Engineered and chimeric nucleases

Abstract

Disclosed herein are engineered nucleases and nuclease systems, including chimeric nucleases and chimeric nuclease systems. Engineered and chimeric nucleases disclosed herein include nucleic acid guided nuclease. Additionally disclosed herein are methods of generating engineered nucleases and methods of using the same.

Description

ENGINEERED AND CHIMERIC NUCLEASESCROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 582,489, filed September 13, 2023, which is incorporated by reference in its entirety herein.BACKGROUND

[0002] 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. While 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.SUMMARY

[0003] Described herein, in certain embodiments, are 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. 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 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.

[0004] Described herein, in certain embodiments, are 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.

[0005] Described herein, in certain embodiments, are 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. 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 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.

[0006] Described herein, in certain embodiments, are 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.

[0007] Described herein, in certain embodiments, are 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 polynucleotidecomprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3749-3787 and 3825-3861. In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3749-3787 and 3825-3861. 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.

[0008] Described herein, in certain embodiments, are 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. In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3710-3748 and 3788-3824. 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.

[0009] Described herein, in certain embodiments, are 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. In some embodiments, 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.

[0010] Described herein, in certain embodiments, are 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. In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 4123-4172. 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.

[0011] Described herein, in certain embodiments, are methods for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system described herein. In some embodiments, modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, the modification is in vitro. In some embodiments, the modification is in vivo. In some embodiments, the modification is ex vivo. In some embodiments, the gRNA is encoded by a sequence having any one of SEQ ID NOs: 251-260, 271-274, and 279-290. In some embodiments, the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 261-270, 275-278, and 291-302.

[0012] Described herein, in certain embodiments, are methods of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system described herein. In some embodiments, the methods further comprise selecting cells comprising the modification.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] Described herein, in certain embodiments, are cells comprising the endonuclease described herein. In some embodiments, 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. In some embodiments, 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. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell.

[0018] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0020] 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 areshown in dark grey. The star represents the breakpoint for swapping the PID and WED domains.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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- round 1 engineering.

[0027] 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.

[0028] 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- round 2 engineering.

[0029] 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.

[0030] FIG. 11 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.

[0031] 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.

[0032] 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).

[0033] 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).

[0034] 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.

[0035] 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. In most cases MG71-56 showed slightly improved levels of editing compared to MG71-54 and MG71-55.

[0036] 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. Other 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 PAHр.R408W target and in Hepal-6 cells showed activity at PAH c.1066-11G>A and PAHС.1315+1G>A position.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] FIGs. 24A-24C depict MG3 chimeras display high specificity in the mammalian cells. Cells were assessed for double strand break (DSB) formation by co-nucleofectionmRNA, 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.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0044] The Sequence Listing filed herewith provides example polynucleotide and polypeptide sequences for use in methods, compositions, and systems according to the disclosure. Below are example descriptions of sequences therein.MG3-6 Chimeras

[0045] SEQ ID NOs: 1-27 and 771-862 show the full-length peptide sequences of MG3-6 chimeric nucleases.

[0046] 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.

[0047] SEQ ID NO: 722 shows the nucleotide sequence of a MG3-6 / 3-4 guide sgRNA scaffold.

[0048] 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.

[0049] SEQ ID NO: 603 shows the DNA coding sequence for MG3-6 / 3-4.

[0050] SEQ ID NO: 604 shows the protein sequence of the MG3-6 / 3-4 cassette coding sequence.MG29-1 Chimeras

[0051] SEQ ID NOs: 109-110 and 2842-2854 show the full-length peptide sequences of MG29-1 chimeric nucleases.

[0052] SEQ ID NOs: 111-113 show the nucleotide sequences of sgRNAs engineered to function with an MG29-1 chimeric nuclease.MG Chimeras

[0053] SEQ ID NO: 696 shows a N-terminal peptide sequence (1-742) of MG3-6.

[0054] SEQ ID NOs: 697-721 show C-terminal peptide sequences.MG71-2 Chimeras

[0055] SEQ ID NOs: 4374-4379 and 3862-3915 show the full-length peptide sequences of MG71-2 chimeric nucleases.

[0056] SEQ ID NOs: 3954-4050 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 chimeric nuclease.MG87 Reverse Chimeras

[0057] SEQ ID NOs: 4051-4078 show the full-length peptide sequences ofMG87 reverse chimeric nucleases.

[0058] SEQ ID NOs: 4094-4105 show the nucleotide sequences of sgRNAs engineered to function with a MG87 reverse chimeric nucleases.Albumin Targeting

[0059] 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.TRAC Targeting

[0060] 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.

[0061] SEQ ID NOs: 139-158 show the DNA sequences of TRAC target sites.

[0062] 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.

[0063] SEQ ID NOs: 925-927 show the DNA sequences of TRAC target sites.

[0064] 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.

[0065] SEQ ID NOs: 992-1011 show the DNA sequences of TRAC target sites.

[0066] 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.

[0067] SEQ ID NOs: 4123-4172 the nucleotide sequences of sgRNAs engineered to function with MG3 chimeras.

[0068] SEQ ID NOs: 1184-1279, 1321-1361, 2486-2581, and 2618-2653 show the DNA sequences of TRAC target sites.B2M Targeting

[0069] 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.

[0070] SEQ ID NOs: 185-210 show the DNA sequences of B2M target sites.TRBC1 Targeting

[0071] 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.

[0072] SEQ ID NOs: 252-292 show the DNA sequences of TRBC1 target sites.TRBC2 Targeting

[0073] 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.

[0074] SEQ ID NOs: 338-382 show the DNA sequences of TRBC2 target sites.ANGPTL3 Targeting

[0075] 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.

[0076] SEQ ID NOs: 478-572 and 1490-1587 show the DNA sequences of ANGPTL3 target sites.

[0077] 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.

[0078] SEQ ID NOs: 2216-2311 and 2351-2389 show the DNA sequences of ANGPTL3 target sites.PCSK9 Targeting

[0079] 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.

[0080] SEQ ID NOs: 588-602 and 1377-1391 show the DNA sequences of PCSK9 target sites.VCP R155 Targeting

[0081] 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.

[0082] SEQ ID NOs: 739-754 show the DNA sequences of VCPR155 target sites.

[0083] 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.

[0084] SEQ ID NOs: 763-770 show the DNA sequences of VCPR155 target sites.AAVS1 Targeting

[0085] 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.

[0086] SEQ ID NOs: 950-971 show the DNA sequences of AAVS1 target sites.

[0087] 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.

[0088] SEQ ID NOs: 1050-1087 show the DNA sequences of AAVS1 target sites.

[0089] SEQ ID NOs: 4106-4122 the nucleotide sequences of sgRNAs engineered to function with MG3 chimeras.GPR146 Targeting

[0090] 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.

[0091] SEQ ID NOs: 1657-1725 show the DNA sequences of M. musculus GPR146 target sites.APOA1 Targeting

[0092] 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.

[0093] SEQ ID NOs: 1745-1763 show the DNA sequences of M. musculus APOA1 target sites.

[0094] 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.

[0095] SEQ ID NOs: 1775-1785 show the DNA sequences of H. sapiens APOA1 target sites.

[0096] 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.

[0097] SEQ ID NOs: 1962-2057 and 2089-2119 show the DNA sequences of mouse APOA1 target sites.HAO1 Targeting

[0098] 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.

[0099] 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 .Other Sequences

[0100] 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.DETAILED DESCRIPTION

[0102] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0103] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0104] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.

[0105] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0106] The term “nucleotide,” as used herein, 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. 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. The term nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of 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). Specific examples of 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 -dATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12- UTP, and Fluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP,BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14- dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein- 12- dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP,tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically- modified nucleotide is biotin-dNTP. Non-limiting examples of 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).

[0107] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to 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. In a polynucleotide when referring to a T, 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.

[0108] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer 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 acidpolymers 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). The terms also encompass an 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. The terms “amino acid” and “amino acids,” as used herein, 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. The term “amino acid” includes both D-amino acids and L-amino acids.

[0109] As used herein, the “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.

[0110] As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof refer 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. For example, two genetic elements are operably linked if movement of the first element causes an activation of the second element. For instance, 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.

[0111] 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.

[0112] 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.

[0113] The terms “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. For example, 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.

[0114] The term “tracrRNA” or “tracr sequence” means trans-activating CRISPR RNA. tracrRNA interacts with the CRISPR (cr) RNA to form guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. If the tracrRNA is engineered, it may have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and / or sequence similarity to a wild type exemplary tracrRNA sequence (e.g., a tracrRNA from S. pyogenes, S. aureus). tracrRNA may refer to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. The term tracrRNA encompasses a nucleic acid that can be at least about 60% identical to awild type exemplary tracrRNA (e.g., a tracrRNA from .S'. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, 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.

[0115] As used herein, 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. The term 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.” If not otherwise specified, 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.”

[0116] The term “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, asmeasured 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 with default parameters.

[0117] As used herein, the term “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).

[0118] As used herein, the term “Wedge” (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.

[0119] As used herein, the term “PAM interacting domain” or “PI 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. Examples of PAM-interacting domains include, but are not limited to, Topoisomerase-homology (TOPO) domains and C-terminal domains (CTD) present in Cas proteins. 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.

[0120] As used herein, the term “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).

[0121] As used herein, the term “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).

[0122] As used herein, the term “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).

[0123] Included in the current disclosure are variants of any of the enzymes described herein with one or more conservative amino acid substitutions. Such conservative 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. In some embodiments, such conservatively substituted variants arefunctional variants. Such functional variants can encompass sequences with substitutions such that the activity of critical active site residues of the endonuclease is not disrupted. In some embodiments, a functional variant of any of the systems described herein lack substitution of at least one of the conserved or functional residues described herein. In some embodiments, a functional variant of any of the systems described herein lacks substitution of all of the conserved or functional residues described herein.

[0124] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for example, Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd Edition (December 1993))). The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M).

[0125] In reference to nucleotide sequences, nucleotide symbols (irrespective of uppercase or lowercase letters) described herein correspond to the following:Overview

[0126] The discovery of new Cas enzymes with unique functionality and structure may offer the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, improving speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. 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.

[0127] CRISPR / Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes. In their natural context, 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. Efficient nuclease targeting of a particular target nucleic acid sequence may involve both (i) complementary hybridization between the first 6-8 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM usually being a sequence not commonly represented within the host genome). Depending on the exact function and organization of the system,CRISPR-Cas systems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.

[0128] Class 1 CRISPR-Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.

[0129] Type I CRISPR-Cas systems are considered of moderate complexity in terms of components. In Type I CRISPR-Cas systems, the array of RNA-targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at repeat elements to liberate short, mature crRNAs that direct the nuclease complex to nucleic acid targets when they are followed by a suitable short consensus sequence called a protospacer-adjacent motif (PAM). This processing occurs via an endoribonuclease subunit (Cas6) of a large endonuclease complex called Cascade, which also comprises a nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nucleases function primarily as DNA nucleases.

[0130] Type III CRISPR systems may be characterized by the presence of a central nuclease, known as Cas 10, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits. Like in Type I systems, the mature crRNA is processed from a pre-crRNA using a Cas6-like enzyme. Unlike type I and II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).

[0131] 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.

[0132] Class 2 CRISPR-Cas systems generally have single -polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.

[0133] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, 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 aRuvC-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.

[0134] 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. 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. Unlike Type II CRISPR-Cas systems, some Type V enzymes (e.g., Casl2a) appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a doublestranded target sequence.

[0135] 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.

[0136] Because of their simpler architecture, Class 2 CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease / genome editing applications.

[0137] 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) Mg2+. 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.

[0138] 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).Engineered Endonucleases

[0139] Described herein, in certain embodiments, are engineered endonucleases. In some embodiments, 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. In some embodiments, 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.

[0141] In some embodiments, 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. In some embodiments, 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.

[0142] In some embodiments, the engineered endonucleases are functional in prokaryotic or eukaryotic cells for in vitro, in vivo, or ex vivo applications.

[0143] In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, 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. In some embodiments, the engineered endonuclease has less than 86% identity to a SpyCas9 endonuclease.

[0144] In some embodiments, 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.

[0145] Described herein, in certain embodiments, are engineered endonucleases (e.g., chimeric endonucleases or fusion endonucleases) comprising an N-terminal portion comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, 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 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 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 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 endonucleasecomprises 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0146] Described herein, in certain embodiments, are engineered endonucleases (e.g., chimeric endonucleases or fusion endonucleases) comprising an N-terminal portioncomprising 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 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: 697-721.

[0147] Further described herein, in certain embodiments, are engineered endonucleases (e.g., chimeric endonucleases or fusion 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: 4222-4224 and 4227; and a C-terminal sequence comprising WED, TOPO, or CTD domains 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: 4173-4221, 4225-4226, 4228-4238. In some embodiments, the N-terminal sequence and the C- terminal sequence do not naturally occur together in a same reading frame.

[0148] Further described herein, in certain embodiments, are engineered endonucleases (e.g., chimeric endonucleases or fusion 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% sequenceidentity to any one of SEQ ID NOs: 4239-4252 and 4255; and a C-terminal sequence comprising WED, TOPO, or CTD domains 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: 4253- 4254. In some embodiments, the N-terminal sequence and the C-terminal sequence do not naturally occur together in a same reading frame.

[0149] In some embodiments, the N-terminal portion of the engineered endonuclease comprises a RuvC domain, a REC domain, an HNH domain, a BH domain, or combinations thereof. In some embodiments, the C-terminal portion of the engineered endonuclease comprises a WED domain, TOPO domain, CTD domain, a PAM-interacting domain, or combinations thereof. In some embodiments, the N-terminal portion of the engineered endonuclease comprises RuvC, REC, and HNH domains. In some embodiments, the N- terminal portion of the engineered endonuclease comprises RuvC and HNH domains. In some embodiments, the N-terminal portion of the engineered endonuclease further comprises RuvC, REC, HNH, RuvC-I, BH, and RuvC-II domains. In some embodiments, 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.

[0150] In some embodiments, 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.

[0151] In some embodiments, the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are fused directly. In some embodiments, the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are joined by a linker. In some embodiments, the linker is a glycine and / or serine-rich linker, a large protein domain, a long helix structure, or a short helix structure. In some embodiments, the linker is (G4S)n (SEQ ID NO: 2950), and wherein n is an integer from 1 to 20. In some embodiments, the linker is GGGGS (SEQ ID NO: 2864). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0152] In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineered endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862. Insome 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.

[0153] In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0154] In some embodiments, 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%, atleast 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0155] In some embodiments, 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). In some embodiments, 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 atleast 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. In some embodiments, 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. In some embodiments, 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.

[0156] In some embodiments, the engineered endonuclease described herein have natural PAM specificities. In some embodiments, the present disclosure provides for the enablement of PAM specificity by protein engineering. In some embodiments, the enablement of PAM specificity is achieved by the domain swapping of RNA guided CRISPR-associated nucleases. In some embodiments, there is an optimal breakpoint in the process of domain swapping and recombination. In some embodiments, the optimal breakpoint is guided by the alignment of multiple sequences described herein.

[0157] In some embodiments, 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 comprising a sequence of any one of nnRGGnT, nnnmtY, nnnCCCy, nnraww, nnRnYAY, nnnctC, nnRGGTY, nnRGGTY, nnnctC, nnRnYAY, nnnMWTY, nnrRTwy, nnRRTWY, nymwYY, nnrCyY, nnncMMc, nnmY CC, nnncMMc, nnrRnCm, nnnYCMw, nnnmCmC, nnmynnn, nnnmCm, nnRnYCYr, nnRAYAC, nnnmCm, nnnRMYT, nnTY Cm, nnTMCy, nnrMTCr, nnnmCCY, nnrmww, nnrRkyY, nnRnYhy, nnnmwTY, nnnmyY, nnRnYhY, nnnmhTY, nnRMAC, nnrRtwy, nnrwwYY, nnrmyY, nnrwY CC, nnRGnCr, nnnMhYy, nnyCCMww, nnnMCMCw, nnRwYhWw, nnRrYCYr, nnrwyhh,ntrMCm, nnrwyhh, nnRRTWY, ntTYCM, nnTCCC, nnmyhh, nnnCCCYR, nnRnYAYn, nnRRnnYn, and nnRRnnYn. 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.Table 1. PAM Sequences

[0158] In some embodiments, 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.

[0159] In some embodiments, the endonuclease or fusion endonuclease described herein comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is at an N- terminus of the endonuclease. In some embodiments, the NLS is at a C-terminus of the endonuclease. In some embodiments, the NLS is at an N-terminus and a C-terminus of the endonuclease.

[0160] In some embodiments, 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. In some cases, 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. In some cases, 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.

[0161] In some embodiments, engineered endonuclease and the NLS are fused directly. In some embodiments, engineered endonuclease and the NLS are joined by a linker. In some embodiments, the linker is a glycine and / or serine-rich linker, a large protein domain, a long helix structure, or a short helix structure. In some embodiments, the linker is (G4S)n (SEQ ID NO: 2950), and wherein n is an integer from 1 to 20. In some embodiments, the linker is GGGGS (SEQ ID NO: 2864). In some embodiments, 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. In some embodiments, 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. In some embodiments, the one or more linkers comprises a sequence selected from the groupconsisting 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. In some embodiments, 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. In some embodiments, 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.Guide Polynucleotides

[0162] Disclosed herein, in certain embodiments, are 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. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA.

[0163] In some embodiments, the engineered guide polynucleotide is configured to form a complex with the engineered endonuclease. In some cases, the engineered guide polynucleotide comprises a spacer sequence. In some cases, the spacer sequence is configured to hybridize to a target nucleic acid sequence. In some cases, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0164] In some embodiments, the guide polynucleotide (e.g., gRNA) targets a gene or locus in a cell. In some embodiments, the guide polynucleotide targets a gene or locus in a mammalian cell. In some embodiments, 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. In some embodiments, thetarget gene or target locus is albumin, TRAC, B2M, TRBC1, TRBC2, ANGPTL3, PCSK9, VCP R155, AAVS1, GPR146, AP0A1, or HA01.

[0165] In some embodiments, the target gene is albumin. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0166] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the albumin gene or within an intronof 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. 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: 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. 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: 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 96% 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 97% 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 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.

[0167] In some embodiments, the target gene is TRAC. In some embodiments, 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. In some embodiments, 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 about55%, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0168] In some embodiments, 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. 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: 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. 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: 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. 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: 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. 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: 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. 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: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, 2582-2617, and 4123-4172.

[0169] In some embodiments, 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 asequence 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0170] In some embodiments, the target gene is B2M. In some embodiments, 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. In some embodiments, 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 about92%, 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. In some embodiments, 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. In some embodiments, 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.

[0171] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 sequencecomplementary 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. In some embodiments, 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. In some embodiments, the guide polynucleotide hybridizes ortargets 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. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence having at least about 85% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes ortargets a sequence having at least about 96% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 185-210. In some embodiments, 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.

[0173] In some embodiments, the target gene is TRBC1. In some embodiments, 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. In some embodiments, 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 leastabout 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. In some embodiments, 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. In some embodiments, 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.

[0174] In some embodiments, 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. 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: 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 sequencecomplementary to a sequence having at least about 96% 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 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.

[0175] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0176] In some embodiments, the target gene is TRBC2. In some embodiments, 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. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about46-80 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. In some embodiments, 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. In some embodiments, 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.

[0177] In some embodiments, 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. 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: 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 sequencecomplementary to a sequence having at least about 95% 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 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. 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: 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0179] In some embodiments, the target gene is ANGPTL3. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and2312-2350 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0180] In some embodiments, 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% sequenceidentity 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 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. 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: 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. 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: 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. 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: 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.

[0181] In some embodiments, 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. In some embodiments, 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 IDNOs: 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. In some embodiments, 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. In some embodiments, 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.

[0182] In some embodiments, the target gene is PCSK9. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the guide polynucleotide is encoded by a sequence having atleast 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. In some embodiments, 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.

[0183] In some embodiments, 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. 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: 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. 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: 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. 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: 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.

[0184] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0185] In some embodiments, the target gene is VCP (e.g., VCP R155). In some embodiments, 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. In some embodiments, 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 leastabout 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. In some embodiments, 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. In some embodiments, the guide polynucleotide is encoded by 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 is encoded by 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 is encoded by a sequence having at least about 98% 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 99% 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 100% identity to any one of SEQ ID NOs: 723-738 and 755-762.

[0186] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene. In some embodiments, 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. 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: 723-738 and 755-762. 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: 723-738 and 755-762. 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: 723-738 and 755-762. 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: 723-738 and 755-762. 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: 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. 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: 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.

[0187] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0189] In some embodiments, 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 least90%, 95%, 97%, 98%, or 99% sequence 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 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. 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: 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. 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: 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.

[0190] In some embodiments, 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. In some embodiments, 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. Insome 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 someembodiments, 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.

[0192] In some embodiments, 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. 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: 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. 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: 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. 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: 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. 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 guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ IDNOs: 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. In some embodiments, 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. In some embodiments, 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.

[0194] In some embodiments, the target gene is APOA1. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0195] In some embodiments, 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. 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: 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. 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: 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. 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: 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. In someembodiments, 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.

[0196] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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: 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. 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: 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. 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: 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. 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: 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. 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: 611-633, 1789-1826, 1827-1865, 3710-3748, and 3788-3824.

[0199] In some embodiments, the guide polynucleotides (e.g., guide RNAs) 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. In someembodiments, the genome editing system comprises a CRISPR guide RNA. In some embodiments, the guide RNA comprises a crRNA comprising a spacer sequence. In some embodiments, the guide RNA additionally comprises a tracrRNA or a modified tracrRNA.

[0200] In some embodiments, the systems provided herein comprise one or more guide RNAs. In some embodiments, the guide RNA comprises a sense sequence. In some embodiments, 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. For example, a crRNA is part or considered part of a guide RNA, or is comprised in a guide RNA, e.g., a crRNA: tracrRNA chimera.

[0201] In some embodiments, the guide RNA comprises synthetic nucleotides or modified nucleotides. In some embodiments, 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. For example, in some embodiments, the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage. In some embodiments, 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).

[0202] In some embodiments, the guide RNA comprises modifications to a ribose sugar or nucleobase. In some embodiments, 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. In some embodiments, 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). In some embodiments, the sugar- modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, 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.

[0203] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, 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. In some embodiments, substituents are introduced at the 2’, 3’, 4’, 5’ positions, or combinations thereof. In some embodiments, 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. In some embodiments, the modification in the ribose group comprises a modification at the 2’ position of the ribose group. In some embodiments, 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) .

[0204] In some embodiments, 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).

[0205] In some embodiments, 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).

[0206] In some cases, the guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, theguide 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.

[0207] In some embodiments, 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. In some embodiments, 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.MG Endonuclease Systems

[0208] Described herein, in certain embodiments, are 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.

[0209] In some embodiments, 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. 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: 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. 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: 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. 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: 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. 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: 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. 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: 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.

[0210] In some embodiments, 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. 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: 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. 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: 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. 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: 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. 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: 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. In some embodiments, 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.

[0211] In some embodiments, 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. 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: 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. 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: 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 anyone 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 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. 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: 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. 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: 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.

[0212] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 theendonuclease 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In someembodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0213] In some embodiments, 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. 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: 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. 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 IDNOs: 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. 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: 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.

[0214] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to anyone 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In someembodiments, 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. In some embodiments, 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.

[0215] 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.

[0216] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 engineeredguide 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identityto 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0217] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineered nuclease system comprises an endonuclease comprising asequence 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 ofthe 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. In some embodiments, 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. 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.

[0218] 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. In some embodiments, 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.

[0219] In some embodiments, 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. In some embodiments, 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 sequencehaving at least about 75% identity to any one of SEQ ID NOs: 159-184. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 toany one of SEQ ID NOs: 159-184. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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.

[0220] In some embodiments, 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. In some embodiments,the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 185-210.

[0221] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, theengineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 211-251. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 asequence having at least about 99% identity to any one of SEQ ID NOs: 211-251. In some embodiments, 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. 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.

[0222] 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 target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 252-292.

[0223] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, theengineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 293-337. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 asequence having at least about 97% identity to any one of SEQ ID NOs: 293-337. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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.

[0224] In some embodiments, 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 target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 338-382.

[0225] In some embodiments, 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 theANGPTL3 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 theendonuclease 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineered nuclease system comprises an endonucleasecomprising 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. 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.

[0226] 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. In some embodiments, 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.

[0227] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 orwithin 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 sequencewithin 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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.

[0228] 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 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.

[0229] In some embodiments, 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 theendonuclease 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 aspacer 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 nucleicacid 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. In some embodiments, 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.

[0230] 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 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.

[0231] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 toform 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 tohybridize 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. In some embodiments, 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. In some embodiments, 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. 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.

[0232] 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 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.

[0233] In some embodiments, 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. In some embodiments, 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 guidepolynucleotide 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 GPR146 gene or within an intron of the GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1588- 1656. In some embodiments, 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 theendonuclease 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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.

[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.

[0235] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In someembodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, theengineered 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. In some embodiments, 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. In some embodiments, 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. 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.

[0236] 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. In some embodiments, 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.

[0237] In some embodiments, 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. In some embodiments, the engineered nuclease system comprises an endonucleasecomprising 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 sequencewithin 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. In some embodiments, 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.Cells

[0238] Described herein, in certain embodiments, is a cell comprising engineered endonuclease systems described herein.

[0239] In some embodiments, 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 virescens cell), a yeast cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g., aE. coli cell, a streptococcus bacterium cell, a streptomyces soil bacteria cell, or an archaea cell). In some embodiments, 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.

[0240] In some embodiments, 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. In some embodiments, the primary cell is a T cell. In some embodiments, the primary cell is a hematopoietic stem cell (HSC).Delivery and Vectors

[0241] Disclosed herein, in some embodiments, are nucleic acid sequences encoding an engineered nuclease system comprising an engineered endonuclease (e.g. a fusion endonuclease or a chimeric nuclease) and a guide polynucleotide, an engineered endonuclease, or a guide polynucleotide.

[0242] In some embodiments, the nucleic acid encoding the engineered endonuclease system is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid encoding the engineered nuclease system is an RNA, for example a mRNA.

[0243] In some embodiments, the nucleic acid encoding the engineered endonuclease system is delivered by a nucleic acid-based vector. In some embodiments, 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. In some embodiments, 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, pSF-Tac, pRI 101-AN DNA, pCambia2301,pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0244] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, 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. In some embodiments the promoter is a U6 promoter. In some embodiments, the promoter is a CAG promoter.

[0245] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, 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. In some embodiments, the virus is an alphavirus. In some embodiments, the virusis a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, 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.

[0246] In some embodiments, 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- HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0247] In some embodiments, 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. In some embodiments, the virus is AAV 12 or a derivative thereof. In some embodiments, the virus is AAV 13 or a derivative thereof. In some embodiments, the virus is AAV 14 or a derivative thereof. In some embodiments, the virus is AAV 15 or a derivative thereof. In some embodiments, the virus is AAV 16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rhlO or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. InI l lsome embodiments, the virus is AAV-rhM4-l or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV- Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV- 2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV- HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, 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.

[0248] In some embodiments, 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.

[0249] In some embodiments, 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). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, 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. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0250] In some embodiments, the engineered endonuclease or the engineered endonuclease system is introduced into the cell in any suitable way, either stably or transiently. In some embodiments, the engineered endonuclease or the engineered endonuclease system is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes the engineered endonuclease or the engineered endonuclease system. For example, 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. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Deliverymethods to cells for polypeptides and / or RNPs are known in the art, for example by electroporation or by cell squeezing.

[0251] Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (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. In some embodiments, the delivery is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.

[0252] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817.

[0253] In some embodiments, the present disclosure provides a cell comprising a vector or a nucleic acid described herein. In some embodiments, the cell expresses a gene editing system or parts thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is genome edited ex vivo. In some embodiments, the cell is genome edited in vivo.Lipid nanoparticles

[0254] Disclosed herein, in certain embodiments, are lipid nanoparticles comprising an engineered endonuclease system of the disclosure for delivery of the engineered endonuclease systems into a cell.

[0255] In some embodiments, 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.

[0257] In some embodiments, the lipid nanoparticle is used to deliver the engineered endonuclease system or components thereof to a cell. In some embodiments, the cell is a specific cell type. In some embodiments, 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 Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or aHeliothis virescens cell), a yeast cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g., a E. coli cell, a streptococcus bacterium cell, a streptomyces soil bacteria cell, or an archaea cell). In some embodiments, 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. In some embodiments, 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. In some embodiments, the primary cell is a T cell. In some embodiments, the primary cell is a hematopoietic stem cell (HS...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered nuclease system, 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.

2. The engineered nuclease system of claim 1, wherein the engineered guide polynucleotide is a single guide nucleic acid.

3. The engineered nuclease system of claim 1, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

4. The engineered nuclease system of any one of claims 1-3, wherein the engineered guide polynucleotide is RNA.

5. The engineered nuclease system of any one of claims 1-4, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3954-4050.

6. The engineered nuclease system of any one of claims 1-5, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

7. The engineered nuclease system of any one of claims 1-5, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

8. The engineered nuclease system of any one of claims 1-5, wherein the endonuclease is fused to the engineered guide polynucleotide.

9. A fusion endonuclease, 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.

10. An engineered nuclease system, comprising: a) an engineered endonuclease comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4051-4078; andb) 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.

11. The engineered nuclease system of claim 10, wherein the engineered guide polynucleotide is a single guide nucleic acid.

12. The engineered nuclease system of claim 10, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

13. The engineered nuclease system of any one of claims 10-12, wherein the engineered guide polynucleotide is RNA.

14. The engineered nuclease system of any one of claims 10-13, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 4094-4105.

15. The engineered nuclease system of any one of claims 10-14, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

16. The engineered nuclease system of any one of claims 10-14, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

17. The engineered nuclease system of any one of claims 10-14, wherein the endonuclease is fused to the engineered guide polynucleotide.

18. A fusion endonuclease, 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.

19. 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 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.

20. The engineered nuclease system of claim 19, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3749-3787 and 3825-3861.

21. The engineered nuclease system of any one of claims 19-20, wherein the engineered guide polynucleotide is a single guide nucleic acid.

22. The engineered nuclease system of any one of claims 19-20, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

23. The engineered nuclease system of any one of claims 19-22, wherein the engineered guide polynucleotide is RNA.

24. The engineered nuclease system of any one of claims 19-23, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

25. The engineered nuclease system of any one of claims 19-23, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

26. The engineered nuclease system of any one of claims 19-23, wherein the endonuclease is fused to the engineered guide polynucleotide.

27. 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.

28. The engineered nuclease system of claim 27, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 3710-3748 and 3788-3824.

29. The engineered nuclease system of any one of claims 27-28, wherein the engineered guide polynucleotide is a single guide nucleic acid.

30. The engineered nuclease system of any one of claims 27-28, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

31. The engineered nuclease system of any one of claims 27-30, wherein the engineered guide polynucleotide is RNA.

32. The engineered nuclease system of any one of claims 27-31, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

33. The engineered nuclease system of any one of claims 27-31, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

34. The engineered nuclease system of any one of claims 27-31, wherein the endonuclease is fused to the engineered guide polynucleotide.

35. 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 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.

36. The engineered nuclease system of claim 35, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 4106-4122.

37. The engineered nuclease system of any one of claims 35-36, wherein the engineered guide polynucleotide is a single guide nucleic acid.

38. The engineered nuclease system of any one of claims 35-38, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

39. The engineered nuclease system of any one of claims 35-38, wherein the engineered guide polynucleotide is RNA.

40. The engineered nuclease system of any one of claims 35-39, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

41. The engineered nuclease system of any one of claims 35-39, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

42. The engineered nuclease system of any one of claims 35-39, wherein the endonuclease is fused to the engineered guide polynucleotide.

43. 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 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.

44. The engineered nuclease system of claim 43, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 4123-4172.

45. The engineered nuclease system of any one of claims 43-44, wherein the engineered guide polynucleotide is a single guide nucleic acid.

46. The engineered nuclease system of any one of claims 43-45, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

47. The engineered nuclease system of any one of claims 43-45, wherein the engineered guide polynucleotide is RNA.

48. The engineered nuclease system of any one of claims 43-47, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

49. The engineered nuclease system of any one of claims 43-47, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

50. The engineered nuclease system of any one of claims 43-47, wherein the endonuclease is fused to the engineered guide polynucleotide.

51. A method for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system of any one of claims 1-50.

52. The method of claim 51, wherein modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.

53. The method of any one of claims 51-52, wherein the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

54. The method of any one of claims 51-53, wherein the modification is in vitro.

55. The method of any one of claims 51-53, wherein the modification is in vivo.

56. The method of any one of claims 51-53, wherein the modification is ex vivo.

57. The method of any one of claims 51-53, wherein the gRNA is encoded by a sequence having any one of SEQ ID NOs: 251-260, 271-274, and 279-290.

58. The method of any one of claims 51-56, wherein the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 261-270, 275-278, and 291-302.

59. A method of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system of any one of claims 1-50.

60. The method of claim 59, further comprising selecting cells comprising the modification.

61. A method 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.

62. A method of modifying a HA01 gene comprising contacting the HAO1 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.

63. A method 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 AAVS 1 gene or within an intron of the AAVS 1 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4106-4122.

64. A method 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; andb) 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.

65. A cell comprising the endonuclease of any one of claims 1-50.

66. The cell of claim 65, wherein the cell is a eukaryotic cell.

67. The cell of claim 65, wherein the cell is a mammalian cell.

68. The cell of claim 65, wherein the cell is an immortalized cell.

69. The cell of claim 65, wherein the cell is an insect cell.

70. The cell of claim 65, wherein the cell is a yeast cell.

71. The cell of claim 65, wherein the cell is a plant cell.

72. The cell of claim 65, wherein the cell is a fungal cell.

73. The cell of claim 65, wherein the cell is a prokaryotic cell.

74. The cell of claim 65, wherein 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.

75. The cell of claim 65, wherein the cell is an engineered cell.

76. The cell of claim 65, wherein the cell is a stable cell.

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