122.
  • the guide polynucleotide hybridizes or targets a sequence within the AAVS1 locus. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 950-971 and 1050-1087 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 950-971 and 1050-1087.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 950-971 and 1050- 1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 950-971 and 1050-1087.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 950-971 and 1050-1087.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. [0191] In some embodiments, the target gene is GPR146. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 1588-1656 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1588- 1656.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1588-1656.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1588-1656.
  • the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1588-1656.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the GPR146 gene or within an intron of the GPR146 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 1588-1656 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1588- 1656. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1588-1656.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1588-1656.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1588-1656.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 1588-1656. [0193] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the GPR146 gene or within an intron of the GPR146 gene.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1657-1725 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1657-1725.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1657-1725.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 1657-1725.
  • the target gene is APOA1.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1726- 1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866- 1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866- 1961, and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the APOA1 gene or within an intron of the APOA1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1726-1744, 1764- 1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1726-1744, 1764- 1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1726-1744, 1764- 1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1726-1744, 1764- 1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866- 1961, and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence within the APOA1 gene or within an intron of the APO Al gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962- 2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1745-1763, 1775- 1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 1745-1763, 1775- 1785, 1962-2057, and 2089-2119. [0197] In some embodiments, the target gene is HAO1.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710- 3748, and 3788-3824.
  • the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 611- 633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710- 3748, and 3788-3824. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 611-633, 1789- 1826, 1827-1865, 3710-3748, and 3788-3824. [0198] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the HAO 1 gene or within an intron of the HA01 gene.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710- 3748, and 3788-3824 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710- 3748, and 3788-3824.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827- 1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotides comprise various structural elements including but not limited to: a spacer sequence which binds to the protospacer sequence (target sequence), a crRNA, and an optional tracrRNA.
  • the genome editing system comprises a CRISPR guide RNA.
  • the guide RNA comprises a crRNA comprising a spacer sequence.
  • the guide RNA additionally comprises a tracrRNA or a modified tracrRNA.
  • the systems provided herein comprise one or more guide RNAs.
  • the guide RNA comprises a sense sequence.
  • the guide RNA comprises an anti-sense sequence. In some embodiments, the guide RNA comprises nucleotide sequences other than the region complementary to or substantially complementary to a region of a target sequence.
  • a crRNA is part or considered part of a guide RNA, or is comprised in a guide RNA, e.g., a crRNA: tracrRNA chimera.
  • the guide RNA comprises synthetic nucleotides or modified nucleotides.
  • the guide RNA comprises one or more inter-nucleoside linkers modified from the natural phosphodiester. In some embodiments, all of the inter- nucleoside linkers of the guide RNA, or contiguous nucleotide sequence thereof, are modified.
  • the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.
  • the guide RNA comprises greater than about 10%, 25%, 50%, 75%, or 90% modified inter- nucleoside linkers. In some embodiments, the guide RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified inter-nucleoside linkers (e.g., phosphorothioate inter-nucleoside linkage).
  • the guide RNA comprises modifications to a ribose sugar or nucleobase.
  • the guide RNA comprises one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA.
  • the modification is within the ribose ring structure.
  • Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g., UNA).
  • the sugar- modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids.
  • the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example peptide nucleic acids (PNA) or morpholino nucleic acids.
  • the guide RNA comprises one or more modified sugars.
  • the sugar modifications comprise modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2 ’-OH group naturally found in DNA and RNA nucleosides.
  • substituents are introduced at the 2’, 3’, 4’, 5’ positions, or combinations thereof.
  • nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substituted nucleosides.
  • a 2’ sugar modified nucleoside in some embodiments, is a nucleoside that has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2’-4’ biradical bridged) nucleosides.
  • Examples of 2 ’-substituted modified nucleosides comprise, but are not limited to, 2’-O-alkyl-RNA, 2’-O-methyl-RNA, 2’-alkoxy-RNA, 2’-O- methoxyethyl- RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleoside.
  • the modification in the ribose group comprises a modification at the 2’ position of the ribose group.
  • the modification at the 2’ position of the ribose group is selected from the group consisting of 2’-O-methyl, 2’-fluoro, 2’-deoxy, and 2 ’ -O-(2-methoxyethyl) .
  • the guide RNA comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In some embodiments, the guide RNA comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2’-O-methyl. In some embodiments, the modified sugar comprises a 2’-fluoro. In some embodiments, the modified sugar comprises a 2’-O- methoxyethyl group. In some embodiments, the guide RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising a 2’-O-methyl or 2’-fluoro).
  • the guide RNA comprises both inter-nucleoside linker modifications and nucleoside modifications. In some embodiments, the guide RNA comprises greater than about 10%, 25%, 50%, 75%, or 90% modified inter-nucleoside linkers and greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the guide RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified inter-nucleoside linkers (e.g., phosphorothioate inter-nucleoside linkage) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising a 2’-O-methyl or 2’-fluoro).
  • modified inter-nucleoside linkers e.g., phosphorothioate inter-nucleoside linkage
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars e.g., comprising a 2’-O-methyl or 2’-fluoro
  • the guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a human genomic polynucleotide sequence.
  • the guide RNA is 30-250 nucleotides in length. In some embodiments, the guide RNA is more than 90 nucleotides in length. In some embodiments, the guide RNA is less than 245 nucleotides in length. In some embodiments, the guide RNA is 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides in length.
  • the guide RNA is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides in length.
  • engineered nuclease systems comprising an engineered endonuclease and an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 109- 110 and 2842-2854 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 109- 110 and 2842-2854 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 109- 110 and 2842-2854 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising 100% identity to any one of SEQ ID NOs: 109-110 and 2842-2854 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 4374- 4379 and 3862-3915 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 4374- 4379 and 3862-3915 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 4374- 4379 and 3862-3915 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising 100% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 4374- 4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 3954-4050.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 4374-4379 and 3862-3915 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 3954- 4050.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 4051- 4078 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 4051- 4078 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising 100% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 4051- 4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 4094- 4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 4051- 4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 4094- 4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 4051- 4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 4051-4078 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 4094-4105.
  • the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the engineered endonuclease binds non-covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 67-86 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1 19- 138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 119- 138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088- 1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 159-184.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 159-184 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 159-184.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 185-210 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 185-210.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 185-210.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 211 -251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC 1 gene or within an intron of the TRBC 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 211-251.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC1 gene or within an intron of the TRBC1 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 211-251 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 211-251.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 252-292 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 252-292.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 252-292.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 293-337.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 293-337 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 293-337.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 338-382 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 338-382.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 338-382.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 383-477, 1392-1489, 2120- 2215, and 2312-2350 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 573-587 and 1362- 1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 573-587 and 1362- 1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 573-587 and 1362- 1376.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a PCSK9 gene or within an intron of the PCSK9 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 573-587 and 1362-1376 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 588-602 and 1377-1391 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 588-602 and 1377-1391.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 588-602 and 1377-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 723- 738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 723- 738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 723- 738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 723- 738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 723- 738 and 755-762.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 723-738 and 755-762 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 739-754 and 763-770 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 739-754 and 763-770.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 739-754 and 763-770.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 928- 949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS 1 locus, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 928- 949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS 1 locus, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106- 4122.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 950-971 and 1050-1087 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 950-971 and 1050-1087.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 950-971 and 1050-1087.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1588-1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1588- 1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1588-1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1588- 1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1588-1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1588-1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1588- 1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1588-1656.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1588- 1656.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 1588-1656.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 1588-1656 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1588-1656. [0234] In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1657-1725 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1657-1725.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961 and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.
  • the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788- 3824.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862 and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO 1 gene or within an intron of the HAO 1 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 611-633, 1789- 1826, 1827-1865, 3710-3748, and 3788-3824 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryo kidney (HEK), mouse myeloma (NS0), or human retinal cells), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, a N2a cell, or a SY5Y cell), an insect cell (e.g., a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or a Heliothis virescen
  • the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.
  • the nucleic acid encoding the engineered endonuclease system is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA.
  • the nucleic acid encoding the engineered nuclease system is an RNA, for example a mRNA.
  • the nucleic acid encoding the engineered endonuclease system is delivered by a nucleic acid-based vector.
  • the nucleic acid-based vector is a plasmid (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), Pl-derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus.
  • cosmid e.g., pWE or sCos vectors
  • HAC human artificial chromosome
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosome
  • PAC Pl-derived artificial chromosomes
  • the nucleic acid-based vector is selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV- PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF- CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-Nl vector, pEFla-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO- NH2-CMYC, pSF-OXB20-BetaGal,pSF-OXB20-Fluc, pSF-OXB20
  • the nucleic acid-based vector comprises a promoter.
  • the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof.
  • the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, pl9, p40, Synapsin, CaMKII, GRK1, and derivatives thereof.
  • the promoter is a U6 promoter.
  • the promoter is a CAG promoter.
  • the nucleic acid-based vector is a virus.
  • the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus.
  • the virus is an alphavirus.
  • the virus is a parvovirus.
  • the virus is an adenovirus.
  • the virus is an AAV.
  • the virus is a baculovirus.
  • the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.
  • the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV- Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV
  • the virus is AAV 1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV 10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof.
  • the virus is AAV-DJ/8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV- NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.
  • the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is NZN or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.
  • the nucleic acid encoding the engineered endonuclease or the engineered endonuclease system is delivered by a non-nucleic acid-based delivery system (e.g., a non-viral delivery system).
  • a non-viral delivery system e.g., a liposome.
  • the nucleic acid is associated with a lipid.
  • the nucleic acid associated with a lipid in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • the nucleic acid is comprised in a lipid nanoparticle (LNP).
  • the engineered endonuclease or the engineered endonuclease system is introduced into the cell in any suitable way, either stably or transiently.
  • the engineered endonuclease or the engineered endonuclease system is transfected into the cell.
  • the cell is transduced or transfected with a nucleic acid construct that encodes the engineered endonuclease or the engineered endonuclease system.
  • a cell is transduced (e.g., with a virus encoding the engineered endonuclease or the engineered endonuclease system), or transfected (e.g., with a plasmid encoding the engineered endonuclease or the engineered endonuclease system) with a nucleic acid that encodes the engineered endonuclease or the engineered endonuclease system, or the translated the engineered endonuclease or the engineered endonuclease system.
  • the transduction is a stable or transient transduction.
  • cells expressing the engineered endonuclease or the engineered endonuclease system or containing the engineered endonuclease or the engineered endonuclease system are transduced or transfected with one or more gRNA molecules, for example, when the engineered endonuclease or the engineered endonuclease system comprises a CRISPR nuclease.
  • a plasmid expressing the engineered endonuclease or the engineered endonuclease system is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction (for example lentivirus or AAV) or other methods known to those of skill in the art.
  • the gene editing system is introduced into the cell as one or more polypeptides.
  • delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and/or RNPs are known in the art, for example by electroporation or by cell squeezing.
  • lipofection reagents are sold commercially (e.g., TransfectamTM, LipofectinTM and SF Cell Line 4D-Nucleofector X KitTM (Lonza)).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91/17424 and WO 91/16024.
  • the delivery is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).
  • the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.
  • the present disclosure provides a cell comprising a vector or a nucleic acid described herein.
  • the cell expresses a gene editing system or parts thereof.
  • the cell is a human cell.
  • the cell is genome edited ex vivo.
  • the cell is genome edited in vivo.
  • lipid nanoparticles comprising an engineered endonuclease system of the disclosure for delivery of the engineered endonuclease systems into a cell.
  • the lipid nanoparticle comprises the engineered endonuclease system. In some embodiments, the lipid nanoparticle comprises the engineered endonuclease or a nucleic acid encoding the engineered endonuclease and an engineered guide polynucleotide. In some embodiments, the lipid nanoparticle comprises the one or more components of the engineered endonuclease system. In some embodiments, the lipid nanoparticle comprises the engineered endonuclease or a nucleic acid encoding the engineered endonuclease. In some embodiments, the lipid nanoparticle comprises the engineered guide polynucleotide. [0256] In some embodiments, the lipid nanoparticle is tethered to the engineered endonuclease system.
  • the lipid nanoparticle is used to deliver the engineered endonuclease system or components thereof to a cell.
  • the cell is a specific cell type.
  • the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryo kidney (HEK), mouse myeloma (NSO), or human retinal cells), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC 12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, a N2a cell, or a SY5Y cell), an insect cell (e.g., a Spodopter
  • a Spodopter e.g.,
  • the cell is an A549, HEK- 293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell, or derivative thereof.
  • the primary cell is a T cell.
  • the primary cell is a hematopoietic stem cell (HSC).
  • Lipid nanoparticles as described herein can be 4-component lipid nanoparticles. Such nanoparticles can be configured for delivery of RNA or other nucleic acids (e.g., synthetic RNA, mRNA, or in w/ro-synthcsizcd mRNA) and can be generally formulated as described in WO2012135805A2. Such nanoparticles can generally comprise: (a) a cationic lipid (e.g., any of the lipids described in FIG.
  • Cationic lipid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotide, primary construct, or RNA (e.g., mRNA).
  • formulations with certain cationic lipids include, but are not limited to, 98N12-5 (or any of the other structures described in FIG. 45) and may contain 42% lipidoid, 48% cholesterol and 10% PEG (Cl 4 or greater alkyl chain length).
  • formulations with certain lipidoids include, but are not limited to, C12-200 and may contain 50% cationic lipid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG- DMG.
  • lipid nanoparticles are formulated as described in US 10709779.
  • the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.
  • the cationic lipid is selected from the group consisting of, or comprising any of, the cationic lipids depicted in FIG. 45.
  • the cationic lipid nanoparticle has a molar ratio of about 20-60% cationic lipid: about 5-25% non-cationic lipid: about 25-55% sterol; and about 0.5-15% PEG-modified lipid.
  • the cationic lipid nanoparticle comprises a molar ratio of about 50% cationic lipid, about 1.5% PEG-modified lipid, about 38.5% cholesterol, and about 10% non-cationic lipid. In some embodiments, the cationic lipid nanoparticle comprises a molar ratio of about 55% cationic lipid, about 2.5% PEG-modified lipid, about 32.5% cholesterol, and about 10% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.
  • the engineered endonucleases e.g., chimeric nucleases or fusion endonucleases
  • the engineered nuclease is a class 2, type II endonuclease.
  • the type II endonuclease has a sequence having at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) identity to SEQ ID NO: 10.
  • the methods are used to introduce a modification in the genome of a cell.
  • the modification is an insertion, deletion, or mutation.
  • the methods are used to introduce site-directed insertions, deletions, and/or mutations in the genome of a cell (for example an insertion and a mutation).
  • the methods are used in combination with a nucleic acid template to facilitate site-directed insertions into the genome of a cell.
  • the cell is a human cell.
  • the cell genome or a vector comprised in the cell is modified.
  • the cell genome is modified ex vivo.
  • the cell genome is modified in vivo.
  • methods described herein are for modifying an albumin gene.
  • methods described herein are for targeting an albumin gene in a cell.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-86, 3749-3787, and 3825-3861.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 67- 86, 3749-3787, and 3825-3861.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or within an intron of the TRAC gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.
  • the gRNA hybridizes or targets a sequence according to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184- 1279, 1321-1361, 2486-2581, and 2618-2653 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.
  • methods described herein are for modifying a B2M gene. In some embodiments, methods described herein are for targeting a B2M gene in a cell.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a B2M gene or within an intron of the B2M gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 159-184.
  • the gRNA hybridizes or targets a sequence according to any one of SEQ ID NOs: 185-210 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 185-210.
  • methods described herein are for modifying a TRBC2 gene. In some embodiments, methods described herein are for targeting a TRBC2 gene in a cell.
  • the engineered guide polynucleotide e.g., guide ribonucleic acid
  • the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 293-337 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 293-337.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 293-337 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 293-337.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a TRBC2 gene or within an intron of the TRBC2 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 293-337.
  • the gRNA hybridizes or targets a sequence according to any one of SEQ ID NOs: 338-382 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 338-382.
  • methods described herein are for modifying an ANGPTL3 gene. In some embodiments, methods described herein are for targeting an ANGPTL3 gene in a cell.
  • the engineered guide polynucleotide e.g., guide ribonucleic acid
  • the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312- 2350 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 383- 477, 1392-1489, 2120-2215, and 2312-2350 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 383-477, 1392- 1489, 2120-2215, and 2312-2350.
  • methods described herein are for modifying a PCSK9 gene. In some embodiments, methods described herein are for targeting a PCSK9 gene in a cell.
  • the engineered guide polynucleotide e.g., guide ribonucleic acid
  • the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 573-587 and 1362-1376 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 573-587 and 1362-1376 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376.
  • the gRNA hybridizes or targets a sequence according to any one of SEQ ID NOs: 588-602 and 1377-1391 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 588-602 and 1377-1391.
  • methods described herein are for modifying a VCP gene. In some embodiments, methods described herein are for targeting a VCP gene in a cell.
  • the engineered guide polynucleotide e.g., guide ribonucleic acid
  • the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 723-738 and 755-762 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106-4122.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an AAVS1 locus, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 928-949, 1012-1049, and 4106- 4122.
  • the gRNA hybridizes or targets a sequence according to any one of SEQ ID NOs: 950-971 and 1050-1087 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 950-971 and 1050-1087.
  • methods described herein are for modifying a GPR146 gene. In some embodiments, methods described herein are for targeting a GPR146 gene in a cell.
  • the engineered guide polynucleotide e.g., guide ribonucleic acid
  • the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 1588-1656 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1588-1656.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 1588-1656 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1588-1656.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1588-1656.
  • the gRNA hybridizes or targets a sequence according to any one of SEQ ID NOs: 1657-1725 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1657-1725.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058- 2088.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an APOA1 gene or within an intron of the APOA1 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.
  • the gRNA hybridizes or targets a sequence according to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.
  • the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 611- 633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.
  • kits comprising one or more nucleic acid constructs encoding the various components of the chimeric nuclease or gene editing system described herein, e.g., comprising a nucleotide sequence encoding the components of the chimeric nuclease or gene editing system capable of modifying a target DNA sequence.
  • the nucleotide sequence comprises a heterologous promoter that drives expression of the gene editing system components.
  • any of the chimeric nuclease or gene editing systems disclosed herein is assembled into a pharmaceutical, diagnostic, or research kit to facilitate its use in therapeutic, diagnostic, or research applications.
  • a kit may include one or more containers housing any of the vectors disclosed herein and instructions for use.
  • the kit may be designed to facilitate use of the methods described herein by researchers and can take many forms.
  • Each of the compositions of the kit may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions in some embodiments, are in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration.
  • Example 1 - Chimeric Type II enzymes of the MG71 family are active nucleases in vitro [0281] In silica identification of breakpoints for MG71-2 chimera generation
  • MG71 chimeric nucleases were expressed using 5 nM of a PCR-generated template. After expression, the nucleases were diluted 10-fold and incubated for 1 hour in a mixture containing 5 nM protospacer adjacent motif (PAM) library plasmid and 50 nM sgRNA in 10 mM Tris pH 7.5, 100 mM NaCl, and 10 rnM MgCh to allow digestion of the plasmid to occur. Hie digest was cleaned using magnetic nucleic acid binding beads and eluted in TE buffer.
  • PAM protospacer adjacent motif
  • the digested PAM plasmids (15 nM) were blunt-end ligated to double-stranded adapter oligos (150 nM) with T4 ligase in IX T4 ligase buffer.
  • Tire ligated product was amplified via PCR.
  • the ligated product was sequenced using 150 bp single read amplicon sequencing.
  • the resulting reads were filtered by a quality- score >20.
  • PAMs were identified by mapping the reads to the PAM plasmid backbones requiring a perfect match of 20-24 nt after the 8N PAM region. SeqLogos of the PAMs were generated and PAM sequences determined by the height of each nucleotide.
  • the cut site was also identified by calculating the distance between the PAM and the ligated adapter.
  • results [0285] The nuclease MG71-2 was used as a base for chimeragenesis. Three breakpoints were selected that retain different amounts of the parent nuclease sequence: a chimerathat swaps at the phosphate loop, a chimera that swaps before the WED domain, and a chimera that swaps at the PID domain. The chimera which swaps the PID would retain the largest amount of parent 1 sequence and thus is the most conservative change. Two homologous nucleases, MG71-1 and MG18-1 were selected for chimeragenesis. The chimeras were tested with active sgRNA for MG71-2, MG71-1, and MG18-1.
  • Table 3 Chimeric architecture with the MG71-2 chassis.
  • Example 2 - MG3-6 chimeras are active nucleases in cells
  • Table 4 In cell editing data of chimeric nucleases.
  • FIG. 7 shows the correlation between relative indel % between different engineered versions of MG3-6/3-4 mouse HA01 guide 7 and mouse ALB guide 34 (SEQ ID NOs: 3710- 3787). Shown is the fold improvement compared to chemistry 119 (SEQ ID NOs: 3714 and 3753) for each guide. Dashed lines indicate a cutoff at 90% relative indel for further engineering efforts. Moderate levels of 2’OMe in the repeat anti-repeat, as well as 2’OMe in the linker regions of the guide (FIG.
  • repeat anti-repeat region is not amenable to modification with 2’0Me, perhaps due to the disruption of critical interactions between the 2 ’OH of the nucleotides and the MG3-6/3-4 nuclease or disruption of the folding of the guide RNA.
  • FIG. 8 shows % indel for the two guides. All three of these regions were highly amenable to modification, with all designs maintaining editing compared to chemistry 119 (SEQ ID NOs: 3714 and 3753) for both spacers, with the exception of chemistries 160, 174, 179, and 186 (SEQ ID NOs: 3798, 3812, 3817, 3824, 3835, 3849, 3854, 3861).
  • FIG. 9 shows the correlation between relative indel % between different engineered versions of MG3-6/3-4 mouse HA01 guide 7 and mouse ALB guide 34 (SEQ ID NOs: 3788- 3861). Shown is the fold improvement compared to chemistry 119 (SEQ ID NOs: 3714 and 3753) for each guide. Dashed lines indicate a cutoff at 90% relative indel. From this round of engineering, we found that we could modify up to 68% of nucleotides with 2’OMe (chemistry 184, SEQ ID NOs: 3822 and 3859) while maintaining 90% relative indel.
  • Breakpoints were determined using the predicted structure of MG71-2 (FIG. 10A). Three breakpoints were chosen in MG71-2: Alal095, Asnl 106, and Seri 133, which roughly break the MG71-2 enzyme at its Phosphate lock-loop, WED domain, and PID respectively, fire corresponding breakpoints in the MG71 homologs were determined using multiple sequence alignment (FIG. 10B).
  • Table 5 Guide RNA sequences for determining in vitro PAMs determination assay of
  • Transduced cells were selected with puromycin from 3 to 10 days posttransduction.
  • gRNAs were designed that targeted AAVS 1 (SEQ ID NOs: 3954-3966) PAH (C.1066-11G>A, C.1315+1G>A, and p.R408W) in these engineered K562 cells (SEQ ID NOs: 3967-4021) as well as mouse Hepal-6 cells (SEQ ID NOs: 4022-4050).
  • chimeras (MG71-80 through MG71-53, MG7I-68 through MG71-73, and MG71-98, MG71-100, MG71-102, MG71-104, MG71-106, MG71-108, MG71-110, MG71-112 were tested with a total of 8 pools (4 targets, two pools per site) but none showed editing >5% at this target.
  • This example describes the “reverse” chimeragenesis strategy for Type II nucleases from the MG87 family. Starting from the PID of MG87-21 (PAM NNNNCCNN) and MG87- 70 (PAM NNNNGTNN) and combining them with the N-terminal RuvC, REC, and HNH domains from closely related homologs with different PAM sequences to yield “reverse” chimeras.
  • the PID boundary and breakpoints for the MG87 nucleases were determined using the predicted structure of MG87-21 (FIG. 18A).
  • the PID of MG87-21 starts at around Asp972 (Asp962 in MG87-70).
  • the corresponding PID breakpoints in MG87 homologs were determined using the multiple sequence alignment of the active MG87 nuclease homologs.
  • the MSA was generated (FIG. 18B).
  • the “reverse” chimeras listed in Table 7 were designed (SEQ ID NOs: 4051-4078).
  • the MG87-21 chimeras were screened in wild type K562 cells using 12 guides that target TRAC or AAVS1 (SEQ ID NOs: 4094-4105). Cells were passaged 2-7 times prior to nucleofection. Cells were nucleofected with 500 ng of mRNA and 150 pmol of guide into 100,000 cells, and recovered for three days at 37 °C in a 96-well format. gDNA was extracted, target regions were amplified, processed for NGS, and percent editing was analyzed. The comparison with MG87-21 WT protein was tested with 750 pmol of guide RNA.
  • Example 8 - MG3 chimeras demonstrate high in-cell activity
  • MG3-6 3-8 3-78 SEQ ID NO: 819
  • MG3-6 3-8 3-90 SEQ ID NO: 821
  • MG3-6_3-8_3-96 SEQ ID NO: 8257
  • guides designed targeting the AAVS1 or TRAC locus SEQ ID NOs: 4106-4172
  • Cells were passaged 2-7 times prior to nucleofection.
  • Cells were nucleofected with 500 ng of mRNA and 150 pmol of guide into 100,000 cells and recovered for three days at 37 °C in a 96-well format.
  • gDNA was extracted, target regions were amplified and processed for NGS. Percent editing was analyzed.
  • Fragmented adapter ligated DNA was size selected using magnetic beads and used as input material for the PCR reaction that enriches for fragments containing the dsODN sequence.
  • This PCR step is carried out in the form of two different reactions: one targeting the plus strand and the second targeting the minus strand, utilizing primers that bind to the dsODN and end ligated adapters, thus enriching for fragments containing the dsODN.
  • the resulting DNA was quantified, normalized, and used for input in an indexing PCR which adds Illumina sequencing adapters and as well as sample barcodes to each library.
  • the final libraries were pooled and purified before being quantified and sequenced. Reads were filtered to a max edit distance of 7 and read count of 10 to call off-targets.
  • MG3-6_3-8_3-78 was tested with 7 highly active guides (SEQ ID NOs: 2957, 2958, 4112, 4113, 4115, 4120, and 4122), which show high activity (>80% indel) to look for off- target cleavage. None of the 7 guides showed reproducible off-targets within our analysis criteria (FIG. 24A). Guide gl-TRAC-PlF7 (SEQ ID NO: 2957) showed one off-target in one replicate, but it was not detected in the other replicates, resulting in 99.78% of reads coming from the on-target.

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Abstract

La divulgation concerne des nucléases et des systèmes de nucléase ingéniérisés, comprenant des nucléases chimériques et des systèmes de nucléases chimériques. Les nucléases ingéniérisées et chimériques de la divulgation comprennent des nucléases guidées par des acides nucléiques. La divulgation concerne en outre des procédés de génération de nucléases ingéniérisées et des méthodes d'utilisation de celles-ci.
PCT/US2024/046782 2023-09-13 2024-09-13 Nucléases ingénierisées et chimériques Pending WO2025059585A1 (fr)

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Citations (3)

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US20200332273A1 (en) * 2019-02-14 2020-10-22 Metagenomi Ip Technologies, Llc Enzymes with ruvc domains
WO2022159758A1 (fr) * 2021-01-22 2022-07-28 Metagenomi, Inc Nouvelles nucléases ingéniérisées et chimériques
WO2022232638A2 (fr) * 2021-04-30 2022-11-03 Metagenomi, Inc. Enzymes à domaines ruvc

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200332273A1 (en) * 2019-02-14 2020-10-22 Metagenomi Ip Technologies, Llc Enzymes with ruvc domains
WO2022159758A1 (fr) * 2021-01-22 2022-07-28 Metagenomi, Inc Nouvelles nucléases ingéniérisées et chimériques
WO2022232638A2 (fr) * 2021-04-30 2022-11-03 Metagenomi, Inc. Enzymes à domaines ruvc

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