Engineered nucleases and guide RNA spacer sequences therefor, and compositions, and methods of use thereof

Abstract

Provided herein are one or more engineered nucleases and systems, compositions, and methods thereof, wherein the one or more engineered nucleases exhibits broadened targeting range. The engineered nuclease can be used to effect binding, cleaving, and / or editing a target polynucleotide sequence. The one or more engineered nucleases can be engineered variants of a small CRISPR / Cas protein. Also provided are methods, systems, and algorithms for identifying guide RNA (gRNA) spacer sequences.

Description

EPICR.026WO PATENT ENGINEERED NUCLEASES AND GUIDE RNA SPACER SEQUENCES THEREFOR, AND COMPOSITIONS, AND METHODS OF USE THEREOF REFERENCE TO PRIORITY APPLICATIONS

[0001] This application claims priority to U.S. provisional application numbers: 63 / 513746, filed July 14, 2023; 63 / 513751, filed July 14, 2023; 63 / 513755, filed July 14, 2023; and 63 / 513757, filed July 14, 2023. The content of each of the aforementioned applications are hereby expressly incorporated herein by reference in their entirety. REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled EPICR.026WOSequenceListing.xml created July 10, 2024, which is 950,171 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND

[0003] Various nucleases (e.g., endonucleases) can be utilized to edit a target sequence in a cell or regulate expression or activity of the target gene in the cell. For example, a heterologous nuclease can be introduced (e.g., delivered, expressed, etc.) to the cell, and the heterologous nuclease, either alone or along with an additional agent, can affect such editing or regulation of the target gene. For example, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) is a family of nucleases that are involved in specifically binding, cleaving, and / or editing a target deoxyribonucleic acid (DNA) sequence or ribonucleic acid (RNA) sequence (e.g., foreign DNA sequence or RNA sequence). The programmable nature of these nucleases has facilitated their use as a versatile technology that is revolutionizing the field of target gene manipulation, e.g., as gene therapy to treat or ameliorate a condition (e.g., a disease) of a subject.

[0004] Designing novel guide RNA for CRISPR Cas remains a challenge due to off- target effects and target specificity, among other difficulties. SUMMARY

[0005] Provided herein is a composition comprising: (a) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acidsequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease; and (b) a non- naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA, wherein the non-naturally occurring guide RNA comprises: (i) a region that binds to the programmable nuclease; and (ii) a guide sequence that binds to a target sequence of a target nucleic acid adjacent to a protospacer-adjacent motif (PAM) sequence that is different than a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. In some embodiments, the canonical PAM sequence is TTTA. In some embodiments, the canonical PAM sequence is TTTG. In some embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In someembodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease is nuclease inactive. In some embodiments, the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease is coupled or fused to a gene modulator. In some embodiments, the gene modulator is a transcriptional activator. In some embodiments, the gene modulator is a transcriptional repressor. In some embodiments, the programmable nuclease is coupled or fused to a gene editing moiety. In some embodiments, the gene editing moiety is a base-editor.

[0006] Provided herein is a system comprising a composition, wherein the programmable nuclease is in a complex with the non-naturally occurring guide RNA.

[0007] Provided herein is a vector comprising the composition described herein or the system described herein. In some embodiments, the vector is a particle. In some embodiments, the particle is a lipid nanoparticle (LNP). In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0008] Provided herein is a guide RNA comprising: (i) a region that binds to a programmable nuclease comprising an amino acid sequence having at least about 80% sequenceidentity to the amino acid sequence of SEQ ID NO: 1; and (ii) a guide sequence that binds to a target sequence of a target nucleic acid adjacent to a protospacer-adjacent motif (PAM) sequence that is different than a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. In some embodiments, the canonical PAM sequence is TTTA. In some embodiments, the canonical PAM sequence is TTTG. In some embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease is nuclease inactive. In some embodiments, the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 74% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 75% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 80% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 85% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 90% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 95% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmablenuclease comprises or consists of a polynucleotide sequence having at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, said scaffold sequence has a length of at most about 110 nucleotides. In some embodiments, said scaffold sequence has a length of at most about 105 nucleotides.

[0009] Provided herein is a nucleic acid encoding the guide RNA described herein.

[0010] Provided herein is a vector comprising the guide RNA described herein or the nucleic acid described herein. In some embodiments, the vector is a particle. In some embodiments, the particle is a lipid nanoparticle (LNP). In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the vector further comprises the programmable nuclease, or a nucleic acid sequence encoding the programmable nuclease.

[0011] Provided herein is a method of modulating one or more target genes in a cell, the method comprising contacting the cell with the composition described herein, the system described herein, or the vector described herein, wherein the programmable nuclease and the guide RNA form a complex and modulate the one or more target genes in the cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the complex binds to the target sequence. In some embodiments, the target sequence is within a coding sequence of the target gene. In some embodiments, the target sequence is within a regulatory region of the target gene. In some embodiments, the target sequence is adjacent to the PAM sequence that is different from the canonical PAM sequence. In some embodiments, the modulating comprises activating the target gene, or a regulatory region thereof. In some embodiments, the modulating comprises repressing the target gene, or a regulatory region thereof. In some embodiments, the modulating comprises editing the target gene, or a regulatory region thereof. In some embodiments, the modulating comprises nicking the target gene, or a regulatory region thereof. In some embodiments, the modulating comprises knocking-in a gene. In some embodiments, the modulating comprises knocking-out the target gene. In some embodiments, the modulating comprises introducing one or more base substitutions, one or more insertions, one or more deletions, or a combination thereof, into the target gene, or a regulatory region thereof. In some embodiments, the modulatingcomprises altering methylation of the target gene, or a regulatory region thereof. In some embodiments, the modulating comprises altering acetylation of the target gene, or a regulatory region thereof. In some embodiments, the modulating comprises altering the acetylation or methylation of at least one histone or nucleosome associated with the target gene, or a regulatory region thereof.

[0012] Described herein is a method of preventing, inhibiting, or treating a genetic disorder in a subject, the method comprising administering to the subject the composition described herein, the system described herein, or the vector described herein, wherein the programmable nuclease and the guide RNA form a complex and modulate one or more target gene associated with the genetic disorder. In some embodiments, the composition, the system, or the vector are administered in an amount sufficient to modulate the one or more target gene associated with the genetic disorder. In some embodiments, the subject is human. In some embodiments, the genetic disorder is selected from the group consisting of: X-linked severe combined immune deficiency, sickle cell anemia, thalassemia, hemophilia, neoplasia, cancer, age-related macular degeneration, schizophrenia, trinucleotide repeat disorders, fragile X syndrome, prion-related disorders, amyotrophic lateral sclerosis, drug addiction, autism, Alzheimer’s disease, Parkinson’s disease, cystic fibrosis, blood and coagulation disease or disorders, inflammation, facioscapulohumeral muscular dystrophy, retinitis pigmentosa, Leber congenital amaurosis, glaucoma, immune-related diseases or disorders, metabolic diseases and disorders, liver diseases and disorders, kidney diseases and disorders, muscular / skeletal diseases and disorders, neurological and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and ocular diseases and disorders. In some embodiments, the complex binds to the target sequence. In some embodiments, the target sequence is within a coding sequence of the target gene. In some embodiments, the target sequence is within a regulatory region of the target gene. In some embodiments, the target sequence is adjacent to the PAM sequence that is different from the canonical PAM sequence. In some embodiments, the modulating comprises activating the one or more target gene, or a regulatory region thereof. In some embodiments, the modulating comprises repressing the one or more target gene, or a regulatory region thereof. In some embodiments, the modulating comprises editing the one or more target gene, or a regulatory region thereof. In some embodiments, the modulating comprises nicking the one or more target gene, or a regulatory region thereof. In some embodiments, themodulating comprises knocking-in a gene into the one or more target gene, or a regulatory region thereof. In some embodiments, the modulating comprises knocking-out the one or more target gene. In some embodiments, the modulating comprises introducing one or more base substitutions, one or more insertions, one or more deletions, or a combination thereof, into the one or more target gene, or a regulatory region thereof. In some embodiments, the modulating comprises altering methylation of the one or more target gene, or a regulatory region thereof. In some embodiments, the modulating comprises altering acetylation of the one or more target gene, or a regulatory region thereof. In some embodiments, the modulating comprises altering the acetylation or methylation of at least one histone or nucleosome associated with the one or more target gene, or a regulatory region thereof.

[0013] Provided herein is a composition comprising: (a) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease; and (b) a non- naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA, wherein the non-naturally occurring guide RNA comprises a spacer sequence that is at least partially complementary to a nucleotide sequence within one or more target genes or a regulatory region thereof, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, and wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes or the regulatory region thereof. In some embodiments, the seed sequence is from nucleotide positions 1-6 from the 5’-end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-7 from the 5’-end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-8 from the 5’-end of the spacer sequence. In some embodiments, the spacer sequence comprises additional nucleotides at the 3’-end of the seed sequence. In some embodiments, the additional nucleotides are 1-20 additional nucleotides. In some embodiments, the additional nucleotides form a nucleotide sequence that is partially complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory region thereof. In some embodiments, the additional nucleotides form a nucleotide sequence that is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory regionsthereof. In some embodiments, the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer-adjacent motif (PAM) sequence. In some embodiments, the PAM sequence is a canonical PAM sequence. In some embodiments, the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence is a non-canonical PAM sequence. In some embodiments, the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. In some embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least onedeletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease is nuclease inactive. In some embodiments, the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease is coupled or fused to a gene modulator. In some embodiments, the gene modulator is a transcriptional activator. In some embodiments, the gene modulator is a transcriptional repressor. In some embodiments, the programmable nuclease is coupled or fused to a gene editing moiety. In some embodiments, the gene editing moiety is a base-editor.

[0014] Provided herein is a composition comprising: (a) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease; and (b) a non- naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA, wherein the non-naturally occurring guide RNA comprises a spacer sequence that is at least partially complementary to a nucleotide sequence within a target allele or a regulatory region thereof comprising a single nucleotide polymorphism (SNP), wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, wherein the seed sequence binds to a corresponding nucleotide sequence of the nucleotide sequence within the target allele or regulatoryregion thereof, wherein the corresponding sequence comprises the SNP, and wherein the seed sequence is 100% complementary to the corresponding nucleotide sequence. In some embodiments, the SNP is a disease-causing SNP. In some embodiments, the seed sequence is from nucleotide positions 1-6 from the 5’-end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-7 from the 5’-end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-8 from the 5’-end of the spacer sequence. In some embodiments, the spacer sequence comprises additional nucleotides at the 3’- end of the seed sequence. In some embodiments, the additional nucleotides are 1-20 additional nucleotides. In some embodiments, the additional nucleotides form a nucleotide sequence that is partially complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory region thereof. In some embodiments, the additional nucleotides form a nucleotide sequence that is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof. In some embodiments, the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer-adjacent motif (PAM) sequence. In some embodiments, the PAM sequence is a canonical PAM sequence. In some embodiments, the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence is a non-canonical PAM sequence. In some embodiments, the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. In some embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease is nuclease inactive. In some embodiments, the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease is coupled or fused to a gene modulator. In some embodiments, thegene modulator is a transcriptional activator. In some embodiments, the gene modulator is a transcriptional repressor. In some embodiments, the programmable nuclease is coupled or fused to a gene editing moiety. In some embodiments, the gene editing moiety is a base-editor.

[0015] Provided herein is a guide RNA comprising: (a) a region that binds to a programmable nuclease comprising an amino acid sequence having at least or at least about 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1; and (b) a guide sequence that binds to a target sequence of a target gene, wherein the guide sequence comprises a spacer sequence that is at least partially complementary to the target sequence, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, and wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the target sequence. In some embodiments, the seed sequence is from nucleotide positions 1-6 from the 5’- end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-7 from the 5’-end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-8 from the 5’-end of the spacer sequence. In some embodiments, the spacer sequence comprises additional nucleotides at the 3’-end of the seed sequence. In some embodiments, the additional nucleotides are 1-20 additional nucleotides. In some embodiments, the additional nucleotides form a nucleotide sequence that is partially complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory region thereof. In some embodiments, the additional nucleotides form a nucleotide sequence that is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof. In some embodiments, the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer-adjacent motif (PAM) sequence. In some embodiments, the PAM sequence is a canonical PAM sequence. In some embodiments, the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence is a non-canonical PAM sequence. In some embodiments, the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is oneor more nucleic acid sequence selected from Table 11. In some embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease isnuclease inactive. In some embodiments, the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least or at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease is coupled or fused to a gene modulator. In some embodiments, the gene modulator is a transcriptional activator. In some embodiments, the gene modulator is a transcriptional repressor. In some embodiments, the programmable nuclease is coupled or fused to a gene editing moiety. In some embodiments, the gene editing moiety is a base-editor. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 74% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 75% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 80% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 85% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 90% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 95% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the scaffold sequence has a length of at most about 110 nucleotides. Insome embodiments, the scaffold sequence has a length of at most about 105 nucleotides.

[0016] Provided herein is a guide RNA comprising: (a) a region that binds to a programmable nuclease comprising an amino acid sequence having at least or at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1; and (b) a guide sequence that binds to a target sequence of a target allele comprising a single nucleotide polymorphism (SNP), wherein the guide sequence comprises a spacer sequence that is at least partially complementary to the target sequence, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the target sequence, and wherein the corresponding nucleotide sequence comprises the SNP. In some embodiments, the SNP is a disease-causing SNP. In some embodiments, the seed sequence is from nucleotide positions 1-6 from the 5’-end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-7 from the 5’-end of the spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-8 from the 5’- end of the spacer sequence. In some embodiments, the spacer sequence comprises additional nucleotides at the 3’-end of the seed sequence. In some embodiments, the additional nucleotides are 1-20 additional nucleotides. In some embodiments, the additional nucleotides form a nucleotide sequence that is partially complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory region thereof. In some embodiments, the additional nucleotides form a nucleotide sequence that is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof. In some embodiments, the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer-adjacent motif (PAM) sequence. In some embodiments, the PAM sequence is a canonical PAM sequence. In some embodiments, the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence is a non-canonical PAM sequence. In some embodiments, the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. Insome embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease is nuclease inactive. In some embodiments, the programmable nuclease comprises an amino acidsubstitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least or at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease is coupled or fused to a gene modulator. In some embodiments, the gene modulator is a transcriptional activator. In some embodiments, the gene modulator is a transcriptional repressor. In some embodiments, the programmable nuclease is coupled or fused to a gene editing moiety. In some embodiments, the gene editing moiety is a base-editor. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 74% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 75% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 80% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 85% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 90% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 95% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 597. In some embodiments, the scaffold sequence has a length of at most about 110 nucleotides. In some embodiments, the scaffold sequence has a length of at most about 105 nucleotides.

[0017] Provided herein is a nucleic acid encoding the guide RNA described herein.

[0018] Provided herein is a system comprising the composition described herein, wherein the programmable nuclease is in a complex with the non-naturally occurring guide RNA.

[0019] Provided herein is a vector comprising the composition described herein, the guide RNA described herein, the nucleic acid described herein, or the system described herein. In some embodiments, the vector is a particle. In some embodiments, the particle is a lipid nanoparticle (LNP). In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0020] Described herein is a method of modulating one or more target genes or a target allele in a cell, the method comprising: contacting the cell with the composition described herein, the system described herein, or the vector described herein, wherein the programmable nuclease and the non-naturally occurring guide RNA form a complex and modulate the one or more target genes or the target allele in the cell. In some embodiments, the method results in enhanced binding of the programmable nuclease to the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof, relative to a method that uses a guide RNA with a seed sequence that is partially complementary to a corresponding nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof. In some embodiments, the method results in enhanced modulation of the one or more target genes or the target allele relative to a method that uses a guide RNA with a seed sequence that is partially complementary to a corresponding nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the modulating comprises activating the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises repressing the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises editing the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises nicking the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises knocking-in a gene. In some embodiments, the modulating comprises knocking-out the one ormore target genes, or the target allele. In some embodiments, the modulating comprises introducing one or more base substitutions, one or more insertions, one or more deletions, or a combination thereof, into the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises altering methylation of the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises altering acetylation of the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises altering the acetylation or methylation of at least one histone or nucleosome associated with the one or more target genes, the target allele, or a regulatory region thereof.

[0021] Provided herein is a nuclease system comprising (i) a programmable nuclease comprising an amino acid sequence having at least or at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease, and (ii) a non-naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA exhibiting specific binding to a target polynucleotide of a target gene, wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced immunogenicity compared to that of a control system that does not comprise the programmable nuclease. In some embodiments, the reduced immunogenicity comprises a decrease in or absence of T cell activation. In some embodiments, the reduced immunogenicity comprises a decrease in or absence production of one or more inflammatory cytokines by T cells. In some embodiments, the T cells comprise cytotoxic T cells, regulatory T cells, T-helper cells, gamma-delta T cells, memory T cells, CD3+ T cells, CD4+ T cells, or CD8+ T cells. In some embodiments, the one or more inflammatory cytokines comprise interleukin (IL)-1, IL-6, IL-8, or IL-17, tumor necrosis factor-alpha (TNF-alpha), or interferon gamma (IFN-Ȗ). In some embodiments, the one or more inflammatory cytokines are IFN-Ȗ. In some embodiments, the decrease in production of one or more inflammatory cytokines is characterized by a reduced IFN-Ȗ production by the T cells, wherein IFN-Ȗ level is reduced by at least or at least about 15%, at least or at least about 20%, at least or at least about 30%, at least or at least about 40%, at least or at least about 50%, at least or at least about 60%, at least or at least about 70%, at least or at least about 80%, at least or at least about 90, at least or at least about 95% or 100% as compared to that of the control system. In some embodiments, the production of one or more inflammatory cytokines is at least or at least about 80%, at least or at least about 90, at least or at least about 95%, or at least or at least about 100%as compared to that of a control subject that is not administered with the nuclease system. In some embodiments, the nuclease system is administered with one or more pharmaceutically acceptable excipients. In some embodiments, the excipient is selected from a solvent, aqueous solvent, non- aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids, liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplex, peptide, protein, cell, hyaluronidase, or mixtures thereof. In some embodiments, the nuclease system is formulated. In some embodiments, the nuclease system is administered by an injection. In some embodiments, the injection is an intradermal injection, a subcutaneous injection, an intramuscular injection, or an intravenous injection. In some embodiments, the non-naturally occurring guide RNA comprises a spacer sequence that is at least partially complementary to a nucleotide sequence within one or more target genes or a regulatory region thereof, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’- end of the spacer sequence, and wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes or the regulatory region thereof. In some embodiments, the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer- adjacent motif (PAM) sequence. In some embodiments, the PAM sequence is a canonical PAM sequence. In some embodiments, the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence is a non-canonical PAM sequence. In some embodiments, the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. In some embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at leastabout 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease is nuclease inactive. In some embodiments, the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least or at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nucleasecomprises an amino acid sequence having at least or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease is coupled or fused to a gene modulator. In some embodiments, the gene modulator is a transcriptional activator. In some embodiments, the gene modulator is a transcriptional repressor. In some embodiments, the programmable nuclease is coupled or fused to a gene editing moiety. In some embodiments, the gene editing moiety is a base-editor.

[0022] Provided herein is a nuclease system comprising: (i) a programmable nuclease comprising an amino acid sequence having at least or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease, and (ii) a non-naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA exhibiting specific binding to a target polynucleotide of a target gene, wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced toxicity compared to that of a control system that does not comprise the programmable nuclease. In some embodiments, the reduced toxicity is assessed by measuring liver function, blood counts, hemoglobin counts, hematocrit counts, or white blood differentiation. In some embodiments, the toxicity of the subjected treated with the nuclease system is reduced by at least or at least about 15%, at least or at least about 20%, at least or at least about 30%, at least or at least about 40%, at least or at least about 50%, at least or at least about 60%, at least or at least about 70%, at least or at least about 80%, at least or at least about 90, at least or at least about 95% or 100% as compared to that of the control system. In some embodiments, the reduced toxicity is sustained for at least about 1 week, at least about 4 weeks, at least about 12 weeks, at least about 24 weeks, or at least about 48 weeks. In some embodiments, the nuclease system is administered with one or more pharmaceutically acceptable excipients. In some embodiments, the excipient is selected from a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids, liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplex, peptide, protein, cell, hyaluronidase, or mixtures thereof. In some embodiments, the nuclease system is formulated. In some embodiments, the nuclease system is administered by an injection. In some embodiments, the injection is an intradermal injection, a subcutaneous injection, an intramuscular injection, or an intravenous injection. In some embodiments, the non-naturally occurring guide RNA comprises a spacer sequence that is at least partially complementary to anucleotide sequence within one or more target genes or a regulatory region thereof, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1- 7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, and wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes or the regulatory region thereof. In some embodiments, the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer-adjacent motif (PAM) sequence. In some embodiments, the PAM sequence is a canonical PAM sequence. In some embodiments, the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence is a non-canonical PAM sequence. In some embodiments, the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. In some embodiments, the programmable nuclease is a Cas nuclease. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence having at least or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848. In some embodiments, the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201. In some embodiments, the amino acid sequence comprises at leastone deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the programmable nuclease has a length of at most about 528 amino acids. In some embodiments, the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the programmable nuclease is nuclease inactive. In some embodiments, the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least or at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease comprises an amino acid sequence having at least or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the programmable nuclease is coupled or fused to a gene modulator. In some embodiments, the gene modulator is a transcriptional activator. In some embodiments, the gene modulator is a transcriptional repressor. In some embodiments, the programmable nuclease is coupled or fused to a gene editing moiety. In some embodiments, the gene editing moiety is a base-editor.

[0023] Provided herein is a vector comprising the system described herein. In some embodiments, the vector is a particle. In some embodiments, the particle is a lipid nanoparticle (LNP). In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0024] Provided herein is a method of modulating one or more target genes or a target allele in a cell, the method comprising: contacting the cell with the nuclease system of any one of claims 1-52 or the vector of any one of claims 53-57, wherein the programmable nuclease and the non-naturally occurring guide RNA form a complex and modulate one or more target genes or the target allele in the cell associated with the one or more disease conditions, wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced immunogenicity compared to that of a control system that does not comprise the programmable nuclease, and wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced toxicity compared to that of a control system that does not comprise the programmable nuclease. In some embodiments, the method results in enhanced binding of the programmable nuclease to the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof, relative to a method that uses a guide RNA with a seed sequence that is partially complementary to a corresponding nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof. In some embodiments, the method results in enhanced modulation of the one or more target genes or the target allele relative to a method that uses a guide RNA with a seed sequence that is partially complementary to a corresponding nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the modulating comprises activating the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises repressing the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises editing the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises nicking the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises knocking- in a gene. In some embodiments, the modulating comprises knocking-out the one or more target genes, or the target allele. In some embodiments, the modulating comprises introducing one or more base substitutions, one or more insertions, one or more deletions, or a combination thereof, into the one or more target genes, the target allele, or a regulatory region thereof. In someembodiments, the modulating comprises altering methylation of the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises altering acetylation of the one or more target genes, the target allele, or a regulatory region thereof. In some embodiments, the modulating comprises altering the acetylation or methylation of at least one histone or nucleosome associated with the one or more target genes, the target allele, or a regulatory region thereof.

[0025] There is an unmet need for methods and systems that identify guide RNAs (gRNAs) for optimal target binding and off-target potential. The methods and systems provided herein meet this unmet need.

[0026] In one aspect, a computer-implemented method for identifying guide RNA (gRNA) spacer sequences is provided, the method comprising: (a) identifying an optimal target region of a target gene, wherein the optimal target region comprises a target nucleotide sequence that can be bound by a Cas protein-gRNA complex to modulate the target gene; (b) identifying one or more protospacer adjacent motif (PAM) sequences present within the optimal target region, wherein the PAM sequence is specific for the Cas protein; (c) extracting a candidate spacer sequence that is immediately 3’ to the PAM sequence identified in (b); and(d) identifying one or more potential off-target binding sites for the candidate spacer sequence based on a pre-defined rule; and(e) generating an association between a candidate spacer sequence extracted in (c) and the one or more potential off-target binding sites identified in (d).

[0027] In some embodiments, the optimal target region is an optimal target region for activating the target gene. In some embodiments, the optimal target region is -1000 bp to +200 bp from the transcription start site (TSS) of the target gene. In some embodiments, the optimal target region is an optimal target region for suppressing the target gene. In some embodiments, the optimal target region is -200 bp to +1000 bp from the TSS of the target gene. In some embodiments, the Cas protein is Cas12f or a variant thereof. In some embodiments, the Cas protein comprises or consists of an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the Cas protein comprises or consists of the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the Cas protein comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 1092-1240. In some embodiments, the Cas protein comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 1000-1091. In some embodiments, the Casprotein comprises or consists of at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 31-40, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 41-60, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the at least one deletion comprises at least one deletion from amino acid residues 61-80, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the at least one deletion is a deletion of at least 30 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the Cas protein has a length of about 350 amino acids to about 900 amino acids. In some embodiments, the Cas protein has a length of at most about 528 amino acids. In some embodiments, the Cas protein comprises an amino acid mutation which results in reduced nuclease activity. In some embodiments, the Cas protein is nuclease inactive. In some embodiments, the Cas protein comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the Cas protein comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1000. In some embodiments, the Cas protein comprises an amino acid sequence having at least or at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 1010. In some embodiments, the Cas protein comprises an amino acid sequence having at least or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1010. In some embodiments, the PAM sequence specific for the Cas protein is a canonical PAM sequence. In some embodiments, the canonical PAM sequence is TTTR, wherein R is A or G. In some embodiments, the PAM sequence specific for the Cas protein is a non- canonical PAM sequence. In some embodiments, the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG. In some embodiments, the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11. In some embodiments, the candidate spacer sequence is from 10-30 nucleotides inlength. In some embodiments, the identifying one or more potential off-target binding sites of (d) comprises mapping the candidate spacer sequence to a reference genome to identify at least one candidate off-target binding site. In some embodiments, the pre-defined rule identifies a candidate off-target binding site as a potential off-target binding site when the candidate off-target binding site has a PAM sequence specific for the Cas protein within 5 nucleotides upstream of the 5’ end of the candidate off-target binding site. In some embodiments, the pre-defined rule eliminates a candidate off-target binding site as a potential off-target binding site when the candidate off-target binding site has one or more nucleotide mismatches within a seed sequence of the candidate spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-6 from the 5’ end of the candidate spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-7 from the 5’ end of the candidate spacer sequence. In some embodiments, the seed sequence is from nucleotide positions 1-8 from the 5’ end of the candidate spacer sequence. In some embodiments, the pre-defined rule eliminates a candidate off-target binding site as a potential off-target binding site when the candidate off-target binding site has more than three nucleotide mismatches within the candidate spacer sequence in a region outside of the seed sequence. In some embodiments, the associating of (e) comprises outputting a list or table of candidate spacer sequences associated with corresponding potential off-target binding sites. In some embodiments, the computer-implemented method further comprises associating each candidate spacer sequence with an indication of whether the candidate spacer sequence binds to the optimal target region for activating the target gene, or binds to the optimal target region for suppressing the target gene.

[0028] In another aspect, a computer-implemented system is provided comprising at least one processor and instructions executable by the at least one processor to provide an application for implementing the computer-implemented method of any one of the preceding.

[0029] In another aspect, a non-transitory computer-readable storage media is provided encoded with instructions executable by one or more processors for implementing the computer- implemented method of any one of the preceding.

[0030] 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

[0031] Non-limiting aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0032] FIG.1 schematically illustrates structural alignment between Un1Cas12f1 and AsCas12f, to identify one or more domains in Un1Cas12f1 that may not be conserved in one or more additional Cas12f homologous structures.

[0033] FIG.2 schematically illustrates structural alignment between Un1Cas12f1 and Un2Cas12f1, to identify one or more domains in Un1Cas12f1 that may not be conserved in one or more additional Cas12f homologous structures.

[0034] FIG.3A schematically illustrates selection of various domains of Un1Cas12f1 that are to be at least partially deleted to generate one or more engineered nucleases. FIG. 3B schematically illustrates deletion landscape approach to generate engineered nuclease variants of Un1Cas12f1.

[0035] FIG. 4 schematically illustrates example deletion landscape of a Cas protein (e.g., dCas9), to identify one or more domains in the Cas protein that may be deleted with minimal or substantially no effect on the Cas protein’s activity (e.g., the ability to induce transcription expression when the modified variant of dCas9 is operatively coupled to a gene repressor).

[0036] FIG. 5 shows enhanced expression of endogenous IFN gamma (top plot) and endogenous CD2 (bottom plot) in cells by various engineered nuclease variants disclosed herein. The engineered nuclease variants were engineered to exhibit reduced nuclease activity (e.g., dCas variants), and were fused with a gene activating modulator.

[0037] FIG.6 shows reduced expression of a target gene (e.g., GFP) in cells by various engineered nuclease variants disclosed herein. The engineered nuclease variants were engineered to exhibit reduced nuclease activity (e.g., dCas variants), and were fused with a gene repressing modulator. Two different gene repressing modulators were used: gene repressor A (top plot) and gene repressor B (bottom plot).

[0038] FIG.7 shows enhanced expression of endogenous CD2 (top plot), endogenous IFN gamma (middle plot), and endogenous CXCR4 (bottom plot) in cells by various engineered nuclease variants disclosed herein. The engineered nuclease variants were engineered to exhibit reduced nuclease activity (e.g., dCas variants), and were fused with a gene activating modulator.

[0039] FIG. 8 schematically illustrates a guide nucleic acid molecule configured to form a complex with a Cas protein.

[0040] FIG. 9 schematically illustrates regions of a scaffold region of a guide nucleic acid molecule that can be modified (e.g., mutated or deleted) to engineer the guide nucleic acid molecule.

[0041] FIG. 10 shows reduced expression of a target gene (e.g., GFP) in cells by a plurality of engineered guide RNA variants disclosed herein. The plurality of engineered guide RNA variants was modified at least in the scaffold region, as compared to a control guide RNA sequence (“SQ”). The reduced gene expression was performed with the same dCas protein coupled to a gene repressing modulator.

[0042] FIG. 11 shows reduced expression of a target gene (e.g., GFP) in cells by an additional plurality of engineered guide RNA variants disclosed herein. The additional plurality of engineered guide RNA variants was modified at least in the scaffold region, as compared to a control guide RNA sequence (“SQ”). The reduced gene expression was performed with the same dCas protein coupled to a gene repressing modulator.

[0043] FIG.12 shows enhanced expression of endogenous CD2 in cells by a different plurality of engineered guide RNA variants disclosed herein. The different plurality of engineered guide RNA variants was modified at least in the scaffold region, as compared to a control guide RNA sequence (“SQ”). The enhanced gene expression was performed with the same dCas protein coupled to a gene activating modulator.

[0044] FIG.13 schematically illustrates examples of the engineered guide nucleic acid molecules disclosed herein.

[0045] FIG. 14 shows comparison of sizes of various DNA vectors, each encoding a Cas protein and a respective single guide nucleic acid molecule.

[0046] FIG.15 shows increased expression of endogenous IFN gamma (IFNȖ) in cells by the truncation nuclease variant t1 disclosed herein. The engineered nuclease variant t1 was fused with a gene activating modulator, VPR.

[0047] FIG. 16 shows reduced expression of endogenous CXCR4 in cells by the chimeric nuclease variants disclosed herein. The engineered nuclease variant t1 was fused with a gene repressing modulator (ZNF10-KRAB-hDNMT3L).

[0048] FIG. 17A shows an experimental timeline for assessing PAM characterization using the engineered reporter system.

[0049] FIG.17B schematically illustrates an engineered reporter system (repression or activation system) with all-in-one plasmid expressing cA2-modulator fusion protein and sgRNA targeting TRE3GS promoter of the engineered reporter system.

[0050] FIG.17C shows GFP expression of cells bearing the repression reporter system with a canonical PAM sequence-TRE3GS promoter targeted by either TRE3G sgRNA or non- target (NT) sgRNA.

[0051] FIG.17D shows GFP expression of cells bearing the activation reporter system with canonical PAM sequence (5’-CGTTTA-3’ or 5’-CGTTTG-3’)-TRE3GS promoter or a non- canonical PAM sequence (5’-CGTCTG-3’)-TRE3GS promoter targeted by either TRE3G sgRNA or NT sgRNA.

[0052] FIG. 18A shows an experimental timeline for PAM characterization using N6 library PAM sequences.

[0053] FIG.18B shows GFP expression of cells bearing the activation reporter system with N6 library PAM sequences-TRE3GS promoter targeted by either TRE3G sgRNA or NT sgRNA on Day 4.

[0054] FIG.18C shows GFP expression of cells bearing the activation reporter system with N6 library PAM sequences-TRE3GS promoter targeted by either TRE3G sgRNA or NT sgRNA on Day11.

[0055] FIG.19A shows the enrichment profile of GFP ON / OFF samples.

[0056] FIG.19B shows the enrichment profile of GFP ON / UNSORTED samples.

[0057] FIG.19C shows the enrichment profile of GFP ON / OFF samples highlighting the 3 individual PAM sequences (5’-CGTTTG-3’, 5’-CGTTTA-3’ and 5’CGTCTG-3’).

[0058] FIG. 19D shows a correlation between N6 library PAM sequences screening results and the 3 individual PAM sequences (5’-CGTTTG-3’, 5’-CGTTTA-3’ and 5’CGTCTG- 3’).

[0059] FIG. 20A shows an enrichment profile of N6 library PAM sequences,highlighting the enriched sequence within the 95th percentile.

[0060] FIG. 20B shows the sequence logo analysis of the enriched PAM sequences within the 95th percentile of N6 library PAM sequences.

[0061] FIG.21A shows an enrichment profile of N4 library PAM sequences.

[0062] FIG. 21B shows the enrichment profile of N4 library PAM sequences, highlighting the enriched sequence within the 99th percentile.

[0063] FIG. 22 shows the PAM wheel generated using the enriched N4 library PAM sequences.

[0064] FIG.23 shows pET Duet-1 expressing the engineered nuclease and sgRNA.

[0065] FIG. 24 shows pACYC expressing target genes (e.g., chloramphenicol resistance gene (CmR) or TRE3G).

[0066] FIG. 25 schematically illustrates an engineered nuclease mediated plasmid interference in E. coli.

[0067] FIG. 26A schematically illustrates a bacterial all-in-one system comprising an engineered nuclease (e.g., CasMINI or cA2) and guide RNA targeting CmR, and a reporter system comprising target genes (TRE3G and CmR).

[0068] FIG.26B shows cA2 or CasMINI mediated plasmid interference assay induced with 0.25, 0.5, or 1 mM IPTG.

[0069] FIG.26C shows cA2 or CasMINI mediated plasmid interference assay induced with 0.025, 0.05. and 0.1 mM IPTG.

[0070] FIG.26D shows cA2 or CasMINI mediated plasmid interference assay induced with 0.025, 0.05.0.1, and 0.25 mM IPTG.

[0071] FIG.26E shows cA2 or CasMINI mediated plasmid interference assay induced with 0.06, 0.07.0.08, and 0.09 mM IPTG.

[0072] FIG. 27A schematically illustrates a bacterial all-in-one system comprising an engineered dead-nuclease (e.g., dead-CasMINI or dead-cA2) and guide RNA targeting CmR, and a reporter system comprising target genes (TRE3G and CmR).

[0073] FIG. 27B shows dead-cA2 or dead-CasMINI mediated plasmid interference assay induced with 0.5mM IPTG.

[0074] FIG. 28A schematically illustrates a bacterial all-in-one system comprising an engineered nuclease (e.g., CasMINI or cA2) and guide RNA targeting TRE3G, and a reportersystem comprising target genes (TRE3G and CmR).

[0075] FIG.28B shows cA2 or CasMINI mediated plasmid interference assay induced with 0.5mM IPTG.

[0076] FIG. 29A schematically illustrates a bacterial all-in-one system comprising an engineered dead-nuclease (e.g., dead-CasMINI or dead-cA2) and guide RNA targeting TRE3G, and a reporter system comprising target genes (TRE3G and CmR).

[0077] FIG. 29B shows dead-cA2 or dead-CasMINI mediated plasmid interference assay induced with 0.5mM IPTG.

[0078] FIG. 30A shows an experimental timeline of screening assay to characterize spacer fidelity for the engineered nuclease.

[0079] FIG. 30B shows reads counts per spacers for cA2-KRAB and dCas9-KRAB (control).

[0080] FIG.30C shows log2 enrichment profile of cA2-KRAB at Day 7, Day 10, and Day 15.

[0081] FIG. 30D shows log2 enrichment profile of dCas9-KRAB at Day 7, Day 10, and Day 15.

[0082] FIG. 31A shows the essential genes pooled from gene-trap mutagenesis in haploid lines, shRNA screens across 72 cancer lines, and candidate genes identified by dCas9- KRAB in K562 cells.

[0083] FIG.31B shows the activation level of all expressed genes and essential genes selected for the screen.

[0084] FIG.31C shows chromatin archetypes assessed by ChromHMM modeling.

[0085] FIG. 31D shows sgRNA spacer sequences selected from the region located between -2000 bp and +1000 bp relative to the transcription start site (TSS).

[0086] FIG. 31E shows the targeting position distribution of the sgRNA spacer sequences.

[0087] FIG.31F shows the distribution of the number of spacers per gene.

[0088] FIG. 31G shows the PAM sequence distribution of the sgRNA spacer sequences.

[0089] FIG. 32 schematically illustrates CD2 spacer single-base mismatches and / or single-base deletion position.

[0090] FIG.33A shows the activation level of dCasMINI or cA2 fusion proteins (e.g., gene activators) against spacer variants.

[0091] FIG. 33B shows the activity heat map of dCasMINI or cA2 fusion proteins (e.g., gene activators) against spacer variants.

[0092] FIG.34A shows the activation level of cA2_21 or cA2_29 fusion proteins (e.g., gene activators) against spacer variants.

[0093] FIG. 34B shows the activity heat map of cA2_21 or cA2_29 fusion proteins (e.g., gene activators) against spacer variants.

[0094] FIG. 35 shows the number of spot-forming cells (SFCs) for IFN-Ȗ cytokine secretion of the donors.

[0095] FIG. 36 shows the serum chemistry on immunocompetent mice administered with an AAV containing the engineered nuclease (e.g., dead-cA2) and guide nucleic acid molecules.

[0096] FIG. 37 shows the histopathology of multiple tissues (e.g., liver, kidney, skeletal muscles, etc.) on immunocompetent mice administered with an AAV containing the engineered nuclease (e.g., dead-cA2) and guide nucleic acid molecules at 3- and 6-months post AAV administration.

[0097] FIG. 38 shows blood chemistry and hematology analyses on non-human primate administered with AAV containing the engineered nuclease (e.g., dead-cA2) and guide nucleic acid molecule.

[0098] FIG.39 depicts a computer system that is programmed or otherwise configured to implement methods provided herein.

[0099] FIG. 40 depicts a non-limiting example characterization of guide RNA sequences as on-target, not off-target and off-target. DETAILED DESCRIPTION OVERVIEW

[0100] Various aspects of the present disclosure can provide engineered nucleases that are smaller, yet effective, in binding, cleaving, and / or editing a target polynucleotide sequence, compositions thereof, and methods of use thereof. Such engineered nucleases (e.g., engineered CRISPR / Cas nuclease) can, for example, effect manipulation of expression or activity of a target gene (e.g., a target endogenous gene) in a cell, e.g., to treat or ameliorate a condition (e.g., adisease) of a subject. In some aspects, the engineered nucleases described herein can recognize a PAM sequence (or a non-canonical PAM sequence) that is different than a canonical PAM sequence (e.g., 5’-TTTG-3’ or 5’-TTTA-3’). In some aspects, the present disclosure provides an engineered nuclease having enhanced spacer fidelity. In some embodiments, the engineered nuclease with high spacer fidelity described herein is characterized by enhanced on-and off-target profiles and precise allele-specific targeting. In some aspects, the present disclosure provides an engineered nuclease with reduced immunogenicity. In some embodiments, the engineered nuclease has reduced immunogenicity characterized by a decrease in or absence of T cell activation. In some embodiments, the engineered nuclease has reduced toxicity. Gene expression can underpin various physiological and pathological effects in cells and tissues, contributing to many diseases and conditions, and thus compositions and methods utilizing the engineered nucleases of the present disclosure can modulate expression of specific genes in a desirable way to have therapeutic benefit.

[0101] Also provided are computer-implemented methods for identifying guide RNA spacer sequences. The guide RNA spacer identified by the present methods find use in engineered nucleases, compositions, systems, and methods described herein. TERMS

[0102] While various embodiments of the invention 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0103] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0104] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equalto 3, less than or equal to 2, or less than or equal to 1.

[0105] The term “about” or “approximately” generally 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, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0106] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and / or” should be understood to mean either one, or both of the alternatives.

[0107] The term “cell” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soybean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non- human primate, a human, etc.), and etcetera. Sometimes a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).

[0108] The term “nucleotide,” as used herein has its ordinary and customary meaningas understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include 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 can include, for example, >ĮS@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 can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2ƍ7ƍ- dimethoxy-4ƍ5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,Nƍ,Nƍ-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4- (4ƍdimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2ƍ-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can 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, Ill.; Fluorescein- 15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2ƍ-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. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can 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).

[0109] The term “polynucleotide,” “oligonucleotide,” or “nucleic acid,” as used interchangeably herein each has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure, and can perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (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. Non-limiting examples of polynucleotides include 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, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0110] The term “gene” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5ƍ and 3ƍ ends. In some uses, the term encompasses the transcribed sequences, including 5ƍ and 3ƍ untranslated regions (5ƍ-UTR and 3ƍ-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and / or deletions (e.g., non-native sequence).

[0111] The term “deletion” generally refers to the removal (or loss) of one or more (or a specified number of) amino acids (e.g., contiguous or non-contiguous amino acids) from a polypeptide sequence, or the removal (or loss) one or more (or a specified number of) nucleic acid bases (e.g., contiguous or non-contiguous nucleic acid bases) from a polynucleotide sequence (e.g., that encodes the polypeptide sequence. The term “internal deletion” generally refers to a deletion that does not include the N- or C-terminus of a polypeptide or the 5ƍ or 3ƍ end of a polynucleotide. A deletion (e.g., an internal deletion) can be identified by comparing to a reference sequence, e.g., by specifying the start and end positions of the deletion relative to the reference sequence. A deletion (e.g., an internal deletion) is different and distinct from a substitution. For example, deletion of at least one amino acid is not followed by an insertion of at least one different amino acid at the same position as the at least one amino acid as compared to a reference polypeptidesequence, such that the size (e.g., a number of the amino acid residue(s)) of a modified (or engineered) polypeptide sequence comprising the deletion of the at least one amino acid is smaller than the reference polypeptide sequence by the size of the at least one amino acid that has been deleted.

[0112] The term “sequence identity” generally refers to an exact nucleotide-to- nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol., 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Ranges of desired degrees of sequence identity are approximately 50% to 100% and integer values therebetween. In general, this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein.

[0113] The term “expression” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequentlytranslated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated,” with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression in a wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0114] The term “expression profile” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to quantitative (e.g., abundance) and qualitative expression of one or more genes in a sample (e.g., a cell). The one or more genes can be expressed and ascertained in the form of a nucleic acid molecule (e.g., an mRNA or other RNA transcript). Alternatively or in addition to, the one or more genes can be expressed and ascertained in the form of a polypeptide (e.g., a protein measured via Western blot). An expression profile of a gene may be defined as a shape of an expression level of the gene over a time period (e.g., at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 11 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 36 hours, at least or up to about 48 hours, at least up to about 3 days, at least up to about 4 days, at least up to about 5 days, at least up to about 6 days, at least up to about 7 days, at least up to about 8 days, at least up to about 9 days, at least up to about 10 days, at least up to about 11 days, at least up to about 12 days, at least up to about 13 days, at least up to about 14 days, etc.). Alternatively, an expression profile of a gene may be defined as an expression level of the gene at a time point of interest (e.g., the expression level ofthe gene measured at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 11 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 36 hours, at least or up to about 48 hours, at least up to about 3 days, at least up to about 4 days, at least up to about 5 days, at least up to about 6 days, at least up to about 7 days, at least up to about 8 days, at least up to about 9 days, at least up to about 10 days, at least up to about 11 days, at least up to about 12 days, at least up to about 13 days, or at least up to about 14 days after treating a cell to induce such expression level.)

[0115] The term “peptide,” “polypeptide,” or “protein,” as used interchangeably herein, each has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and / 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, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.

[0116] The term “derivative,” “variant,” or “fragment,” as used herein with reference to a polypeptide, each has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a polypeptide related to awild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide.

[0117] The term “engineered,” “chimeric,” or “recombinant,” as used herein with respect to a polypeptide molecule (e.g., a protein), has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the polypeptide molecule, as well as cells or organisms which express the polypeptide molecule. The term “engineered” or “recombinant,” as used herein with respect to a polynucleotide molecule (e.g., a DNA or RNA molecule), generally refers to a polynucleotide molecule having a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.

[0118] For example, an engineered nuclease (e.g., an engineered Cas protein) as disclosed herein is not a naturally occurring nuclease (e.g., not a naturally occurring Cas protein). The terms “engineered nuclease” and “engineered nuclease variant” may be used interchangeable herein.

[0119] The terms “engineered” and “modified” are used interchangeably herein. The terms “engineering” and “modifying” are used interchangeably herein. The terms “engineered cell” or “modified cell” are used interchangeably herein. The terms “engineered characteristic” and “modified characteristic” are used interchangeably herein.

[0120] The term “enhanced expression,” “increased expression,” or “upregulated expression” each has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of themoiety of interest in a host strain (e.g., a host cell). The normal level of expression can be substantially zero (or null) or higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. The moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.

[0121] The term “enhanced activity,” “increased activity,” or “upregulated activity” each has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity can be substantially zero (or null) or higher than zero. The moiety of interest can comprise a polypeptide construct of the host strain. The moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.

[0122] The term “reduced expression,” “decreased expression,” or “downregulated expression” each has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to a production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is below a normal level of expression of the moiety of interest in a host strain (e.g., a host cell). The normal level of expression is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced expression of the moiety of interest can include a complete inhibition of such expression in the host strain.

[0123] The term “reduced activity,” “decreased activity,” or “downregulated activity” each has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is below a normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct ofthe host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced activity of the moiety of interest can include a complete inhibition of such activity in the host strain.

[0124] The term “subject,” “individual,” or “patient,” as used interchangeably herein, generally refers to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0125] The term “treatment” or “treating” generally refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0126] The term “effective amount” or “therapeutically effective amount” generally refers to the quantity of a composition, for example a composition comprising heterologous polypeptides, heterologous polynucleotides, and / or modified cells (e.g., modified stem cells), that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” generally refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure. ENGINEERED NUCLEASES, COMPOSITIONS, AND METHODS THEREOF

[0127] In some aspects, the present disclosure provides an engineered nuclease comprising an amino acid sequence that is at least 50% identical to the polypeptide sequence of SEQ ID NO: 1.

[0128] In some aspects, the present disclosure provides an engineered nuclease having enhanced fidelity comprising an amino acid sequence that is at least 50% identical to the polypeptide sequence of SEQ ID NO: 1.

[0129] In some embodiments, the engineered nuclease is characterized by enhancedon- and off-target profiles and precise allele-specific targeting. In some embodiments, the engineered nuclease can distinguish single-base pair mismatches.

[0130] In some aspects, the present disclosure provides an engineered nuclease having reduced immunogenicity, wherein the engineered nuclease comprises an amino acid sequence that is at least 50% identical to the polypeptide sequence of SEQ ID NO: 1.

[0131] In some embodiments, the amino acid sequence of the engineered nuclease comprises at least one deletion, as compared to (e.g., when aligned to) the polypeptide sequence of SEQ ID NO: 1 (or SEQ ID NO: 10). The at least one deletion can be selectively removed in accordance with the present disclosure. As disclosed herein, SEQ ID NO: 1 encodes the polypeptide sequence of Un1Cas12f1 (or Cas14a1). As disclosed herein, SEQ ID NO: 10 encodes an engineered variant of Un1Cas12f1 with reduced nuclease activity. Thus, the amino acid sequence of the engineered nuclease as disclosed herein can be a mutant sequence (or a mutant variant) of Un1Cas12f1 (or a deactivated variant thereof).

[0132] Un1Cas12f1 (or Cas14a1 SEQ ID NO: 1) or an engineered variant of Un1Cas12f1 with reduced nuclease activity (SEQ ID NO: 10) is characterized to recognize T-rich PAM sequences (e.g., 5’-TTTR-3’, wherein R is A or G) positioned 5’ to the spacer sequence. Such strict PAM requirement limits the targeting range and utility of the enzyme for genome editing. In some aspects, the present disclosure provides an engineered nuclease that recognizes PAM sequence that is different than the canonical PAM sequence (e.g., TTTR), thereby increasing the targeting range of the engineered nuclease described herein. The engineered nuclease described herein (e.g., having at least one deletion, as compared to (e.g., when aligned to) the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10) exhibits broadened targeting range, while also enabling more potent multiplex editing, gene modulation (e.g., activation and suppression), DNA detection, and base editing applications. In some embodiments, the engineered nuclease can be configured to bind to its target sequence (e.g., by forming a complex with a non-naturally occurring guide RNA comprising a region that binds to the engineered nuclease and a spacer sequence that binds to a target sequence of a target nucleic acid) by recognizing PAM sequence that is different than a canonical PAM sequence. In some embodiments, the canonical PAM sequence is 5’-TTTR- 3’, wherein R is A or G. In some embodiments, the canonical PAM is 5’-TTTG-3’. In some embodiments the canonical PAM sequence is 5’-TTTA-3’. In some embodiments, the engineered nuclease described herein can be configured to recognize a broader range of PAM sequences (e.g.,non-canonical PAM sequence or relaxed PAM sequence). In some embodiments, the engineered nuclease described herein can recognize a broader range of PAM sequences comprising one or more nucleic acid sequence selected from the group consisting of 5’-CCCC-3', 5’-TTAT-3', 5’- GTCC-3', 5’-TTTC-3', 5’-TCAT-3', 5’-ACCC-3', 5’-TCTG-3', 5’-TTAG-3', 5’-GTTA-3', 5’- ACTC-3', 5’-GCCA-3', 5’-TTTT-3', 5’-TTGG-3', 5’-GTTG-3', 5’-ATTG-3', 5’-TTAA-3', 5’- CTTA-3', 5’-TTGA-3', 5’-GCTC-3', 5’-GCCC-3', 5’-TTCG-3', 5’-CTTG-3', 5’-TTCA-3', and 5’- TCTA-3'. In some embodiments, the engineered nuclease described herein can recognize a broader range of PAM sequences comprising one or more nucleic acid sequence selected from the group consisting of 5’-NCCCC-3', 5’-NTTAT-3', 5’-NGTCC-3', 5’-NTTTC-3', 5’-NTCAT-3', 5’- NACCC-3', 5’-NTCTG-3', 5’-NTTAG-3', 5’-NGTTA-3', 5’-NACTC-3', 5’-NGCCA-3', 5’- NTTTT-3', 5’-NTTGG-3', 5’-NGTTG-3', 5’-NATTG-3', 5’-NTTAA-3', 5’-NCTTA-3', 5’- NTTGA-3', 5’-NGCTC-3', 5’-NGCCC-3', 5’-NTTCG-3', 5’-NCTTG-3', 5’-NTTCA-3', and 5’- NTCTA-3', wherein “N” can be any nucleotide (A, T, C, or G). In some embodiments, the engineered nuclease described herein can recognize a broader range of PAM sequences comprising one or more nucleic acid sequence selected from the group consisting of 5’-NNCCCC-3', 5’- NNTTAT-3', 5’-NNGTCC-3', 5’-NNTTTC-3', 5’-NNTCAT-3', 5’-NNACCC-3', 5’-NNTCTG-3', 5’-NNTTAG-3', 5’-NNGTTA-3', 5’-NNACTC-3', 5’-NNGCCA-3', 5’-NNTTTT-3', 5’- NNTTGG-3', 5’-NNGTTG-3', 5’-NNATTG-3', 5’-NNTTAA-3', 5’-NNCTTA-3', 5’-NNTTGA-3', 5’-NNGCTC-3', 5’-NNGCCC-3', 5’-NNTTCG-3', 5’-NNCTTG-3', 5’-NNTTCA-3', and 5’- NNTCTA-3', wherein “N” can be any nucleotide (A, T, C, or G). In some embodiments, the engineered nuclease can recognize one or more PAM sequences selected from TABLE 11.

[0133] Without wishing to be bound by theory, the at least one deletion of the amino acid sequence of the engineered nuclease, as disclosed herein, can be found in one or more regions of the native Un1Cas12f1 nuclease that do not structurally align to a control CRISPR / Cas protein. The control CRISPR / Cas protein can be from Class 1 CRISPR system or Class 2 CRISPR system (e.g., as a wild type CRISPR / Cas protein). Class 1 CRISPR system can be divided into types I, III, and IV, and Class 2 CRISPR system can be divided into types II, V, and VI. In some cases, the control CRISPR / Cas protein can be a type V Cas protein, e.g., a type V-A Cas protein, a type V-B Cas protein, a type V-C Cas protein, a type V-D Cas protein, a type V-E Cas protein, a type V-F Cas protein, a type V-G Cas protein, a type V-H Cas protein, a type V-I Cas protein, a type V-J Cas protein, a type V-K Cas protein, or a type V-U Cas protein. In some cases, the controlCRISPR / Cas protein can be a type V-J protein, such as a wild-type Cas^ (Cas 12J) protein. In some cases, the control CRISPR / Cas protein can be Un2Cas12f1 (SEQ ID NO: 2) or AsCas12f (SEQ ID NO: 3).

[0134] Without wishing to be bound by theory, the at least one deletion of the amino acid sequence of the engineered nuclease, as disclosed herein, can be determined by performing a deletion landscape study (e.g., iterative and / or comprehensive deletion) of the Cas nuclease encoded by the polypeptide sequence of SEQ ID NO: 1 (or SEQ ID NO: 10).

[0135] Without wishing to be bound by theory, a plurality of different variants of the engineered nuclease, as disclosed herein, can exhibit different activities (e.g., different binding affinities to a control sgRNA, different binding affinities to a control target gene, different target gene cleaving level, different target gene activation level, different target gene repression level, etc.). In some embodiments, a first variant of the plurality of different variants can comprise the at least one deletion at the amino acid resides 1-100 (e.g., when aligned to the polypeptide sequence of SEQ ID NO: 1), and a second variant of the plurality of different variants can comprise the at least one deletion at the amino acid resides 101-529 (e.g., when aligned to the polypeptide sequence of SEQ ID NO: 1), and the first variant and the second variant can exhibit different activities (e.g., the first variant can effect enhanced target gene activation and / or expression as compared to that of the second variant, or vice versa). Alternatively, the first variant and the second variant can exhibit comparable activities. In some embodiments, a first variant of the plurality of different variants can comprise a single deletion at the amino acid resides 1-100 (e.g., when aligned to the polypeptide sequence of SEQ ID NO: 1), and a second variant of the plurality of different variants can comprise a plurality of deletions at the amino acid resides 1-100 (e.g., when aligned to the polypeptide sequence of SEQ ID NO: 1), and the first variant and the second variant can exhibit different activities (e.g., the second variant can effect enhanced target gene activation and / or expression as compared to that of the first variant, or vice versa). Alternatively, the first variant and the second variant can exhibit comparable activities. SEQ ID NO: 1 (Un1Cas12f1) 1 MAKNTITKTL KLRIVRPYNS AEVEKIVADE KNNREKIALE KNKDKVKEAC 51 SKHLKVAAYC TTQVERNACL FCKARKLDDK FYQKLRGQFP DAVFWQEISE 101 IFRQLQKQAA EIYNQSLIEL YYEIFIKGKG IANASSVEHY LSDVCYTRAA 151 ELFKNAAIAS GLRSKIKSNF RLKELKNMKS GLPTTKSDNF PIPLVKQKGG 201 QYTGFEISNH NSDFIIKIPF GRWQVKKEID KYRPWEKFDF EQVQKSPKPI 251 SLLLSTQRRK RNKGWSKDEG TEAEIKKVMN GDYQTSYIEV KRGSKIGEKS 301 AWMLNLSIDV PKIDKGVDPS IIGGIDVGVK SPLVCAINNA FSRYSISDND351 LFHFNKKMFA RRRILLKKNR HKRAGHGAKN KLKPITILTE KSERFRKKLI 401 ERWACEIADF FIKNKVGTVQ MENLESMKRK EDSYFNIRLR GFWPYAEMQN 451 KIEFKLKQYG IEIRKVAPNN TSKTCSKCGH LNNYFNFEYR KKNKFPHFKC 501 EKCNFKENAD YNAALNISNP KLKSTKEEP SEQ ID NO: 10 (deactivated nuclease variant of Un1Cas12f1, i.e., dCasMINI) 1 MAKNTITKTL KLRIVRPYNS AEVEKIVADE KNNREKIALE KNKDKVKEAC 51 SKHLKVAAYC TTQVERNACL FCKARKLDDK FYQKLRGQFP DAVFWQEISE 101 IFRQLQKQAA EIYNQSLIEL YYEIFIKGKG IANASSVEHY LSRVCYRRAA 151 ELFKNAAIAS GLRSKIKSNF RLKELKNMKS GLPTTKSDNF PIPLVKQKGG 201 QYTGFEISNH NSDFIIKIPF GRWQVKKEID KYRPWEKFDF EQVQKSPKPI 251 SLLLSTQRRK RNKGWSKDEG TEAEIKKVMN GDYQTSYIEV KRGSKICEKS 301 AWMLNLSIDV PKIDKGVDPS IIGGIAVGVR SPLVCAINNA FSRYSISDND 351 LFHFNKKMFA RRRILLKKNR HKRAGHGAKN KLKPITILTE KSERFRKKLI 401 ERWACEIADF FIKNKVGTVQ MENLESMKRK EDSYFNIRLR GFWPYAEMQN 451 KIEFKLKQYG IEIRKVAPNN TSKTCSKCGH LNNYFNFEYR KKNKFPHFKC 501 EKCNFKENAA YNAALNISNP KLKSTKERP SEQ ID NO: 2 (Un2Cas12f1) 1 MEVQKTVMKT LSLRILRPLY SQEIEKEIKE EKERRKQAGG TGELDGGFYK 51 KLEKKHSEMF SFDRLNLLLN QLQREIAKVY NHAISELYIA TIAQGNKSNK 101 HYISSIVYNR AYGYFYNAYI ALGICSKVEA NFRSNELLTQ QSALPTAKSD 151 NFPIVLHKQK GAEGEDGGFR ISTEGSDLIF EIPIPFYEYN GENRKEPYKW 201 VKKGGQKPVL KLILSTFRRQ RNKGWAKDEG TDAEIRKVTE GKYQVSQIEI 251 NRGKKLGEHQ KWFANFSIEQ PIYERKPNRS IVGGLDVGIR SPLVCAINNS 301 FSRYSVDSND VFKFSKQVFA FRRRLLSKNS LKRKGHGAAH KLEPITEMTE 351 KNDKFRKKII ERWAKEVTNF FVKNQVGIVQ IEDLSTMKDR EDHFFNQYLR 401 GFWPYYQMQT LIENKLKEYG IEVKRVQAKY TSQLCSNPNC RYWNNYFNFE 451 YRKVNKFPKF KCEKCNLEIS ADYNAARNLS TPDIEKFVAK ATKGINLPEK SEQ ID NO: 3 (AsCas12f) 1 MIKVYRYEIV KPLDLDWKEF GTILRQLQQE TRFALNKATQ LAWEWMGFSS 51 DYKDNHGEYP KSKDILGYTN VHGYAYHTIK TKAYRLNSGN LSQTIKRATD 101 RFKAYQKEIL RGDMSIPSYK RDIPLDLIKE NISVNRMNHG DYIASLSLLS 151 NPAKQEMNVK RKISVIIIVR GAGKTIMDRI LSGEYQVSAS QIIHDDRKNK 201 WYLNISYDFE PQTRVLDLNK IMGIDLGVAV AVYMAFQHTP ARYKLEGGEI 251 ENFRRQVESR RISMLRQGKY AGGARGGHGR DKRIKPIEQL RDKIANFRDT 301 TNHRYSRYIV DMAIKEGCGT IQMEDLTNIR DIGSRFLQNW TYYDLQQKII 351 YKAEEAGIKV IKIDPQYTSQ RCSECGNIDS GNRIGQAIFK CRACGYEANA 401 DYNAARNIAI PNIDKIIAES IK

[0136] Throughout the present disclosure, (i) a sequence comparison between the amino acid sequence of the engineered nuclease disclosed herein and the polypeptide sequence of SEQ ID NO: 1 may be comparable (e.g., substantially identical) to (ii) a sequence comparison between the amino acid sequence of the engineered nuclease disclosed herein and the polypeptide sequence of SEQ ID NO: 10.

[0137] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein can be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99% identical to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2. For example, the amino acid sequence of the engineered nuclease can be between about 80% and about 100% identical to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2.

[0138] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein is not identical to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2.

[0139] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can comprise an N-terminus deletion, a C-terminus deletion, and / or an internal deletion. For example, the at least one deletion may not comprise an N-terminus deletion, but rather an internal deletion and / or a C-terminus deletion.

[0140] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can be from the amino acid residues 1-100 (e.g., the amino acid residues 2-100), the amino acid resides 101-429, the amino acid residues 101-200, the amino acid residues 201-300, the amino acid residues 301-400, the amino acid residues 401-500, or the amino acid residues 500-529, and / or amino acid residues 430-529, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0141] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can be from at least one deletion from the amino acidresides 2-100, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0142] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from the amino acid residues 2-98, the amino acid residues 2-96, the amino acid residues 2-95, the amino acid residues 2-94, the amino acid residues 2-92, the amino acid residues 2-90, the amino acid residues 2-88, the amino acid residues 2-86, the amino acid residues 2-85, the amino acid residues 2-84, the amino acid residues 2-82, the amino acid residues 2-80, the amino acid residues 2-78, the amino acid residues 2-76, the amino acid residues 2-75, the amino acid residues 2-74, the amino acid residues 2-72, the amino acid residues 2-70, the amino acid residues 2-68, the amino acid residues 2-66, the amino acid residues 2-65, the amino acid residues 2-64, the amino acid residues 2-62, the amino acid residues 2-60, the amino acid residues 2-58, the amino acid residues 2-56, the amino acid residues 2-55, the amino acid residues 2-54, the amino acid residues 2-52, the amino acid residues 2-50, the amino acid residues 2-48, the amino acid residues 2-46, the amino acid residues 2-45, the amino acid residues 2-44, the amino acid residues 2-42, the amino acid residues 2-40, the amino acid residues 2-38, the amino acid residues 2-36, the amino acid residues 2-35, the amino acid residues 2-34, the amino acid residues 2-32, the amino acid residues 2-30, the amino acid residues 2-28, the amino acid residues 2-26, the amino acid residues 2-25, the amino acid residues 2-24, the amino acid residues 2-22, the amino acid residues 2-20, the amino acid residues 2-18, the amino acid residues 2-16, the amino acid residues 2-15, the amino acid residues 2-14, the amino acid residues 2-12, the amino acid residues 2-10, the amino acid residues 2-8, the amino acid residues 2-6, the amino acid residues 2-5, or the amino acid residues 2-4, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0143] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from the amino acid residues 2-100, the amino acid residues 4-100, the amino acid residues 5-100, the amino acid residues 6-100, the amino acid residues 8-100, the amino acid residues 10-100, the amino acid residues 12-100, the amino acid residues 14-100, the amino acid residues 15-100, the amino acid residues 16-100, the amino acid residues 18-100, the amino acid residues 20-100, the amino acid residues 22-100, the amino acid residues 24-100, the amino acid residues 25-100, the amino acid residues 26-100, the amino acid residues 28-100, theamino acid residues 30-100, the amino acid residues 32-100, the amino acid residues 34-100, the amino acid residues 35-100, the amino acid residues 36-100, the amino acid residues 38-100, the amino acid residues 40-100, the amino acid residues 42-100, the amino acid residues 44-100, the amino acid residues 45-100, the amino acid residues 46-100, the amino acid residues 48-100, the amino acid residues 50-100, the amino acid residues 52-100, the amino acid residues 54-100, the amino acid residues 55-100, the amino acid residues 56-100, the amino acid residues 58-100, the amino acid residues 60-100, the amino acid residues 62-100, the amino acid residues 64-100, the amino acid residues 65-100, the amino acid residues 66-100, the amino acid residues 68-100, the amino acid residues 70-100, the amino acid residues 72-100, the amino acid residues 75-100, the amino acid residues 76-100, the amino acid residues 78-100, the amino acid residues 80-100, the amino acid residues 82-100, the amino acid residues 84-100, the amino acid residues 85-100, the amino acid residues 86-100, the amino acid residues 88-100, the amino acid residues 90-100, the amino acid residues 92-100, the amino acid residues 94-100, the amino acid residues 95-100, the amino acid residues 96-100, or the amino acid residues 98-100, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0144] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from the amino acid residues 30-100, the amino acid residues 30- 98, the amino acid residues 30-96, the amino acid residues 30-95, the amino acid residues 30-94, the amino acid residues 30-92, the amino acid residues 30-90, the amino acid residues 30-88, the amino acid residues 30-86, the amino acid residues 30-85, the amino acid residues 30-84, the amino acid residuesamino acid residues 30-80, the amino acid residues 30-78, the amino acid residues 30-76, the amino acid residues 30-75, the amino acid residues 30-74, the amino acid residuesamino acid residues 30-70, the amino acid residues 30-68, the amino acid residues 30-66, the amino acid residues 30-65, the amino acid residues 30-64, the amino acid residuesamino acid residues 30-60, the amino acid residues 30-58, the amino acid residues 30-56, the amino acid residues 30-55, the amino acid residues 30-54, the amino acid residues 30-52, the amino acid residues 30-50, the amino acid residues 30-48, the amino acid residues 30-46, the amino acid residues 30-45, the amino acid residues 30-44, the amino acid residues 30-42, the amino acid residues 30-40, the amino acid residues 30-38, the amino acid residues 30-36, the amino acid residues 30-34, or the amino acid residues 30-32, ascompared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0145] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can comprise one or more deletions from (e.g., deletions of substantially all of the amino acid residues from) the amino acid residues 55-56, the amino acid residues 54-57, the amino acid residues 54-58, the amino acid residues 53-59, the amino acid residues 52-60, the amino acid residues 51-61, the amino acid residues 50-62, the amino acid residues 49-63, the amino acid residues 48-64, the amino acid residues 47-65, the amino acid residues 46-66, the amino acid residues 45-67, the amino acid residues 44-68, the amino acid residues 43-69, the amino acid residues 42-70, the amino acid residues 41-71, the amino acid residues 40-72, the amino acid residues 39-73, the amino acid residues 38-74, the amino acid residues 37-73, the amino acid residues 36-74, or the amino acid residues 35-75, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0146] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more) selected from the group consisting of the amino acid resides 2-10, the amino acid resides 11-20, the amino acid resides 21-30, the amino acid resides 31-40, the amino acid resides 41-50, the amino acid resides 51-60, the amino acid resides 61-70, and the amino acid resides 71-80, as compared to the polypeptide sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more) selected from the group consisting of the amino acid resides 2-5, the amino acid resides 6-10, the amino acid resides 11-15, the amino acid resides 16-20, the amino acid resides 21-25, the amino acid resides 26-30, the amino acid resides 31-35, the amino acid resides 36-40, the amino acid resides 41-45, the amino acid resides 46-50, the amino acid resides 51-55, the amino acid resides 56-60, the amino acid resides 61-65, the amino acid resides 66-70, the amino acid resides 71-75, and the amino acid resides 76-80, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0147] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can be from one or more members (e.g., two or more members, three or more members, four or more members, five or more members, etc.) selected from the group consisting of the amino acid resides 10-90, the amino acid residues 15-85, the amino acid residues 20-80, the amino acid residues 25-75, the amino acid residues 30-70, the amino acid residues 35-75, the amino acid residues 40-70, the amino acid residues 45-65, or the amino acid residues 50-60, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0148] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can be from one or more members (e.g., two or more members, three or more members, four or more members, five or more members, etc.) selected from the group consisting of the amino acid residues 20-30, the amino acid resides 25-35, the amino acid residues 30-40, the amino acid residues 35-45, the amino acid residues 40-50, the amino acid residues 45-55, the amino acid residues 50-60, the amino acid residues 55-65, the amino acid residues 60-70, the amino acid residues 65-75, the amino acid residues 70-80, the amino acid residues 75-85, and the amino acid residues 80-90, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0149] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can be from one or more members (e.g., two or more members, three or more members, four or more members, five or more members, etc.) selected from the group consisting of the amino acid residues 20-25, the amino acid residues 25-30, the amino acid residues 30-35, the amino acid residues 35-40, the amino acid residues 40-45, the amino acid residues 45-50, the amino acid residues 50-55, the amino acid residues 55-60, the amino acid residues 60-65, the amino acid residues 65-70, the amino acid residues 70-75, the amino acid residues 75-80, and the amino acid residues 85-90, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0150] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can be from the amino acid resides 430-529, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one ofthe Cas proteins selected from TABLE 2 (when applicable).

[0151] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from the amino acid residues 430-528, the amino acid residues 430-526, the amino acid residues 430-525, the amino acid residues 430-524, the amino acid residues 430-522, the amino acid residues 430-520, the amino acid residues 430-518, the amino acid residues 430-516, the amino acid residues 430-515, the amino acid residues 430-514, the amino acid residues 430-512, the amino acid residues 430-510, the amino acid residues 430-508, the amino acid residues 430-506, the amino acid residues 430-505, the amino acid residues 430- 504, the amino acid residues 430-502, the amino acid residues 430-500, the amino acid residues 430-498, the amino acid residues 430-496, the amino acid residues 430-495, the amino acid residues 430-494, the amino acid residues 430-492, the amino acid residues 430-490, the amino acid residues 430-488, the amino acid residues 430-486, the amino acid residues 430-485, the amino acid residues 430-484, the amino acid residues 430-482, the amino acid residues 430-480, the amino acid residues 430-478, the amino acid residues 430-476, the amino acid residues 430- 475, the amino acid residues 430-474, the amino acid residues 430-472, the amino acid residues 430-470, the amino acid residues 430-468, the amino acid residues 430-466, the amino acid residues 430-465, the amino acid residues 430-464, the amino acid residues 430-462, the amino acid residues 430-460, the amino acid residues 430-458, the amino acid residues 430-456, the amino acid residues 430-455, the amino acid residues 430-454, the amino acid residues 430-452, the amino acid residues 430-450, the amino acid residues 430-448, the amino acid residues 430- 446, the amino acid residues 430-445, the amino acid residues 430-444, the amino acid residues 430-442, the amino acid residues 430-440, the amino acid residues 430-438, the amino acid residues 430-436, the amino acid residues 430-435, the amino acid residues 430-434, or the amino acid residues 430-432, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0152] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from the amino acid residues 430-529, the amino acid residues 432-529, the amino acid residues 434-529, the amino acid residues 435-529, the amino acid residues 436-529, the amino acid residues 438-529, the amino acid residues 440-529, the amino acid residues 442-529, the amino acid residues 444-529, the amino acid residues 445-529, the amino acid residues 446-529, the amino acid residues 448-529, the amino acid residues 450-529,the amino acid residues 452-529, the amino acid residues 454-529, the amino acid residues 455- 529, the amino acid residues 456-529, the amino acid residues 458-529, the amino acid residues 460-529, the amino acid residues 462-529, the amino acid residues 464-529, the amino acid residues 465-529, the amino acid residues 466-529, the amino acid residues 468-529, the amino acid residues 470-529, the amino acid residues 472-529, the amino acid residues 474-529, the amino acid residues 475-529, the amino acid residues 476-529, the amino acid residues 478-529, the amino acid residues 480-529, the amino acid residues 482-529, the amino acid residues 484- 529, the amino acid residues 485-529, the amino acid residues 486-529, the amino acid residues 488-529, the amino acid residues 490-529, the amino acid residues 492-529, the amino acid residues 494-529, the amino acid residues 495-529, the amino acid residues 496-529, the amino acid residues 498-529, the amino acid residues 500-529, the amino acid residues 502-529, the amino acid residues 504-529, the amino acid residues 505-529, the amino acid residues 506-529, the amino acid residues 508-529, the amino acid residues 510-529, the amino acid residues 512- 529, the amino acid residues 514-529, the amino acid residues 515-529, the amino acid residues 516-529, the amino acid residues 518-529, the amino acid residues 520-529, the amino acid residues 522-529, the amino acid residues 524-529, the amino acid residues 525-529, the amino acid residues 526-529, or the amino acid residues 528-529, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0153] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more) selected from the group consisting of the amino acid resides 450-459, the amino acid resides 460-469, the amino acid resides 470-479, the amino acid resides 480-489, the amino acid resides 490-499, the amino acid resides 500-509, the amino acid resides 510-519, and the amino acid resides 520-529, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more) selected from the group consisting of the amino acid resides 450-459, the amino acid resides 460-465, the amino acid resides 466-469, the amino acid resides 470-475,the amino acid resides 476-479, the amino acid resides 480-485, the amino acid resides 486-489, the amino acid resides 490-495, the amino acid resides 496-499, the amino acid resides 500-505, the amino acid resides 506-509, the amino acid resides 510-515, the amino acid resides 516-519, the amino acid resides 520-525, and the amino acid resides 526-529, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0154] In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can comprise deletion of a single amino acid residue, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). In some embodiments, the at least one deletion of the amino acid sequence of the engineered nuclease can comprise deletion of a plurality of amino acid residues, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). The plurality of amino acid residues that are deleted can be adjacent to each other (e.g., consecutive) when aligned to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). Alternatively or in addition to, the plurality of amino acid residues that are deleted may not be adjacent to each other, when aligned to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). The plurality of amino acid residues can comprise at least or up to about 2 amino acid residues, at least or up to about 3 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 6 amino acid residues, at least or up to about 7 amino acid residues, at least or up to about 8 amino acid residues, at least or up to about 9 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 11 amino acid residues, at least or up to about 12 amino acid residues, at least or up to about 13 amino acid residues, at least or up to about 14 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 16 amino acid residues, at least or up to about 17 amino acid residues, at least or up to about 18 amino acid residues, at least or up to about 19 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 22 amino acid residues, at least or up to about 24 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 26 amino acid residues, at least or up to about 28 amino acidresidues, at least or up to about 30 amino acid residues, at least or up to about 32 amino acid residues, at least or up to about 34 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 36 amino acid residues, at least or up to about 38 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 42 amino acid residues, at least or up to about 44 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 46 amino acid residues, at least or up to about 48 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 52 amino acid residues, at least or up to about 54 amino acid residues, at least or up to about 55 amino acid residues, at least or up to about 56 amino acid residues, at least or up to about 58 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 62 amino acid residues, at least or up to about 64 amino acid residues, at least or up to about 65 amino acid residues, at least or up to about 66 amino acid residues, at least or up to about 68 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 72 amino acid residues, at least or up to about 74 amino acid residues, at least or up to about 75 amino acid residues, at least or up to about 76 amino acid residues, at least or up to about 78 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 82 amino acid residues, at least or up to about 84 amino acid residues, at least or up to about 85 amino acid residues, at least or up to about 86 amino acid residues, at least or up to about 88 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 92 amino acid residues, at least or up to about 94 amino acid residues, at least or up to about 95 amino acid residues, at least or up to about 96 amino acid residues, at least or up to about 98 amino acid residues, or at least or up to about 100 amino acid residues.

[0155] In some embodiments, the deletion of the plurality of amino acid residues to generate the amino acid sequence of the engineered nuclease can comprise deletion of a plurality of non-consecutive amino acid residues, e.g., as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). In some cases, the deletion of the plurality of non-consecutive amino acid residues in the amino acid sequence of the engineered nuclease can comprise deletion of a first amino acid residue (e.g., a first single amino acid residue or a first set of multiple amino acid residues, such as a first set of consecutive amino acid residues) and a second amino acid residue (e.g., a second single amino acid residue or a second set of multiple amino acid residues, such asa second set of consecutive amino acid residues) that are not consecutive to each other, when aligned to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). For example, the position of the first amino acid residue and the second amino acid residue (e.g., when aligned to the polypeptide sequence of SEQ ID NO: 1) can be separated by at least or up to about 1, at least or up to about 2, at least or up to about 3, at least or up to about 4, at least or up to about 5, at least or up to about 10, at least or up to about 15, at least or up to about 20, at least or up to about 30, at least or up to about 40, at least or up to about 50, at least or up to about 60, at least or up to about 70, at least or up to about 80, at least or up to about 90, at least or up to about 100, at least or up to about 120, at least or up to about 150, at least or up to about 200, at least or up to about 250, at least or up to about 300, at least or up to about 350, at least or up to about 400, at least or up to about 450, at least or up to about 500, at least or up to about 510, at least or up to about 520, or at least or up to about 525 amino acid residues.

[0156] In some embodiments, the deletion of the plurality of amino acid residues to generate the amino acid sequence of the engineered nuclease can comprise deletion of a plurality of consecutive amino acid residues, e.g., as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable). In some cases, the deletion of the plurality of consecutive amino acid residues can comprise deletion of at least or up to about 2 consecutive amino acid residues, at least or up to about 3 consecutive amino acid residues, at least or up to about 4 consecutive amino acid residues, at least or up to about 5 consecutive amino acid residues, at least or up to about 6 consecutive amino acid residues, at least or up to about 7 consecutive amino acid residues, at least or up to about 8 consecutive amino acid residues, at least or up to about 9 consecutive amino acid residues, at least or up to about 10 consecutive amino acid residues, at least or up to about 11 consecutive amino acid residues, at least or up to about 12 consecutive amino acid residues, at least or up to about 13 consecutive amino acid residues, at least or up to about 14 consecutive amino acid residues, at least or up to about 15 consecutive amino acid residues, at least or up to about 16 consecutive amino acid residues, at least or up to about 17 consecutive amino acid residues, at least or up to about 18 consecutive amino acid residues, at least or up to about 19 consecutive amino acid residues, at least or up to about 20 consecutive amino acid residues, at least or up to about 21 consecutive amino acid residues, at least or up to about 22 consecutive amino acidresidues, at least or up to about 23 consecutive amino acid residues, at least or up to about 24 consecutive amino acid residues, at least or up to about 25 consecutive amino acid residues, at least or up to about 26 consecutive amino acid residues, at least or up to about 27 consecutive amino acid residues, at least or up to about 28 consecutive amino acid residues, at least or up to about 29 consecutive amino acid residues, at least or up to about 30 consecutive amino acid residues, at least or up to about 31 consecutive amino acid residues, at least or up to about 32 consecutive amino acid residues, at least or up to about 34 consecutive amino acid residues, at least or up to about 35 consecutive amino acid residues, at least or up to about 36 consecutive amino acid residues, at least or up to about 37 consecutive amino acid residues, at least or up to about 38 consecutive amino acid residues, at least or up to about 39 consecutive amino acid residues, at least or up to about 40 consecutive amino acid residues, at least or up to about 45 consecutive amino acid residues, at least or up to about 50 consecutive amino acid residues, at least or up to about 55 consecutive amino acid residues, at least or up to about 60 consecutive amino acid residues, at least or up to about 65 consecutive amino acid residues, at least or up to about 70 consecutive amino acid residues, at least or up to about 75 consecutive amino acid residues, at least or up to about 80 consecutive amino acid residues, at least or up to about 85 consecutive amino acid residues, at least or up to about 80 consecutive amino acid residues 90, at least or up to about 95 consecutive amino acid residues, or at least or up to about 100 consecutive amino acid residues, e.g., as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0157] For example, the deletion can comprise a single deletion of a plurality of consecutive amino acid residues. In another example, the deletion can comprise a first deletion of a first plurality of consecutive amino acid resides and a second deletion of a second plurality of consecutive amino acid resides, and the first plurality of consecutive amino acid resides and the second plurality of consecutive amino acid resides may not be consecutive (e.g., may not be adjacent to each other), when aligned to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable).

[0158] In some embodiments, the amino acid sequence of the engineered nuclease as disclosed herein can comprise addition of one or more heterologous amino acid residues (e.g., one or more polypeptide sequences), as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (whenapplicable). The one or more heterologous amino acid residues may be at the position of the at least one deletion of the engineered nuclease. Alternatively or in addition to, the one or more heterologous amino acid residues may not be at the position of the at least one deletion of the engineered nuclease. For example, the one or more heterologous amino acid residues may be upstream and / or downstream of the position of the at least one deletion of the engineered nuclease.

[0159] In some embodiments, the one or more heterologous amino acid residues of the engineered nuclease may comprise a single amino acid residue. In some embodiments, the one or more heterologous amino acid residues of the engineered nuclease may comprise a plurality of amino acid residues, such as, at least or up to about 2 amino acid residues, at least or up to about 3 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 6 amino acid residues, at least or up to about 7 amino acid residues, at least or up to about 8 amino acid residues, at least or up to about 9 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 11 amino acid residues, at least or up to about 12 amino acid residues, at least or up to about 13 amino acid residues, at least or up to about 14 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 16 amino acid residues, at least or up to about 17 amino acid residues, at least or up to about 18 amino acid residues, at least or up to about 19 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 22 amino acid residues, at least or up to about 24 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 26 amino acid residues, at least or up to about 28 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 32 amino acid residues, at least or up to about 34 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 36 amino acid residues, at least or up to about 38 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 42 amino acid residues, at least or up to about 44 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 46 amino acid residues, at least or up to about 48 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 52 amino acid residues, at least or up to about 54 amino acid residues, at least or up to about 55 amino acid residues, at least or up to about 56 amino acid residues, at least or up to about 58 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 62 amino acid residues, at least or up to about 64 amino acid residues, at least or up to about 65 amino acid residues, at least or up to about 66 amino acidresidues, at least or up to about 68 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 72 amino acid residues, at least or up to about 74 amino acid residues, at least or up to about 75 amino acid residues, at least or up to about 76 amino acid residues, at least or up to about 78 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 82 amino acid residues, at least or up to about 84 amino acid residues, at least or up to about 85 amino acid residues, at least or up to about 86 amino acid residues, at least or up to about 88 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 92 amino acid residues, at least or up to about 94 amino acid residues, at least or up to about 95 amino acid residues, at least or up to about 96 amino acid residues, at least or up to about 98 amino acid residues, or at least or up to about 100 amino acid residues.

[0160] In some embodiments, the plurality of amino acid residues of the one or more heterologous amino acid residues may be consecutive amino acid residues. In some embodiments, the plurality of amino acid residues of the one or more heterologous amino acid residues may comprise a plurality of non-consecutive amino acid residues.

[0161] In some embodiments, the one or more heterologous amino acid residues of the engineered nuclease may comprise a heterologous polypeptide sequence (e.g., heterologous to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 (when applicable)). The heterologous polypeptide sequence can exhibit a net positive charge (e.g., net +1 charge, net +2 charge, net +3 charge, net +4 charge, net +5 charge, etc., e.g., as measured in a buffer at about pH 7.4). The heterologous polypeptide sequence can exhibit a net negative charge (e.g., net -1 charge, net -2 charge, net -3 charge, net -4 charge, net -5 charge, etc., e.g., as measured in a buffer at about pH 7.4). The heterologous polypeptide sequence can exhibit a neutral charge, e.g., as measured in a buffer at about pH 7.4. In some cases, the heterologous polypeptide sequence can comprise an amino acid sequence that is at least about 50%, 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 95%, at least about 99%, or about 100% identical to the polypeptide sequence of SEQ ID NO: 11. SEQ ID NO: 11 (heterologous polypeptide) ERRKQAGGTG E

[0162] In some embodiments, the amino acid sequence of the engineered nuclease as disclosed herein can comprise deletion of one or more amino acid residues from (i) the amino acidresides 2-100 as compared to the polypeptide sequence of SEQ ID NO: 1 and / or (ii) the amino acid resides 430-529 as compared to the polypeptide sequence of SEQ ID NO: 1, and the amino acid sequence can further comprise deletion of one or more additional amino acid residues (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more additional amino acid residues) from the amino acid residues 101-429 as disclosed herein, as compared to the polypeptide sequence of SEQ ID NO: 1.

[0163] In some embodiments, the amino acid sequence of the engineered nuclease as disclosed herein can comprise deletion of one or more amino acid residues from (i) the amino acid resides 2-100 as compared to the polypeptide sequence of any one of the Cas proteins selected from TABLE 2 and / or (ii) the last 100 C-terminal amino acid resides as compared to the polypeptide sequence of any one of the Cas proteins selected from TABLE 2, and the amino acid sequence can further comprise deletion of one or more additional amino acid residues (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more additional amino acid residues) from the amino acid residues there between, as compared to the polypeptide sequence of any one of the Cas proteins selected from TABLE 2.

[0164] In some embodiments, the engineered polypeptide as disclosed herein can comprise one or more heterologous amino acid residues (e.g., other than mutations) as compared to the native Un1Cas12f1 nuclease as provided in SEQ ID NO: 1. The presence of the one or more heterologous amino acid residues can enhance, for example, activity, stability, expression, binding to the respective guide nucleic acid molecule, etc. of the engineered polypeptide.

[0165] In some embodiments, of the engineered polypeptide as disclosed herein (e.g., a deactivated Cas nuclease variant) can comprises an amino acid sequence that is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% identical to the polypeptide sequence of SEQ ID NO: 12. SEQ ID NO: 12 (example engineered nuclease polypeptide) 1 MAKNTITKTL KLRIVRPYNS AEVEKIVADE KERRKQAGGT GELDDKFYQK 51 LRGQFPDAVF WQEISEIFRQ LQKQAAEIYN QSLIELYYEI FIKGKGIANA101 CYRRAAELFK NAAIASGLRS KIKSNFRLKE LKNMKSGLPT 151 TKSDNFPIPL VKQKGGQYTG FEISNHNSDF IIKIPFGRWQ VKKEIDKYRP 201 WEKFDFEQVQ KSPKPISLLL STQRRKRNKG WSKDEGTEAE IKKVMNGDYQ 251 TSYIEVKRGS KICEKSAWML NLSIDVPKID KGVDPSIIGG IAVGVRSPLV 301 CAINNAFSRY SISDNDLFHF NKKMFARRRI LLKKNRHKRA GHGAKNKLKP 351 ITILTEKSER FRKKLIERWA CEIADFFIKN KVGTVQMENL ESMKRKEDSY 401 FNIRLRGFWP YAEMQNKIEF KLKQYGIEIR KVAPNNTSKT CSKCGHLNNY 451 FNFEYRKKNK FPHFKCEKCN FKENAAYNAA LNISNPKLKS TKERP

[0166] In some embodiments, the engineered polypeptide as disclosed herein can comprise an amino acid sequence that is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% identical to the polypeptide sequence of an engineered nuclease variant from TABLE 3B (e.g., one or more of SEQ ID NOs: 20-27), TABLE 4B (e.g., one or more of SEQ ID NOs: 28-111), and / or TABLE 5C (e.g., one or more of SEQ ID NOs: 112-201).

[0167] In some embodiments, the engineered polypeptide as disclosed herein may not be identical to any one of: SEQ ID NO: 1 and any Cas protein from TABLE 2 (SEQ ID NOs: 2, 3, and 13-19).

[0168] In some embodiments, the engineered polypeptide as disclosed herein can comprise at least one amino acid modification as compared to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10. The at least one amino acid modification can comprise one or more members selected from the group consisting of A21Q, V23I, N32E, D29E, N33R, E35K, K36Q, I37A, A38G, E40D, K73G, A74T, R75G, K76E, Q83K, G87K, E151A, A340S, H353K, A374K, I387E, N423D, K473Q, T474L, T474R, H497K, L515R, N519T, K521D, K521N, L522I, and any deletion of one or more amino acid residues relative to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10 as disclosed herein (e.g., at least one deletion from the amino acid residues 400-529 of SEQ ID NO: 1 or SEQ ID NO: 10). The one or more members can comprise at least or up to about 1, at least or up to about 2, at least or up to about 3, at least or up to about 4, at least or up to about 5, at least or up to about 6, at least or up to about 7, at least or up to about 8, at least or up to about 9, at least or up to about 10, at least or up to about 11, at least or up to about 12, atleast or up to about 13, at least or up to about 14, at least or up to about 15, at least or up to about 20 amino acid, at least or up to about 25 amino acid, or at least or up to about 30 amino acid modifications selected from the group consisting of A21Q, V23I, N32E, D29E, N33R, E35K, K36Q, I37A, A38G, E40D, K73G, A74T, R75G, K76E, Q83K, G87K, E151A, A340S, H353K, A374K, I387E, N423D, K473Q, T474L, T474R, H497K, L515R, N519T, K521D, K521N, L522I, as compared to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10. In some cases, the at least one amino acid modification can comprise at least one set of modifications selected from TABLE 5A. In some cases, the at least one amino acid modification can comprise at least one combination of modifications selected from TABLE 5B. For example, the at least one combination of modifications selected from TABLE 5B may not be cA2.55 or cA2.84.

[0169] In some embodiments, the engineered polypeptide as disclosed herein can comprise at least one amino acid modification as compared to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10. The at least one amino acid modification can comprise one or more members (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more members) selected from the group consisting of A21Q, V23I, D29E, N33R, E40D, Q83K, G87K, E151A, A340S, H353K, A374K, I387E, N423D, K473Q, T474L, T474R, H497K, L515R, N519T, K521D, K521N, and L522I.

[0170] In some embodiments, the engineered polypeptide as disclosed herein can comprise at least one amino acid modification as compared to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10. The at least one amino acid modification can comprise one or more members (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 members) selected from the group consisting of N32E, N33R, E35K, K36Q, I37A, A38G, K73G, A74T, R75G, and K76E.

[0171] In some embodiments, the engineered nuclease variant of the engineered polypeptide as disclosed herein can be a chimeric polypeptide comprising different polynucleotide sequence domains derived from different Cas proteins. The chimeric polypeptide can comprise a first polypeptide sequence comprising at least 3 contiguous amino acid residues in common with a first Cas protein and a second polypeptide sequence comprising at least 3 contiguous amino acid residues in common with a second Cas protein, which second Cas protein is different from the first Cas protein. The first Cas protein and the second Cas protein can be different naturally occurring Cas proteins. The first Cas protein and the second Cas protein can have a size (or amino acid sequence length) that is different from each other by no more than 500, no more than 400, no morethan 350, no more than 300, no more than 250, no more than 200, no more than 150, no more than 140, no more than 130, no more than 120, no more than 110, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, or no more than 20 amino acid residues.

[0172] In some cases, a length of the first polypeptide sequence can be substantially the same as a length of the second polypeptide sequence. Alternatively, the length of the first polypeptide sequence can be different from the length of the second polypeptide sequence. The length of the second polypeptide sequence can be less than or equal to about 90%, less than or equal to about 85%, less than or equal to about 80%, less than or equal to about 75%, less than or equal to about 70%, less than or equal to about 65%, less than or equal to about 60%, less than or equal to about 55%, less than or equal to about 50%, less than or equal to about 45%, less than or equal to about 40%, less than or equal to about 35%, less than or equal to about 30%, less than or equal to about 25%, less than or equal to about 20%, less than or equal to about 18%, less than or equal to about 16%, less than or equal to about 15%, less than or equal to about 14%, less than or equal to about 12%, less than or equal to about 10%, less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 4%, less than about 3%, less than or equal to about 2%, or less than or equal to about 1% of the length of the first polypeptide sequence. The length of the second polypeptide sequence can be at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 6%, at least or up to about 7%, at least or up to about 8%, at least or up to about 9%, at least or up to about 10%, at least or up to about 12%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 18%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, or at least or up to about 95% of the length of the first polypeptide sequence.

[0173] In some cases, the first Cas protein and the second Cas protein can be two different members selected from: SEQ ID NO: 1 and any Cas protein from TABLE 2 (SEQ ID NOs: 2, 3, and 13-19). The first Cas protein or the second Cas protein can comprise an amino acid sequence that is at least or up to about 5%, at least or up to about 10%, at least or up to about 15%,at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 92%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% identical to the polypeptide sequence of a member selected from: SEQ ID NO: 1 and any Cas protein from TABLE 2 (SEQ ID NOs: 2, 3, and 13-19). For example, such member can be SEQ ID NO: 1 or SEQ ID NO: 2.

[0174] In some cases, the first polypeptide of the chimeric polypeptide of the engineered nuclease variant can comprise at least or up to about 4 continuous amino acid residues, at least or up to about 5 contiguous amino acid residues, at least or up to about 6 continuous amino acid residues, at least or up to about 7 continuous amino acid residues, at least or up to about 8 continuous amino acid residues, at least or up to about 9 continuous amino acid residues, at least or up to about 10 contiguous amino acid residues, at least or up to about 11 continuous amino acid residues, at least or up to about 12 continuous amino acid residues, at least or up to about 13 continuous amino acid residues, at least or up to about 14 continuous amino acid residues, at least or up to about 15 continuous amino acid residues, at least or up to about 18 continuous amino acid residues, at least or up to about 20 contiguous amino acid residues, at least or up to about 30 contiguous amino acid residues, at least or up to about 40 contiguous amino acid residues, or at least or up to about 50 contiguous amino acid residues in common with the first Cas protein.

[0175] In some cases, the second polypeptide of the chimeric polypeptide of the engineered nuclease variant can comprise at least or up to about 4 continuous amino acid residues, at least or up to about 5 contiguous amino acid residues, at least or up to about 6 continuous amino acid residues, at least or up to about 7 continuous amino acid residues, at least or up to about 8 continuous amino acid residues, at least or up to about 9 continuous amino acid residues, at least or up to about 10 contiguous amino acid residues, at least or up to about 11 continuous amino acid residues, at least or up to about 12 continuous amino acid residues, at least or up to about 13 continuous amino acid residues, at least or up to about 14 continuous amino acid residues, at least or up to about 15 continuous amino acid residues, at least or up to about 18 continuous amino acid residues, at least or up to about 20 contiguous amino acid residues, at least or up to about 30 contiguous amino acid residues, at least or up to about 40 contiguous amino acid residues, or atleast or up to about 50 contiguous amino acid residues in common with the second Cas protein.

[0176] In some cases, in the second polypeptide of the chimeric polypeptide of the engineered nuclease variant, a length of the first polypeptide can be greater than a length of the second polypeptide by at least or up to about 1 amino acid, at least or up to about 2 amino acids, at least or up to about 5 amino acids, at least or up to about 10 amino acids, at least or up to about 15 amino acids, at least or up to about 20 amino acids, at least or up to about 25 amino acids, at least or up to about 30 amino acids, at least or up to about 35 amino acids, at least or up to about 40 amino acids, at least or up to about 45 amino acids, at least or up to about 50 amino acids, at least or up to about 55 amino acids, at least or up to about 60 amino acids, at least or up to about 70 amino acids, at least or up to about 80 amino acids, at least or up to about 90 amino acids, at least or up to about 100 amino acids, at least or up to about 110 amino acids, at least or up to about 120 amino acids, at least or up to about 130 amino acids, at least or up to about 140 amino acids, at least or up to about 150 amino acids, at least or up to about 200 amino acids, at least or up to about 250 amino acids, at least or up to about 300 amino acids, at least or up to about 350 amino acids, at least or up to about 400 amino acids, at least or up to about 450 amino acids, or at least or up to about 500 amino acids.

[0177] In some cases, the first polypeptide can be derived from the N-terminal 50%, the N-terminal 45%, the N-terminal 40%, the N-terminal 35%, the N-terminal 30%, the N-terminal 25%, the N-terminal 20%, the N-terminal 15%, the N-terminal 10%, or the N-terminal 5% of the first Cas protein. Alternatively or in addition to, the first polypeptide can be derived from the C- terminal 50%, the C-terminal 45%, the C-terminal 40%, the C-terminal 35%, the C-terminal 30%, the C-terminal 25%, the C-terminal 20%, the C-terminal 15%, the C-terminal 10%, or the C- terminal 5% of the first Cas protein. In some cases, the second polypeptide can be derived from the N-terminal 50%, the N-terminal 45%, the N-terminal 40%, the N-terminal 35%, the N-terminal 30%, the N-terminal 25%, the N-terminal 20%, the N-terminal 15%, the N-terminal 10%, or the N-terminal 5% of the second Cas protein. Alternatively or in addition to, the second polypeptide can be derived from the C-terminal 50%, the C-terminal 45%, the C-terminal 40%, the C-terminal 35%, the C-terminal 30%, the C-terminal 25%, the C-terminal 20%, the C-terminal 15%, the C- terminal 10%, or the C-terminal 5% of the second Cas protein.

[0178] In some cases, the first polypeptide can be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acidresidues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues from the N-terminus of the first Cas protein. Alternatively or in addition to, the first polypeptide can be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues from the C- terminus of the first Cas protein.

[0179] In some cases, the second polypeptide can be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues from the N-terminus of the second Cas protein. Alternatively or in addition to, the second polypeptide can be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues from the C-terminus of the second Cas protein.

[0180] In some cases, the engineered nuclease variant of the engineered polypeptide as disclosed herein can comprise a third polypeptide sequence comprising at least 3 contiguous amino acid residues (or more as disclosed herein) in common with the first Cas protein. In such cases, the first polypeptide sequence and the third polypeptide sequence may or may not be contiguous to each other within the chimeric polypeptide. Alternatively or in addition to, the third polypeptidesequence can comprise at least 3 contiguous amino acid residues (or more as disclosed herein) in common with the second Cas protein. The first polypeptide sequence and the third polypeptide sequence may not be contiguous to each other in the chimeric polypeptide. Alternatively, the first polypeptide sequence and the third polypeptide sequence may be contiguous to each other in the chimeric polypeptide. The second polypeptide sequence and the third polypeptide sequence may not be contiguous to each other in the chimeric polypeptide. Alternatively, the second polypeptide sequence and the third polypeptide sequence may be contiguous to each other in the chimeric polypeptide. Yet in another alternative or additionally, the third polypeptide sequence can comprise at least 3 contiguous amino acid residues in common with a third Cas protein that is different from the first Cas protein and the second Cas protein. For example, the first Cas protein, the second Cas protein, and the third Cas protein can be three different members selected from SEQ ID NO: 1 and any Cas protein selected from TABLE 2.

[0181] In some cases, the third polypeptide of the chimeric polypeptide of the engineered nuclease variant can comprise at least or up to about 4 continuous amino acid residues, at least or up to about 5 contiguous amino acid residues, at least or up to about 6 continuous amino acid residues, at least or up to about 7 continuous amino acid residues, at least or up to about 8 continuous amino acid residues, at least or up to about 9 continuous amino acid residues, at least or up to about 10 contiguous amino acid residues, at least or up to about 11 continuous amino acid residues, at least or up to about 12 continuous amino acid residues, at least or up to about 13 continuous amino acid residues, at least or up to about 14 continuous amino acid residues, at least or up to about 15 continuous amino acid residues, at least or up to about 18 continuous amino acid residues, at least or up to about 20 contiguous amino acid residues, at least or up to about 30 contiguous amino acid residues, at least or up to about 40 contiguous amino acid residues, or at least or up to about 50 contiguous amino acid residues in common with the third Cas protein.

[0182] In some cases, the third polypeptide can be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues from the N-terminus of the third Cas protein. Alternatively or in addition to,the third polypeptide can be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues from the C- terminus of the third Cas protein.

[0183] In some cases, within the chimeric polypeptide of the engineered nuclease variant as disclosed herein, the first polypeptide sequence, the second polypeptide sequence, and the third polypeptide sequence can be arranged in any of the following structures, from N-terminus to C-terminus of the chimeric polypeptide: CP1-CP2-CPx (I), CP1-CPx-CP2 (II), CP2-CP1-CPx (III), CP2-CPx-CP1 (IV), CPx-CP1-CP2 (V), or CPx-CP2-CP1 (VI), in which “-“ can be either an amino acid linker (e.g., comprising one or more amino acid sequences) or a direct covalent bond. The amino acid linker as disclosed herein can comprise a single amino acid, at least or up to about 2 amino acids, at least or up to about 3 amino acids, at least or up to about 4 amino acids, at least or up to about 5 amino acids, at least or up to about 8 amino acids, at least or up to about 10 amino acids, at least or up to about 12 amino acids, at least or up to about 15 amino acids, at least or up to about 16 amino acids, or at least or up to about 20 amino acids. The amino acid linker can comprise at least one Glycine, at least one Serine, or at least one Glycine-Serine dipeptide.

[0184] In some embodiments, the amino acid sequence of the engineered nuclease as disclosed herein (e.g., the chimeric polypeptide as disclosed herein) can have a length of at most 528 amino acids, at most 527 amino acids, at most 526 amino acids, at most 525 amino acids, at most 524 amino acids, at most 523 amino acids, at most 522 amino acids, at most 521 amino acids, at most 520 amino acids, at most 519 amino acids, at most 518 amino acids, at most 517 amino acids, at most 516 amino acids, at most 515 amino acids, at most 514 amino acids, at most 513 amino acids, at most 512 amino acids, at most 511 amino acids, at most 510 amino acids, at most 509 amino acids, at most 508 amino acids, at most 507 amino acids, at most 506 amino acids, at most 505 amino acids, at most 504 amino acids, at most 503 amino acids, at most 502 amino acids, at most 501 amino acids, at most about 500 amino acids, at most about 495 amino acids, at most about 490 amino acids, at most about 485 amino acids, at most about 480 amino acids, at most about 475 amino acids, at most about 470 amino acids, at most about 465 amino acids, at mostabout 460 amino acids, at most about 455 amino acids, at most about 450 amino acids, at most about 445 amino acids, at most about 440 amino acids, at most about 435 amino acids, at most about 430 amino acids, at most about 425 amino acids, at most about 420 amino acids, at most about 415 amino acids, at most about 410 amino acids, at most about 405 amino acids, at most about 400 amino acids, at most about 395 amino acids, at most about 390 amino acids, at most about 385 amino acids, at most about 380 amino acids, at most about 375 amino acids, at most about 370 amino acids, at most about 365 amino acids, at most about 360 amino acids, at most about 355 amino acids, at most about 350 amino acids, at most about 345 amino acids, at most about 340 amino acids, at most about 335 amino acids, at most about 330 amino acids, at most about 325 amino acids, at most about 320 amino acids, at most about 315 amino acids, at most about 310 amino acids, at most about 305 amino acids, or at most about 300 amino acids.

[0185] In some embodiments, the engineered nuclease comprising the amino acid sequence as disclosed herein can have a length of at most about 1000 amino acids, at most about 950 amino acids, at most about 900 amino acids, at most about 850 amino acids, at most about 800 amino acids, at most about 750 amino acids, at most about 700 amino acids, at most about 650 amino acids, at most about 640 amino acids, at most about 630 amino acids, at most about 620 amino acids, at most about 610 amino acids, at most about 600 amino acids, at most about 590 amino acids, at most about 580 amino acids, at most about 570 amino acids, at most about 560 amino acids, at most about 550 amino acids, at most about 540 amino acids, at most about 530 amino acids, at most about 520 amino acids, at most about 510 amino acids, at most about 500 amino acids, at most about 490 amino acids, at most about 480 amino acids, at most about 470 amino acids, at most about 460 amino acids, at most about 450 amino acids, at most about 440 amino acids, at most about 430 amino acids, at most about 420 amino acids, at most about 410 amino acids, at most about 400 amino acids, at most about 350 amino acids, or at most about 300 amino acids.

[0186] In some embodiments, at least a portion of the engineered nuclease variant as disclosed herein may be derived from (e.g., via engineering of) a naturally occurring Cas protein (e.g., the first Cas protein, the second Cas protein, or the third Cas protein as described herein). In some cases, the naturally occurring Cas protein can have a length of at most about 800 amino acids, at most about 750 amino acids, at most about 700 amino acids, at most about 650 amino acids, at most about 600 amino acids, at most about 550 amino acids, at most about 540 aminoacids, at most about 530 amino acids, at most about 510 amino acids, at most about 500 amino acids, at most about 490 amino acids, at most about 480 amino acids, at most about 470 amino acids, at most about 460 amino acids, at most about 450 amino acids, or at most about 400 amino acids. The naturally occurring Cas protein, for example, can be a member from SEQ ID NO: 1 and any Cas protein selected from TABLE 2.

[0187] In some embodiments, the first polypeptide sequence, the second polypeptide sequence, and / or the third polypeptide sequence of the chimeric polypeptide of the engineered nuclease variant as disclosed herein may not be derived from Cas12a. In some embodiments, the chimeric polypeptide of the engineered nuclease variant may not be derived from Cas12a. In some embodiments, the chimeric polypeptide of the engineered nuclease variant may be entirely derived from one or more Cas12f type orthologous (e.g., SEQ ID NO:1 or a Cas protein selected from TABLE 2).

[0188] In some embodiments, the engineered nuclease comprising the amino acid sequence as disclosed herein can be is mutated and / or modified to yield a nuclease deficient protein or a protein with decreased nuclease activity relative to a wild-type Cas protein. A nuclease deficient protein can retain the ability to bind a target gene (e.g., DNA), but may lack or have reduced nucleic acid cleavage activity. In some embodiments, the engineered nuclease comprising the amino acid sequence as disclosed herein can exhibit reduced nuclease activity (e.g., nuclease deficient or nuclease null) as compared to the Cas nuclease encoded by SEQ ID NO: 1 or a Cas protein selected from TABLE 2. The reduced nuclease activity can be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than that of the Cas nuclease encoded by SEQ ID NO: 1 or that of the Cas protein selected from TABLE 2. In some cases, the engineered nuclease can comprise a substitution at D326 and / or D510, as compared to the polypeptide sequence of SEQ ID NO: 1. For example, the D326 and / or the D510 substitution(s) can be alanine substitutions (e.g., D326A and / or D510A).

[0189] In some embodiments, the amino acid sequence of the engineered nuclease as disclosed herein can comprise one or more substitutions in the native amino acid sequence, where the positions of at least some of these substitutions follow one or more particular rules determined to have surprising advantages for the engineered nuclease. In some cases, the particularsubstitution rules have been selected for their ability to produce variants of the engineered nuclease, e.g., that can be capable of functioning within eukaryotic cells. According to these particular rules, all or some of the one or more substitutions in the native amino acid sequence are either (1) within or no more than 30 amino acids downstream of a (D / E / K / N)X(R / F)(E / K)N motif of the native amino acid sequence, (2) at or no more than 30 amino acids upstream or downstream of position 241 of the native amino acid sequence, (3) at or no more than 30 amino acids upstream or downstream of position 516 of the native amino acid sequence, and / or (4) having an electrically charged amino acid in the native amino acid sequence.

[0190] In some embodiments, the amino acid sequence of the engineered nuclease as disclosed herein can comprise one or more substitutions at amino acid positions within or no more than a threshold length (e.g., 30 amino acid residues) upstream and / or downstream of a (D / E / K / N)X(R / F)(E / K)N motif, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the amino acid sequence of the engineered nuclease as disclosed herein without the one or more substitutions. In some cases, at least one of the one or more substitutions can be, for example, within or no more than 28 amino acids, 26 amino acids, 24 amino acids, 22 amino acids, 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12 amino acids, or 10 amino acids of the motif. In some cases, at least one of the one or more substitutions can be to an R, A, S, or G amino acid residue. In some cases, the one or more substitutions can include substitutions are at one or more positions selected from the group consisting of D143, T147, E151, and K154 (e.g., D143R, T147R, E151R, and / or K154R). In some cases, the one or more substitutions can include substitutions are at one or more positions selected from the group consisting of N504, E507, N516, N519, E527, and E528 (e.g., N504R, E507R, N516R, N519R, E527R, and / or E528R). In some cases, the one or more substitutions can include substitutions are at one or more positions selected from the group consisting of K11, K73, D143, T147, E151, K154, E241, D318, K330, K457, E425, E462, N504, E507, N516, N519, E527, and E528 (e.g., K11R, K73R, D143R, T147R, E151R, K154R, E241R, D318R, K330R, E425N, K457R, E462R, N504R, E507R, N516R, N519R, E527R, and / or E528R).

[0191] In some embodiments, the amino acid sequence of the engineered nuclease comprising the one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif, as disclosed herein, can exhibit a cationic charge (e.g., a positive) that is greater than that of a control amino acid sequence of the engineered nuclease lacking theone or more substitutions, by at least or up to about 1 cationic charge, at least or up to about 2 cationic charges, at least or up to about 3 cationic charges, at least or up to about 4 cationic charges, at least or up to about 5 cationic charges, at least or up to about 6 cationic charges, at least or up to about 7 cationic charges, at least or up to about 8 cationic charges, at least or up to about 9 cationic charges, at least or up to about 10 cationic charges, at least or up to about 11 cationic charges, at least or up to about 12 cationic charges, at least or up to about 13 cationic charges, at least or up to about 14 cationic charges, at least or up to about 15 cationic charges, at least or up to about 16 cationic charges, at least or up to about 17 cationic charges, or at least or up to about 18 cationic charges.

[0192] Without wishing to be bound by theory, the amino acid sequence of the engineered nuclease comprising the one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif, as disclosed herein, can exhibit enhanced (e.g., higher) binding affinity to (i) a guide nucleic acid sequence (e.g., a guide RNA sequence) and / or (ii) a target polynucleotide sequence (e.g., a target gene in a cell, such as a target endogenous gene) of the Cas / guide nucleic complex, as compared to (A) a control amino acid sequence of the engineered nuclease lacking the one or more substitutions and / or (B) the CRISPR / Cas protein encoded by SEQ ID NO: 1, by at least or up to about 1%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 110%, at least or up to about 120%, at least or up to about 125%, at least or up to about 150%, at least or up to about 200%, at least or up to about 250%, at least or up to about 300%, at least or up to about 350%, at least or up to about 400%, at least or up to about 450%, or at least or up to about 500%, as ascertained by surface plasmon resonance (SPR) or isothermal titration calorimetry (IRC) assays.

[0193] Without wishing to be bound by theory, the amino acid sequence of the engineered nuclease comprising the one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif, as disclosed herein, can exhibit enhanced (e.g., higher) binding affinity to (i) a guide nucleic acid sequence (e.g., a guide RNA sequence) and / or (ii) a target polynucleotide sequence (e.g., a target gene in a cell, such as a target endogenous gene) of the Cas / guide nucleic complex, as compared to (A) a control amino acid sequence of the engineerednuclease lacking the one or more substitutions and / or (B) the CRISPR / Cas protein encoded by SEQ ID NO: 1, by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.5-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 25-fold, at least or up to about 30-fold, at least or up to about 35-fold, or at least or up to about 40-fold, as ascertained by surface plasmon resonance (SPR) or isothermal titration calorimetry (IRC) assays.

[0194] In some embodiments, the present disclosure provides a system comprising the engineered nuclease as disclosed herein. In some embodiments, the system can comprise recombinantly expressed (or generated) form of the engineered nuclease. In some embodiments, the system can comprise one or more polynucleotides encoding at least the engineered nuclease. In some embodiments, the system can comprise a cell (or a population of cells) comprising (e.g., engineered to comprise, such as transfected or transduced to express) at least the engineered nuclease. METHODS OF USE OF THE ENGINEERED NUCLEASE

[0195] In some embodiments, the engineered nuclease as disclosed herein can be used to effect binding, cleaving, and / or editing a target polynucleotide sequence, e.g., to regulate expression and / or activity level of the target polynucleotide sequence of a polypeptide (e.g., a protein) encoded by the target polynucleotide sequence or operatively coupled to the target polynucleotide sequence. In some cases, a heterologous polypeptide comprising the engineered nuclease can be introduced to a cell (e.g., a mammalian cell) to effect binding, cleaving, and / or editing a target polynucleotide sequence of the cell (e.g., endogenous gene or heterologous gene of the cell). In some embodiments, the engineered nuclease as disclosed herein, or a protein comprising the engineered nuclease can be referred to as an actuator moiety.

[0196] In some embodiments, the engineered nuclease as disclosed herein can retain at least a portion (e.g., substantially all of) of the nuclease activity of the nuclease encoded by the polypeptide sequence of SEQ ID NO: 1 or that of the nuclease activity of a Cas protein selected from TABLE 2.

[0197] In some embodiments, is the engineered nuclease as disclosed herein can benuclease-deficient. In some embodiments, the engineered nuclease can be a nuclease-null DNA binding protein that does not induce transcriptional activation or repression of a target DNA sequence unless it is present in a complex with one or more heterologous gene effectors of the disclosure. In some embodiments, the engineered nuclease can be a nuclease-null DNA binding protein that can induce transcriptional activation or repression of a target DNA sequence (e.g., which can be altered or augmented by the presence of a heterologous gene effector as provided herein). The terms “gene effector” and “gene modulator” may be used interchangeably herein. The terms “gene effector polypeptide” and “gene modulator polypeptides” may be used interchangeably herein.

[0198] In some embodiments, the engineered nuclease as disclosed herein can be an RNA nuclease such as an engineered (e.g., programmable or targetable) RNA nuclease. In some embodiments, the engineered nuclease as disclosed herein can be a nuclease-null RNA binding protein that does not induce transcriptional activation or repression of a target RNA sequence unless it is present in a complex with one or more heterologous gene effectors of the disclosure. In some embodiments, the engineered nuclease as disclosed herein can be a nuclease-null RNA binding protein that can induce transcriptional activation or repression of a target RNA sequence (e.g., which can be altered or augmented by the presence of a heterologous gene effector as provided herein).

[0199] In some embodiments, the engineered nuclease can be a nucleic acid-guided targeting system. In some embodiments, the engineered nuclease can be a DNA-guided targeting system. In some embodiments, the engineered nuclease can be an RNA-guided targeting system. The nucleic acid-guided targeting system can comprise and utilize, for example, a guide nucleic acid sequence that facilitates specific binding of a CRISPR-Cas system (e.g., a nuclease deficient form thereof, such as dCas9 or dCas14) to a target gene (e.g., target endogenous gene) or target gene regulatory sequence. For example, the target gene may be any one of the genes listed in TABLE 1, and the target gene regulatory sequence may be operatively coupled to any one of the genes listed in TABLE 1. Binding specificity can be determined by use of a guide nucleic acid, such as a single guide RNA (sgRNA) or a part thereof. In some embodiments, the use of different sgRNAs allows the compositions and methods of the disclosure to be used with (e.g., targeted to) different target genes (e.g., target endogenous genes) or target gene regulatory sequences.

[0200] In some embodiments, the engineered nuclease can form a complex with aguide nucleic acid, such as a guide RNA or a part thereof. In some embodiments, the engineered nuclease can form a complex with a single guide nucleic acid, such as a single guide RNA (sgRNA). In some embodiments, the engineered nuclease can be a RNA-binding protein (RBP) optionally complexed with a guide nucleic acid, such as a guide RNA (e.g., sgRNA), which is able to form a complex with a Cas protein. In some embodiments, the engineered nuclease can be a nuclease-null DNA binding protein that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the engineered nuclease can be a nuclease-null RNA binding protein derived from a RNA.

[0201] A guide nucleic acid used in compositions and methods of the disclosure can be, for example, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 nucleotides.

[0202] In some embodiments, a guide nucleic acid used in compositions and methods of the disclosure is at most at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, or at most 40 nucleotides.

[0203] In some embodiments, a guide nucleic acid used in compositions and methods of the disclosure is between about 8 and about 40 nucleotides, between about 10 and about 40 nucleotides, between aboutand about 40 nucleotides, between aboutand about 40 nucleotides, between about 13 and about 40 nucleotides, between about 14 and about 40 nucleotides, between aboutand about 40 nucleotides, between aboutand about 40 nucleotides, between aboutand about 40 nucleotides, between aboutand about 40 nucleotides, between about 19 and about 40 nucleotides, between about 20 and about 40 nucleotides, between aboutand about 40 nucleotides, between about 24 and about 40 nucleotides, between about 26 and about 40 nucleotides, between about 28 and about 40 nucleotides, between about 30 and about 40 nucleotides, between about 8 and about 30 nucleotides, between about 10 and about 30 nucleotides, between about 11 and about 30 nucleotides, betweenabout 12 and about 30 nucleotides, between about 13 and about 30 nucleotides, between about 14 and about 30 nucleotides, between about 15 and about 30 nucleotides, between about 16 and about 30 nucleotides, between about 17 and about 30 nucleotides, between about 18 and about 30 nucleotides, between about 19 and about 30 nucleotides, between about 20 and about 30 nucleotides, between about 22 and about 30 nucleotides, between about 24 and about 30 nucleotides, between about 26 and about 30 nucleotides, between about 28 and about 30 nucleotides, between about 8 and about 25 nucleotides, between about 10 and about 25 nucleotides, between about 11 and about 25 nucleotides, between about 12 and about 25 nucleotides, between about 13 and about 25 nucleotides, between about 14 and about 25 nucleotides, between about 15 and about 25 nucleotides, between about 16 and about 25 nucleotides, between about 17 and about 25 nucleotides, between about 18 and about 25 nucleotides, between about 19 and about 25 nucleotides, between about 20 and about 25 nucleotides, between about 22 and about 25 nucleotides, between about 24 and about 25 nucleotides, between about 8 and about 20 nucleotides, between about 10 and about 20 nucleotides, between about 11 and about 20 nucleotides, between about 12 and about 20 nucleotides, between about 13 and about 20 nucleotides, between about 14 and about 20 nucleotides, between about 15 and about 20 nucleotides, between about 16 and about 20 nucleotides, between about 17 and about 20 nucleotides, between about 18 and about 20 nucleotides, between about 19 and about 20 nucleotides, between about 8 and about 18 nucleotides, between about 10 and about 18 nucleotides, between about 11 and about 18 nucleotides, between about 12 and about 18 nucleotides, between about 13 and about 18 nucleotides, between about 14 and about 18 nucleotides, between about 15 and about 18 nucleotides, between about 16 and about 18 nucleotides, between about 8 and about 16 nucleotides, between about 10 and about 16 nucleotides, between about 11 and about 16 nucleotides, between about 12 and about 16 nucleotides, between about 13 and about 16 nucleotides, between about 14 and about 16 nucleotides, or between about 15 and about 16 nucleotides. In some embodiments, a guide nucleic acid can be a guide RNA or a part thereof.

[0204] The engineered nuclease as disclosed herein can be modified to enhance regulation of gene expression by compositions and methods of the disclosure, e.g., as part of a complex disclosed herein. The engineered nuclease can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, enzymatic activity, and / or binding to other factors, such as heterodimerization or oligomerization domains and induce ligands. The engineerednuclease can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the engineered nuclease can be modified, deleted, or inactivated, or at least a portion of the engineered nuclease can be truncated to remove domains that are not essential for the desired function of the protein or complex. The engineered nuclease can be modified to modulate (e.g., enhance or reduce) the activity of the engineered nuclease for regulating gene expression by a complex of the disclosure that comprises a heterologous gene effector.

[0205] For example, the engineered nuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a heterologous gene effector (e.g., an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repressor domain). The engineered nuclease can be coupled (e.g., fused, covalently coupled, or non- covalently coupled) to an oligomerization or dimerization domain as disclosed herein (e.g., a heterodimerization domain). The engineered nuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a heterologous polypeptide that provides increased or decreased stability. The engineered nuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a sequence that can facilitate degradation of the engineered nuclease or a complex containing the engineered nuclease. The engineered nuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a gene editing moiety (e.g., heterologous protein, or domain or functional fragment thereof), that edits, mutates, or modifies (either directly or indirectly) a target polynucleotide sequence.

[0206] The engineered nuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to any suitable number of partners, for example, at least one, at least two, at least three, at least four, or at least five, at least six, at least seven, or at least 8 partners. In some embodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, or at most ten partners. In some embodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to 1 – 5, 1 – 4, 1 – 3, 1 – 2, 2 – 5, 2 – 4, 2 – 3, 3 – 5, 3 – 4, or 4 – 5 partners. In some embodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to one partner. In some embodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to two partners. In someembodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to three partners. In some embodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to four partners. In some embodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to five partners. In some embodiments, the engineered nuclease of the disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to six partners.

[0207] The engineered nuclease as disclosed herein can be a fusion protein, e.g., a fusion comprising the engineered nuclease and one or more of the partners as disclosed herein. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the engineered nuclease.

[0208] A partner of the engineered nuclease (e.g., covalently or non-covalently coupled to a nuclease deficient or null variant of the engineered nuclease as disclosed herein) can be a transcriptional effector (e.g., a transcriptional activator or a transcriptional repressor). The transcriptional effector can be heterologous to the cell as provided herein.

[0209] In some embodiments, the transcriptional effector can be a histone epigenetic modifier (or a histone modifier). In some cases, the histone epigenetic modifier can modulate histones through methylation (e.g., a histone methylation modifier, such as an amino acid methyltransferase, e.g., KRAB). In some cases, the histone epigenetic modifier can modulate histones through acetylation. In some cases, the histone epigenetic modifier can modulate histones through phosphorylation. In some cases, the histone epigenetic modifier can modulate histones through ADP-ribosylation. In some cases, the histone epigenetic modifier can modulate histones through glycosylation. In some cases, the histone epigenetic modifier can modulate histones through SUMOylation. In some cases, the histone epigenetic modifier can modulate histones through ubiquitination. In some cases, the histone epigenetic modifier can modulate histones by remodeling histone structure, e.g., via an ATP hydrolysis-dependent process.

[0210] In some embodiments, the transcriptional effector can be a gene epigenetic modifier (or a gene modifier). In some cases, a gene modifier can modulate genes through methylation (e.g., a gene methylation modifier, such as a DNA methyltransferase or DNMT). In some cases, a gene modifier can modulate genes through acetylation.

[0211] In some embodiments, the transcriptional effector is from a family of relatedhistone acetyltransferases. Non-limiting examples of histone acetyltransferases include GNAT subfamily, MYST subfamily, p300 / CBP subfamily, HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0212] In some embodiments, the transcriptional effector can comprise an epigenetic modifier. In some embodiments, the transcriptional effector comprises a histone epigenetic modifier (e.g., a histone lysine methyltransferase., a histone lysine demethylase, or a DNA methylase). Non-limiting examples of an epigenetic modifier can include EZH subfamily, Non- SET subfamily, Other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, PRDM12, PRDM13, PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4, and SYMD5.

[0213] Examples of proteins (or fragments thereof) that can be used as a fusion partner to increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g.,, for activity in plants), SET1A, SET1B, MLLl to 5, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI 60, CLOCK, Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc. An additional example of such gene activating modulator is VP64-p65-Rta fusion polypeptide (VPR).

[0214] Examples of proteins (or fragments thereof) that can be used as a fusion partner to decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g, for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4- 20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD 1 A / RBP2, JARIDlB / PLU-1, JARID 1C / SMCX, JARIDID / SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as Hhal DNAm5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0215] In various aspects, an engineered nuclease provided herein may affect editing or mutating of a target polynucleotide, as described herein. In some embodiments, editing or mutating a target polynucleotide sequence involves changing one or more nucleotides in a target polynucleotide to one or more different nucleotides. In some embodiments, editing or mutating a target polynucleotide involves changing a guanine (G) to a different nucleotide. In some cases, a guanine (G) may be changed to an adenine (A). In some cases, a guanine (G) may be changed to a thymine (T). In some cases, a guanine (G) may be changed to a cytosine (C). In some cases, a guanine (G) may be changed to a uracil (U). In some cases, a guanine (G) may be changed to an inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing a cytosine (C) to a different nucleotide. In some cases, a cytosine (C) may be changed to a guanine (G). In some cases, a cytosine (C) may be changed to an adenine (A). In some cases, a cytosine (C) may be changed to a thymine (T). In some cases, a cytosine (C) may be changed to a uracil (U). In some cases, a cytosine (C) may be changed to an inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing a thymine (T) to a different nucleotide. In some cases, a thymine (T) may be changed to a cytosine (C). In some cases, a thymine (T) may be changed to a guanine (G). In some cases, a thymine (T) may be changed to an adenine (A). In some cases, a thymine (T) may be changed to a uracil (U). In some cases, a thymine (T) may be changed to an inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing an adenine (A) to a different nucleotide. In some cases, an adenine (A) may be changed to a guanine (G). In some cases, an adenine (A) may be changed to a cytosine (C). In some cases, an adenine (A) may be changed to a thymine (T). In some cases, an adenine (A) may be changed to a uracil (U). In some cases, an adenine (A) may be changed to an inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing a uracil (U) to a different nucleotide. In some cases, a uracil (U) may be changed to a guanine (G). In some cases, a uracil (U) may be changed to a cytosine (C). In some cases, a uracil (U) may be changed to a thymine (T). In some cases, a uracil (U) may be changed to an adenine (A). In some cases, a uracil (U) may be changed to an inosine (I). In some embodiments, editing or mutating a targetpolynucleotide involves changing an inosine (I) to a different nucleotide. In some cases, an inosine (I) may be changed to a guanine (G). In some cases, an inosine (I) may be changed to a cytosine (C). In some cases, an inosine (I) may be changed to a thymine (T). In some cases, an inosine (I) may be changed to an adenine (A). In some cases, an inosine (I) may be changed to a uracil (U).

[0216] In some embodiments, editing or mutating a target polynucleotide involves introducing one or more point mutations into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more deletions (e.g., of one or more nucleotides) into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more insertions (e.g., of one or more nucleotides) into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more inversions (e.g., of two or more nucleotides) in a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more translocations (e.g., of one or more nucleotides) in a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more transpositions in a target polynucleotide, e.g., one or more DNA transpositions in a target gene.

[0217] In some cases, an engineered nuclease as described herein may be coupled to a partner (e.g., a gene editing moiety) that effects editing or mutating of a target polynucleotide, as described herein. In some cases, a nuclease-deficient or nuclease-null engineered nuclease as provided herein is covalently or non-covalently coupled to a gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide. In some embodiments, the gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide is a gene editing moiety that changes one or more nucleotides to a different nucleotide. In some embodiments, the gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide is a gene editing moiety that changes a guanine (G) to a different nucleotide. In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a guanine (G) to a cytosine (C). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a guanine (G) to a thymine (T). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a guanine (G) to an adenine (A). In some cases, thegene editing moiety that effects editing or mutating of a target polynucleotide changes a guanine (G) to a uracil (U). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a guanine (G) to an inosine (I). In some embodiments, the gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide sequence is a gene editing moiety that changes a cytosine (C) to a different nucleotide. In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a cytosine (C) to a guanine (G). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a cytosine (C) to a thymine (T). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a cytosine (C) to an adenine (A). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a cytosine (C) to a uracil (U). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a cytosine (C) to an inosine (I). In some embodiments, the gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide sequence is a gene editing moiety that changes a thymine (T) to a different nucleotide. In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a thymine (T) to a cytosine (C). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a thymine (T) to a guanine (G). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a thymine (T) to an adenine (A). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a thymine (T) to a uracil (U). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a thymine (T) to an inosine (I). In some embodiments, the gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide sequence is a gene editing moiety that changes an adenine (A) to a different nucleotide. In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an adenine (A) to a cytosine (C). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an adenine (A) to a thymine (T). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an adenine (A) to a guanine (G). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an adenine (A) to a uracil (U). In somecases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an adenine (A) to an inosine (I). In some embodiments, the gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide sequence is a gene editing moiety that changes a uracil (U) to a different nucleotide. In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a uracil (U) to a cytosine (C). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a uracil (U) to a thymine (T). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a uracil (U) to an adenine (A). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a uracil (U) to a guanine (G). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes a uracil (U) to an inosine (I). In some embodiments, the gene editing moiety (e.g., a protein, or functional domain or functional fragment thereof) that effects editing or mutating of a target polynucleotide sequence is a gene editing moiety that changes an inosine (I) to a different nucleotide. In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an inosine (I) to a cytosine (C). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an inosine (I) to a thymine (T). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an inosine (I) to an adenine (A). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an inosine (I) to a uracil (U). In some cases, the gene editing moiety that effects editing or mutating of a target polynucleotide changes an inosine (I) to a guanine (G).

[0218] In some embodiments, the engineered nuclease as described herein (e.g., a nuclease-deficient or nuclease-null engineered nuclease, as described herein) may be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing moiety) that introduces one or more point mutations into a target polynucleotide. In some embodiments, the engineered nuclease as described herein (e.g., a nuclease-deficient or nuclease-null engineered nuclease, as described herein) may be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing moiety) that introduces one or more deletions (e.g., of one or more nucleotides) into a target polynucleotide. In some embodiments, the engineered nuclease as described herein (e.g., a nuclease-deficient or nuclease-null engineered nuclease, as described herein) may be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing moiety) that introduces one or moreinsertions (e.g., of one or more nucleotides) into a target polynucleotide. In some embodiments, the engineered nuclease as described herein (e.g., a nuclease-deficient or nuclease-null engineered nuclease, as described herein) may be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing moiety) that introduces one or more inversions (e.g., of two or more nucleotides) in a target polynucleotide. In some embodiments, the engineered nuclease as described herein (e.g., a nuclease-deficient or nuclease-null engineered nuclease, as described herein) may be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing moiety) that introduces one or more translocations (e.g., of one or more nucleotides) in a target polynucleotide. In some embodiments, the engineered nuclease as described herein (e.g., a nuclease-deficient or nuclease-null engineered nuclease, as described herein) may be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing moiety) that introduces one or more transpositions in a target polynucleotide, e.g., one or more DNA transpositions in a target gene.

[0219] In some embodiments, the gene editing moiety may be a base-editing protein or a base-editing enzyme. In some embodiments, the base-editing protein or base-editing enzyme is a deaminase. In some cases, the deaminase is a cytidine deaminase. In some cases, the cytidine deaminase catalyzes the reaction of a cytosine (C) to a uracil (U), which has the base-pairing properties of thymine. In some embodiments, for example where the polynucleotide is double- stranded (e.g., double-stranded DNA), the uridine base can then be substituted with a thymidine base (e.g., by cellular repair machinery) to give rise to a C•G to a T•A transition. In some embodiments, the deaminase is an adenine deaminase. In some cases, the adenine deaminase catalyzes the reaction of an adenosine (A) to an inosine (I). Non-limiting examples of deaminases suitable for use herein include, without limitation, APOBEC l deaminase, APOBEC2 deaminase, APOBEC3 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3E deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, activation-induced cytidine deaminase (AID), adenosine deaminase 1 (ADAR1), adenosine deaminase 2 (ADAR2), adenosine deaminase 3 (ADAR3), or TadA.

[0220] In some embodiments, the engineered nucleases described herein may be used for prime editing. For example, an engineered nuclease as described herein may be coupled to a reverse transcriptase enzyme (e.g., an engineered M-MLV reverse transcriptase) and a prime editing RNA (pegRNA). In such cases, the engineered nuclease may comprise nickase activity.In some embodiments, prime editing may be used to mediate targeted insertions, deletions, or base- to-base conversions.

[0221] In some embodiments, the engineered nucleases described herein may be used for gene writing.

[0222] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that methylates a target substrate. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that methylates a target substrate is a methyltransferase. In some cases, the methyltransferase is a DNA methyltransferase, a histone methyltransferase, or an RNA methyltransferase. In some cases, the DNA methyltransferase is DNMT1 or DNMT3.

[0223] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has demethylase activity (e.g., can remove methyl groups from nucleic acids, proteins, or other molecules). In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has demethylase activity is a histone lysine demethylase, such as, but not limited to KDM1, KDM2, KDM3, KDM4, KDM5, and KDM6.

[0224] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has dismutase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has dismutase activity is superoxide dismutase, formaldehyde dismutase, or chlorite dismutase.

[0225] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has alkylation activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has alkylation activity is a prenyltransferase, a terpene cyclase, or a terpene synthase.

[0226] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has depurination activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has depurination activity is DNA glycosylase.

[0227] In some embodiments, the engineered nucleases described herein may becoupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has oxidation activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has oxidation activity is a peroxidase or an oxidase.

[0228] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has pyrimidine dimer forming activity.

[0229] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has integrase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has integrase activity is retroviral integrase or HIV integrase.

[0230] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has transposase activity. In some embodiments, the partner is a transposase (or functional domain or functional fragment thereof). In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has transposase activity is ty1, Mariner transposase, Tn3, transposase (Tnp) Tn5, or Tn7 transposon.

[0231] In some embodiments, the engineered nucleases described herein may be coupled to a partner e.g., a protein, or functional domain or functional fragment thereof) that has recombinase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has recombinase activity is tyrosine recombinase, Rad51 recombinase, RecA recombinase, or Dmc1 recombinase.

[0232] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has polymerase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has polymerase activity is DNA polymerase, RNA polymerase, reverse transcriptase, or RdRp replicase.

[0233] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has ligase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has ligase activity is ubiquitin ligases, glutamate–cysteine ligase, aminoacyl tRNA synthetase, succinyl coenzyme A synthetase, acetyl-CoA synthetase, pyruvate carboxylase,acetyl-CoA carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, DNA ligase, magnesium chelatase, cobalt chelatase, or DNA synthetase.

[0234] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has helicase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has helicase activity is a DNA helicase, an RNA helicase, chromodomain helicase, or DEAD box / DEAD / DEAH box helicase.

[0235] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has photolyase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has photolyase activity is photoreactivating enzyme, DNA photolyase, DNA-photoreactivating enzyme, DNA cyclobutane dipyrimidine photolyase, DNA photolyase, deoxyribonucleic photolyase, deoxyribodipyrimidine photolyase, photolyase, PRE, PhrB photolyase, deoxyribonucleic cyclobutane dipyrimidine photolyase, phr A photolyase, dipyrimidine photolyase (photosensitive), or deoxyribonucleate pyrimidine dimer lyase (photosensitive).

[0236] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has glycosylase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has glycosylase activity is N-methylpurine DNA glycosylase, UNG, hOGG1, hNTH1, hNEIL1, hMYH, hSMUG1, TDG, MBD4, Mag1, Ung1, Ogg1, Ntg1, AlkE, Ntg2, hNEIL2, hNEIL3, AlkC, AlkD, MutY, Nei, Nth, Fpg, or UDG.

[0237] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has acetyltransferase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has acetyltransferase activity is CBP histone acetyltransferase, choline acetyltransferase, chloramphenicol acetyltransferase, serotonin N-acetyltransferase, NatA Acetyltransferase, or NatB acetyltransferase.

[0238] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has deacetylase activity. In some cases, the partner (e.g., a protein, or functional domain or functionalfragment thereof) that has deacetylase activity is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC- 5, HDAC-6, HDAC-7, or HDAC- 8.

[0239] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has kinase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has kinase activity is Ca2+ / calmodulin-dependent protein kinase, cyclin- dependent kinase, nucleoside-diphosphate kinase, a phosphatidylinositol phosphate kinase, thymidine kinase, thymidylate kinase, or wall-associated kinase.

[0240] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has phosphatase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has phosphatase activity is acid phosphatase, alkaline phosphatase, endonuclease / exonuclease / phosphatase family, kinase, phosphatome, phosphotransferase, protein phosphatase, or protein phosphatase 2.

[0241] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has ubiquitin ligase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has ubiquitin ligase activity is E3A, mdm2, Anaphase-promoting complex (APC), UBR5 (EDD1), SOCS / BC-box / eloBC / CUL5 / RING, LNXp80, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HERC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, VHL, WWP1, WWP2, Parkin, or MKRN1.

[0242] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has deubiquitinating activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has deubiquitinating activity is a deubiquitinating peptidase, a deubiquitinating isopeptidase, a deubiquitinase, a ubiquitin protease, a ubiquitin hydrolase, or a ubiquitin isopeptidase.

[0243] In some embodiments, the engineered nucleases described herein may becoupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has adenylation activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has adenylation activity is carboxylic acid reductase.

[0244] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has deadenylation activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has deadenylation activity is 5'-deadenylase, CNOT6 deadenylase, CNOT6L deadenylase, or CCR4-NOT deadenylase,

[0245] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has SUMOylating activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has SUMOylating activity is small ubiquitin-related modifier (SUMO-1), SUMO-2, or SUMO-3.

[0246] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has deSUMOylating activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has deSUMOylating activity is SENP1, SENP2, SENP3, or SENP5.

[0247] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has ribosylation activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has ribosylation activity is a mono(ADP-ribosyl)transferase, a poly(ADP- ribose)polymerase, or histone ribosylase.

[0248] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has deribosylation activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has deribosylation activity is histone lysine deribosylase or ADP- ribose deribosylase.

[0249] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has myristoylation activity. In some cases, the partner (e.g., a protein, or functional domain orfunctional fragment thereof) that has myristoylation activity is N-myristoltransferase (NMT) 1, N- myristoltransferase (NMT) 2, or glycylpeptide N-tetradecanoyltransferase.

[0250] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has remodeling activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has remodeling activity is a histone acetyltransferase (HAT), a deacetylase, or a methyltransferase.

[0251] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has protease activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has protease activity is trypsin, chymotrypsin, elastase, papain, bromelain, a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamic protease, a metalloprotease, or an asparagine peptide lyase.

[0252] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has oxidoreductase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has oxidoreductase activity is donor dehydrogenase, peroxidase, reductase, dehydrogenase, oxidase, oxygenase, hydroxylase, luciferase, DMSO reductase, glucose oxidase, L-gulonolactone oxidase, thiamine oxidase, xanthine oxidase, acetaldehyde dehydrogenase, pyruvate dehydrogenase, oxoglutarate dehydrogenase, monoamine oxidase, biliverdin reductase, dihydrofolate reductase, methylenetetrahydrofolate reductase, sarcosine oxidase, or dihydrobenzophenanthridine oxidase.

[0253] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has transferase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has transferase activity is coenzyme A transferase, acyl transferase, peptidyl transferase, N-acetyltransferase, or pyruvate dehydrogenase.

[0254] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has hydrolase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has hydrolase activity is an esterase, a protease, a glycosidase, or a lipase.

[0255] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has lyase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has lyase activity is phenylalanine ammonia-lyase, citrate lyase, isocitrate lyase, hydroxynitrile, pectate lyase, argininosuccinate lyase, pyruvate formate lyase, alginate lyase, or pectin lyase.

[0256] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has isomerase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has isomerase activity is ribose phosphate isomerase, bisphosphoglycerate mutase, or photoisomerase.

[0257] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has synthase activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has synthase activity is ATP synthase, citrate synthase, tryptophan synthase, pseudouridine synthase, or fatty acid synthase.

[0258] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has demyristoylation activity. In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has demyristoylation activity is T3SS effector protein.

[0259] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) that has transposition activity. In some embodiments, the partner is a transposase (or functional domain or functional fragment thereof). In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) that has transposition activity is transposase Tn5 or Sleeping Beauty transposase.

[0260] In some embodiments, the engineered nucleases described herein may be coupled to a partner (e.g., a protein, or functional domain or functional fragment thereof) for visualization and localization of a target (e.g., a target protein, a target sequence etc.). In some cases, the partner (e.g., a protein, or functional domain or functional fragment thereof) for visualization and localization of a target is a gold nanoparticle, a fluorophore, fluorescent proteinssuch as green fluorescent proteins (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP).

[0261] The engineered nuclease as disclosed herein can be provided in any form. For example, the engineered nuclease can be provided in the form of a protein, such as the engineered nuclease alone or complexed with a guide nucleic acid as a ribonucleoprotein. The engineered nuclease can be provided in a complex, for example, complexed with a guide nucleic acid and / or one or more heterologous gene effectors of the disclosure. The engineered nuclease can be provided in the form of a nucleic acid encoding at least the engineered nuclease, such as an RNA (e.g., messenger RNA (mRNA)), or DNA. The nucleic acid encoding at least the engineered nuclease can be codon optimized for efficient translation into protein in a particular cell or organism (e.g., human codon optimized).

[0262] Nucleic acids encoding at least the engineered nuclease as disclosed herein, fragments, or derivatives thereof can be stably integrated in the genome of a cell. Nucleic acids encoding at least the engineered nuclease can be operably linked to a promoter, for example, a promoter that is constitutively or inducibly active in the cell. Nucleic acids encoding at least the engineered nuclease can be operably linked to a promoter in an expression construct. Expression constructs can include any nucleic acid constructs capable of directing expression of a gene or other nucleic acid sequence of interest (e.g., at least the engineered nuclease) and which can transfer such a nucleic acid sequence of interest to a target cell.

[0263] In some embodiments, the engineered nuclease as disclosed herein can associate with a single guide RNA (sgRNA) to activate or repress transcription of a target gene (e.g., target endogenous gene), for example, in combination with heterologous gene effector(s) disclosed herein. sgRNAs can be introduced into cells expressing the engineered nuclease or variant thereof, as provided herein. In some cases, such cells can contain one or more different sgRNAs that target the same target gene (e.g., target endogenous gene) or target gene regulatory sequence. In other cases, the sgRNAs target different nucleic acids in the cell (e.g., different target genes, different target gene regulatory sequences, or different sequences within the same target gene or target gene regulatory sequence).

[0264] Enzymatically inactive (e.g., nuclease deficient) can refer to a nuclease that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner but may not cleave a target polynucleotide or will cleave it at a substantially reduced frequency. Anenzymatically inactive guide moiety can comprise an enzymatically inactive domain (e.g., nuclease domain). Enzymatically inactive can refer to no activity. Enzymatically inactive can refer to substantially no activity. Enzymatically inactive can refer to essentially no activity. Enzymatically inactive can refer to an activity no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, or no more than 10% activity compared to a comparable wild-type activity (e.g., nucleic acid cleaving activity, wild-type Cas activity).

[0265] In some embodiments, the target nucleic acid of the engineered nuclease as disclosed herein can be dsDNA. In such embodiments, dsDNA-targeting specificity is determined, at least in part, by two parameters: the gRNA spacer targeting a protospacer in the target dsDNA (the sequence in the target dsDNA corresponding to the gRNA spacer on the non-complementary DNA strand) and a short sequence, the protospacer-adjacent motif (PAM), located immediately 5' (upstream) of the protospacer on the non-complementary DNA strand. In some embodiments, the PAM is 5'-TTTG-3', 5'-TTTA-3', or 5'-TTTR-3'. In some embodiments, the PAM is 5'-TTTG-3'. In some embodiments, the PAM is 5'-TTTA-3'. In some embodiments, the PAM is 5'-TTTR-3'. In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from the group consisting of 5’-CCCC-3', 5’-TTAT-3', 5’-GTCC-3', 5’-TTTC-3', 5’-TCAT-3', 5’- ACCC-3', 5’-TCTG-3', 5’-TTAG-3', 5’-GTTA-3', 5’-ACTC-3', 5’-GCCA-3', 5’-TTTT-3', 5’- TTGG-3', 5’-GTTG-3', 5’-ATTG-3', 5’-TTAA-3', 5’-CTTA-3', 5’-TTGA-3', 5’-GCTC-3', 5’- GCCC-3', 5’-TTCG-3', 5’-CTTG-3', 5’-TTCA-3', and 5’-TCTA-3'. In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from the group consisting of 5’- NCCCC-3', 5’-NTTAT-3', 5’-NGTCC-3', 5’-NTTTC-3', 5’-NTCAT-3', 5’-NACCC-3', 5’- NTCTG-3', 5’-NTTAG-3', 5’-NGTTA-3', 5’-NACTC-3', 5’-NGCCA-3', 5’-NTTTT-3', 5’- NTTGG-3', 5’-NGTTG-3', 5’-NATTG-3', 5’-NTTAA-3', 5’-NCTTA-3', 5’-NTTGA-3', 5’- NGCTC-3', 5’-NGCCC-3', 5’-NTTCG-3', 5’-NCTTG-3', 5’-NTTCA-3', and 5’-NTCTA-3', wherein “N” can be any nucleotide (A, T, C, or G). In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from the group consisting of 5’-NNCCCC-3', 5’- NNTTAT-3', 5’-NNGTCC-3', 5’-NNTTTC-3', 5’-NNTCAT-3', 5’-NNACCC-3', 5’-NNTCTG-3', 5’-NNTTAG-3', 5’-NNGTTA-3', 5’-NNACTC-3', 5’-NNGCCA-3', 5’-NNTTTT-3', 5’- NNTTGG-3', 5’-NNGTTG-3', 5’-NNATTG-3', 5’-NNTTAA-3', 5’-NNCTTA-3', 5’-NNTTGA-3', 5’-NNGCTC-3', 5’-NNGCCC-3', 5’-NNTTCG-3', 5’-NNCTTG-3', 5’-NNTTCA-3', and 5’-NNTCTA-3', wherein “N” can be any nucleotide (A, T, C, or G). In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from TABLE 11.

[0266] In some embodiments, the target nucleic acid of the engineered nuclease as disclosed herein can be RNA. In such embodiments, RNA-targeting specificity is determined, at least in part, by the gRNA spacer targeting a protospacer-like sequence in the target RNA (the sequence in the target RNA complementary to the gRNA spacer), and is independent of the sequence located immediately 5' (upstream) of the protospacer-like sequence. In some embodiments, the engineered nuclease can be further capable of targeting a dsDNA molecule, wherein the gRNA spacer is selected such that it targets a protospacer in the target dsDNA molecule having a PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTR-3'. In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from the group consisting of 5’-CCCC-3', 5’-TTAT-3', 5’-GTCC-3', 5’-TTTC-3', 5’-TCAT-3', 5’-ACCC- 3', 5’-TCTG-3', 5’-TTAG-3', 5’-GTTA-3', 5’-ACTC-3', 5’-GCCA-3', 5’-TTTT-3', 5’-TTGG-3', 5’-GTTG-3', 5’-ATTG-3', 5’-TTAA-3', 5’-CTTA-3', 5’-TTGA-3', 5’-GCTC-3', 5’-GCCC-3', 5’- TTCG-3', 5’-CTTG-3', 5’-TTCA-3', and 5’-TCTA-3'. In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from the group consisting of 5’-NCCCC-3', 5’- NTTAT-3', 5’-NGTCC-3', 5’-NTTTC-3', 5’-NTCAT-3', 5’-NACCC-3', 5’-NTCTG-3', 5’- NTTAG-3', 5’-NGTTA-3', 5’-NACTC-3', 5’-NGCCA-3', 5’-NTTTT-3', 5’-NTTGG-3', 5’- NGTTG-3', 5’-NATTG-3', 5’-NTTAA-3', 5’-NCTTA-3', 5’-NTTGA-3', 5’-NGCTC-3', 5’- NGCCC-3', 5’-NTTCG-3', 5’-NCTTG-3', 5’-NTTCA-3', and 5’-NTCTA-3', wherein “N” can be any nucleotide (A, T, C, or G). In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from the group consisting of 5’-NNCCCC-3', 5’-NNTTAT-3', 5’- NNGTCC-3', 5’-NNTTTC-3', 5’-NNTCAT-3', 5’-NNACCC-3', 5’-NNTCTG-3', 5’-NNTTAG-3', 5’-NNGTTA-3', 5’-NNACTC-3', 5’-NNGCCA-3', 5’-NNTTTT-3', 5’-NNTTGG-3', 5’- NNGTTG-3', 5’-NNATTG-3', 5’-NNTTAA-3', 5’-NNCTTA-3', 5’-NNTTGA-3', 5’-NNGCTC-3', 5’-NNGCCC-3', 5’-NNTTCG-3', 5’-NNCTTG-3', 5’-NNTTCA-3', and 5’-NNTCTA-3', wherein “N” can be any nucleotide (A, T, C, or G). In some embodiments, the PAM sequence can be one or more nucleic acid sequence selected from TABLE 11.

[0267] In some embodiments, the heterologous polypeptide comprising the engineered nuclease (e.g., and / or a complex comprising the heterologous polypeptide) can regulate expression and / or a complex comprising the heterologous polypeptide) can regulate expression and / or activityof a target gene (e.g., target endogenous gene). In some embodiments, the heterologous polypeptide and / or a complex thereof can edit the sequence of a nucleic acid (e.g., a gene and / or gene product). A nuclease-active variant of the engineered nuclease can edit a nucleic acid sequence by generating a double-stranded break or single-stranded break in a target polynucleotide.

[0268] In some embodiments, the heterologous polypeptide comprising the engineered nuclease (e.g., and / or a complex comprising the heterologous polypeptide) can generate a double- strand break in a target polynucleotide, such as DNA. A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). In some embodiments, a nuclease induces site-specific single-strand DNA breaks or nicks, thus resulting in HDR.

[0269] A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide that contains homology arms flanking sites of the target DNA can be provided.

[0270] In some embodiments, the heterologous polypeptide comprising the engineered nuclease (e.g., and / or a complex comprising the heterologous polypeptide) does not generate a double-strand break in a target polynucleotide, such as DNA. Binding of the heterologous polypeptide or the complex comprising the heterologous polypeptide (e.g., a complex comprising a nuclease deficient variant of the engineered nuclease and a guide RNA) without a nucleic acid break can be sufficient to regulate expression (e.g., enhance or suppress) of a target gene (e.g., endogenous target gene). TARGET GENE

[0271] The disclosure provides compositions, methods, and systems for modulating expression of one or more target genes. The target gene(s) can be one or more endogenous target genes, such as (i) a disease-causing allele, e.g., a mutant allele, and / or (ii) a non-disease-causing allele, e.g., a wild type allele. For example, disclosed herein are one or more complexes that comprise a guide moiety and one or more heterologous polypeptides comprising the engineered polypeptide (e.g., comprising the engineered nuclease) as disclosed herein that can modulate (e.g., increase or decrease) an activity or expression level of a target gene (e.g., in a cell). Such complexcomprising a guide moiety (e.g., small guide RNA) and an engineered nuclease can affect the modulation of expression of target gene(s) via cleavage of the target gene(s). Alternatively or in addition to, such complex comprise a gene modulator operatively coupled to (e.g., fused to) the engineered nuclease, such that the complex can affect the modulation of expression of target gene(s) without cleaving the target gene(s). In some cases, the gene modulator may affect an increase in expression of the target gene. In some cases, the gene modulator may affect a decrease in expression of the target gene. In some cases, the gene modulator (e.g., a gene editing moiety, as described herein) may affect editing of a target gene, for example, to correct an undesirable mutation in a target gene such that expression of the mutated gene is decreased, and expression of the corrected gene is increased.

[0272] In some embodiments, a target gene or regulatory sequence thereof is endogenous to a cell, for example, present in the cell’s genome, or endogenous to a subject, for example, present in the subject’s genome. In some embodiments, a target gene or regulatory sequence thereof is not part of an engineered reporter system.

[0273] In some embodiments, a target gene is exogenous to a host subject, for example, a pathogen target gene or an exogenous gene expressed as a result of a therapeutic intervention, such as a gene therapy and / or cell therapy. In some embodiments, a target gene is an exogenous reporter gene. In some embodiments, a target gene is an exogenous synthetic gene.

[0274] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the heterologous polypeptide into a cell or population of cells) or a duration thereof. In some embodiments, an expression level is an RNA expression level can be measured by, for example, RNAseq, qPCR, microarray, gene array, FISH, etc. In some embodiments, an expression level is a protein expression level can be measured by, for example, Western Blot, ELISA, multiplex immunoassay, mass spectrometry, NMR, proteomics, flow cytometry, mass cytometry, etc.

[0275] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the heterologous polypeptide into a cell or population of cells) or a duration thereof by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at leastabout 2-fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14, at least fold about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 150 fold, at least about 200 fold, at least about 250 fold, at least about 300 fold, at least about 350 fold, at least about 400 fold, at least about 500 fold, at least about 600 fold, at least about 700 fold, at least about 800 fold, at least about 900 fold, at least about 1000 fold, at least about 1500 fold, at least about 2000 fold, or at least about 3000 fold.

[0276] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the heterologous polypeptide into a cell or population of cells) or a duration thereof by at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 2-fold, at most about 3 fold, at most about 4 fold, at most about 5 fold, at most about 6 fold, at most about 7 fold, at most about 8 fold, at most about 9 fold, at most about 10 fold, at most about 11 fold, at most about 12 fold, at most about 13 fold, at most about 14, at most fold about 15 fold, at most about 20 fold, at most about 30 fold, at most about 40 fold, at most about 50 fold, at most about 60 fold, at most about 70 fold, at most about 80 fold, at most about 90 fold, at most about 100 fold, at most about 150 fold, at most about 200 fold, at most about 250 fold, at most about 300 fold, at most about 350 fold, at most about 400 fold, at most about 500 fold, at most about 600 fold, at most about 700 fold, at most about 800 fold, at most about 900 fold, at most about 1000 fold, at most about 1500 fold, at most about 2000 fold, at most about 3000 fold, at most about 5000 fold, or at most about 10000 fold.

[0277] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the heterologous polypeptide into a cell or population of cells) or a duration thereof by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 2-fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14, about 15 fold, about 20 fold, about 30 fold, about 40 fold, about 50 fold, about 60 fold, about 70 fold, about 80 fold, about 90 fold, about 100 fold, about 150 fold, about 200 fold, about 250fold, about 300 fold, about 350 fold, about 400 fold, about 500 fold, about 600 fold, about 700 fold, about 800 fold, about 900 fold, about 1000 fold, about 1500 fold, about 2000 fold, about 3000 fold, about 5000 fold, or about 10000 fold.

[0278] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the heterologous polypeptide into a cell or population of cells) or a duration thereof from below a limit of detection to a detectable level.

[0279] In some embodiments, the degree in change of expression or duration thereof is relative to before introducing the system of the present disclosure (e.g., a complex comprising the heterologous polypeptide) into the cell or population of cells. In some embodiments, the degree in change of expression or duration thereof is relative to a corresponding control cell or population of cells that are not treated with the system of the present disclosure. In some embodiments, the degree in change of expression or duration thereof is relative to a corresponding control cell or population of cells that are treated with an alternative to the system of the present disclosure.

[0280] In some embodiments, the degree in change of expression or duration thereof is relative to a control nuclease. The control nuclease can comprise a naturally occurring nuclease (e.g., (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2) or any modification thereof (e.g., a variant thereof with reduced nuclease activity and operatively coupled to a gene modulator). For example, the control nuclease can be dCasMINI that is coupled to (e.g., fused to) the same gene modulator, which same gene modulator is coupled to the engineered nuclease as disclosed herein.

[0281] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) an activity level of a target gene (e.g., upon introducing a complex comprising the heterologous polypeptide comprising the engineered nuclease as disclosed herein into a cell or population of cells) or a duration thereof. An activity level can be determined by a suitable functional assay for the target gene in question depending on the functional characteristics of the target gene. For example, an activity level of a target gene that is a mitogen could be determined by measuring cell proliferation; an activity level of a target gene that induces apoptosis could be measured by an annexin V assay or other suitable cell death assay; an activity level of an anti-inflammatory cytokine could be measured by an LPS-induced cytokine release assay.

[0282] The compositions, systems and methods of the present disclosure can, in some cases, elicit changes in expression and / or activity level of a target gene (e.g., target endogenous gene) that persists for longer than can be achieved with alternative compositions and methods (e.g., suppression via RNAi, e.g., using siRNA). In some embodiments, persistent modulation of gene expression is advantageous as compared to transient modulation.

[0283] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression and / or activity level of a target gene for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 18 weeks, at least about 20 weeks, at least about 26 weeks, or at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, or more.

[0284] In some embodiments the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression and / or activity level of a target gene (e.g., target endogenous gene) to above a certain threshold for at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 12 hours, at least or up to about 14 hours, at least or up to about 18 hours, at least or up to about 20 hours, at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 14 days, at least or up to about 21 days, at least or up to about 28 days, at least or up to about 5 weeks, at least or up to about 6 weeks, at least or up to about 7 weeks, at least or up to about 8 weeks, at least or up to about 9 weeks, at least or up toabout 10 weeks, at least or up to about 12 weeks, at least or up to about 14 weeks, at least or up to about 18 weeks, at least or up to about 20 weeks, at least or up to about 26 weeks, or at least or up to about 5 months, at least or up to about 6 months, at least or up to about 9 months, or at least or up to about 12 months.

[0285] In some embodiments, the compositions, systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression and / or activity level of a target gene (e.g., target endogenous gene) to above a certain threshold for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 18 hours, about 20 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 14 days, about 21 days, about 28 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 18 weeks, about 20 weeks, about 26 weeks, about 5 months, about 6 months, about 9 months, or about 12 months.

[0286] In some embodiments, the systems as disclosed herein, upon administration to a subject, can have reduced immunogenicity (e.g., no immunogenicity), as compared to that of a control system that does not comprise the engineered nuclease as disclosed herein. For example, the immunogenicity can be measured subsequent to administration of the system to the subject, to effect modification of the target gene in a cell in the subject. Non-limiting examples of the immunogenicity measurements can include T cell response (e.g., T cell activation), cytokine production (e.g., interleukin (IL)-1, IL-6, IL-8, or IL-17, tumor necrosis factor-alpha (TNF-alpha), or interferon gamma (IFN-Ȗ)), antibody production, cellular immune response (e.g., proliferation and activation of immune cells), and immunoglobulin isotype profiling. In some embodiments, the T cell response of the subject treated by the compositions, systems or methods as provided herein can be reduced by at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95% as compared to that of a control subject treated with a control system that does not comprise the engineered nuclease. In someembodiments, the compositions, systems or methods disclosed herein can result in the absence of T cell response in the subject treated. In some embodiments, the cytokine production (e.g., interleukin (IL)-1, IL-6, IL-8, or IL-17, tumor necrosis factor-alpha (TNF-alpha), or interferon gamma (IFN-Ȗ)) by T cells of the subject treated by the compositions, systems or methods as provided herein can be reduced by at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95% as compared to that of a control subject treated with a control system that does not comprise the engineered nuclease.

[0287] Subsequent to the treatment, the reduced immunogenicity observed in the subject treated by the compositions, systems or methods as provided herein can be sustained for (and / or measured on) at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks.

[0288] In some embodiments, the compositions, systems as disclosed herein, upon administration to a subject, can have reduced toxicity, as compared to that of a control system that does not comprise the engineered nuclease as disclosed herein. For example, the health conditions that correlate with the toxicity in the subject treated can be measured subsequent to administration of the system to the subject. Non-limiting examples of the health conditions that correlate with the toxicity in the subject treated can include food intake (e.g., grams per subject per day), body weight (e.g., grams), body dimension (e.g., length, height, or circumference in meters), body mass index (e.g., grams per centimeter squared), body fat (e.g., milligrams of fat per grams of body weight), organ weight (e.g., grams of liver, kidney, spleen, small intestine, etc. per grams of body weight), clinical chemistry, tissue function or integrity (e.g., structure as ascertained by biopsy and / or histology), body injury, distress, grooming, etc. Clinical chemistry can be ascertained bymeasuring one or more markers from a bodily fluid (e.g., blood), and non-limiting examples of the marker(s) for clinical chemistry can include electrolytes (e.g., sodium, potassium, chloride, bicarbonate), kidney markers (e.g., creatinine, blood urea nitrogen), liver function markers (e.g., albumin, globulin, albumin / globulin ratio, bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP)), cardiac markers (e.g., H-FABP, troponin, myoglobin, CK-MB, B-type natriuretic peptide (BNP)), minerals (e.g., calcium, magnesium, phosphate, potassium), blood disorder markers (e.g., iron, transferrin, TIBC, vitamin B12, vitamin D, folic acid), and others (e.g., glucose, C-reactive protein, glycated hemoglobin, uric acid, arterial blood gases, adrenocorticotropic hormone (ACTH), neuron-specific enolase (NSE), fecal occult blood test (FOBT), etc.). For example, a panel of clinical chemistry markers can be used, including one or more members consisting of sodium, potassium, chloride, bicarbonate, blood urea nitrogen (BUN), creatinine, glucose, calcium, total protein, albumin, alkaline phosphatase (ALP), alanine amino transferase (ALT), aspartate amino transferase (AST), and bilirubin. In some embodiments, the toxicity of the subject treated by the compositions, systems or methods as provided herein can be reduced by at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95% as compared to that of a control subject treated with a control system that does not comprise the engineered nuclease. Subsequent to the treatment, the reduced toxicity can be sustained for (and / or measured on) at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks.

[0289] In some embodiments, the engineered polypeptide as disclosed herein (e.g., an engineered nuclease operatively coupled to a gene modulator) can be capable of or can affectenhanced modulation of a target gene, as compared to modulation of the target gene by a control polypeptide (e.g., a control nuclease operatively coupled to the same gene modulator). The control nuclease can be a naturally occurring nuclease (e.g., (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from TABLE 2) or any modification thereof (e.g., dCasMINI as disclosed herein).

[0290] In some cases, the enhanced modulation of the target gene can be characterized by a change in expression level of the target gene that is greater than that by the control polypeptide. In some examples, such change can be increased expression level of the target gene. The increased expression level of the target gene by the engineered polypeptide as disclosed herein can be greater than that by the control polypeptide, by at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 110%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%. The increased expression level of the target gene by the engineered polypeptide as disclosed herein can be greater than that by the control polypeptide, by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.5-fold, at least or up to about 1- fold, at least or up to about 1.5-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, or at least or up to about 100-fold. In some examples, such change can be decreased (or reduced) expression level of the target gene. The decreased expression level of the target gene by the engineered polypeptide as disclosed herein can be less than that by the control polypeptide, by at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at leastor up to about 110%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%. The decreased (or reduced) expression level of the target gene by the engineered polypeptide as disclosed herein can be less than that by the control polypeptide, by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.5-fold, at least or up to about 1-fold, at least or up to about 1.5-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, or at least or up to about 100-fold.

[0291] In some cases, the enhanced modulation of the target gene can be characterized by a prolonged change in expression level of the target gene (e.g., increased expression or decreased expression above certain threshold as disclosed herein) that is longer than that by the control polypeptide. The prolonged change in the expression level of the target gene by the engineered polypeptide as disclosed herein can be longer than that by the control polypeptide, by at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 110%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%. The prolonged change in the expression level of the target gene by the engineered polypeptide as disclosed herein can be longer than that by the control polypeptide, by at least or up to about 0.1-fold, at least or up to about 0.2- fold, at least or up to about 0.5-fold, at least or up to about 1-fold, at least or up to about 1.5-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40- fold, or at least or up to about 100-fold. For example, the threshold level can be relative to (i) expression level of the target gene prior to the enhanced modulation, (ii) the greatest increase inthe expression level of the target gene for activating the target gene or a portion thereof (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% thereof), or (iii) the greatest decrease in the expression level of the target gene for repressing the target gene or a portion thereof (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% thereof). In some embodiments, the target gene (e.g., endogenous target gene) can be a disease-causing allele, such as a mutant variant of a wild type allele. The disease can be a genetic disease, such as a hereditary disorder. Non-limiting examples of the genetic disorder can include X-linked severe combined immune deficiency, thalassemia, Duchenne muscular dystrophy (DMD), hemophilia, cystic fibrosis, Huntington's chorea, age-related muscular degeneration, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch Nyhan syndrome, sickle cell anemia, thalassaemias, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, schizophrenia, trinucleotide repeat disorders, fragile X syndrome, prion-related disorders...

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: (a) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease; and (b) a non-naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA, wherein the non-naturally occurring guide RNA comprises: (i) a region that binds to the programmable nuclease; and (ii) a guide sequence that binds to a target sequence of a target nucleic acid adjacent to a protospacer-adjacent motif (PAM) sequence (or a non-canonical PAM sequence) that is different than a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G.

2. A composition comprising: (a) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease; and (b) a non-naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA, wherein the non-naturally occurring guide RNA comprises a spacer sequence that is at least partially complementary to a nucleotide sequence within one or more target genes or a regulatory region thereof, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, and wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes or the regulatory region thereof.

3. A composition comprising: (a) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease; and(b) a non-naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA, wherein the non-naturally occurring guide RNA comprises a spacer sequence that is at least partially complementary to a nucleotide sequence within a target allele or a regulatory region thereof comprising a single nucleotide polymorphism (SNP), wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, wherein the seed sequence binds to a corresponding nucleotide sequence of the nucleotide sequence within the target allele or regulatory region thereof, wherein the corresponding sequence comprises the SNP, and wherein the seed sequence is 100% complementary to the corresponding nucleotide sequence.

4. The composition of claim 3, wherein the SNP is a disease-causing SNP.

5. The composition of any one of claims 2-4, wherein the spacer sequence comprises additional nucleotides at the 3’-end of the seed sequence, optionally wherein the additional nucleotides are 1-20 additional nucleotides.

6. The composition of claim 5, wherein the additional nucleotides form a nucleotide sequence that is partially or 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory region thereof.

7. A nuclease system comprising: (i) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease, and (ii) a non-naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA exhibiting specific binding to a target polynucleotide of a target gene, wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced immunogenicity compared to that of a control system that does not comprise the programmable nuclease.

8. The nuclease system of claim 7, wherein the reduced immunogenicity comprises a decrease in or absence of T cell activation.

9. The nuclease system of claim 7, wherein the reduced immunogenicity comprises a decreasein or absence of production of one or more inflammatory cytokines by T cells.

10. The nuclease system of claim 9, wherein the T cells comprise cytotoxic T cells, regulatory T cells, T-helper cells, gamma-delta T cells, memory T cells, CD3+ T cells, CD4+ T cells, or CD8+ T cells.

11. The nuclease system of claim 9, wherein the one or more inflammatory cytokines comprise interleukin (IL)-1, IL-6, IL-8, or IL-17, tumor necrosis factor-alpha (TNF-alpha), or interferon gamma (IFN-Ȗ).

12. The nuclease system of claim 9, wherein the one or more inflammatory cytokines are IFN- Ȗ.

13. The nuclease system of claim 9, wherein the decrease in production of one or more inflammatory cytokines is characterized by a reduced IFN-Ȗ production by the T cells, wherein IFN-Ȗ level is reduced by at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90, at least about 95% or 100% as compared to that of the control system.

14. The nuclease system of claim 9, wherein the production of one or more inflammatory cytokines is at least about 80%, at least about 90, at least about 95%, or at least about 100% as compared to that of a control subject that is not administered with the nuclease system.

15. A nuclease system comprising: (i) a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the programmable nuclease, and (ii) a non-naturally occurring guide RNA, or a nucleic acid encoding the non-naturally occurring guide RNA exhibiting specific binding to a target polynucleotide of a target gene, wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced toxicity compared to that of a control system that does not comprise the programmable nuclease.

16. The nuclease system of claim 15, wherein the reduced toxicity is assessed by measuring liver function, blood counts, hemoglobin counts, hematocrit counts, or white blood differentiation.

17. The nuclease system of claim 15, wherein the toxicity of the subjected treated with the nuclease system is reduced by at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at leastabout 90, at least about 95% or 100% as compared to that of the control system.

18. The nuclease system of any one of claims 15-17, wherein the reduced toxicity is sustained for at least about 1 week, at least about 4 weeks, at least about 12 weeks, at least about 24 weeks, or at least about 48 weeks.

19. The nuclease system of any one of claims 15-18, wherein the nuclease system is administered with a pharmaceutically acceptable excipient.

20. The nuclease system of claim 19, wherein the excipient is selected from a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids, liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplex, peptide, protein, cell, hyaluronidase, and mixtures thereof.

21. The nuclease system of any one of claims 15-20, wherein the nuclease system is formulated.

22. The nuclease system of any one of claims 15-21, wherein the nuclease system is administered by an injection.

23. The nuclease system of claim 22, wherein the injection is an intradermal injection, a subcutaneous injection, an intramuscular injection, or an intravenous injection.

24. The nuclease system of any one of claims 15-23, wherein the non-naturally occurring guide RNA comprises a spacer sequence that is at least partially complementary to a nucleotide sequence within one or more target genes or a regulatory region thereof, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, and wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes or the regulatory region thereof.

25. The composition of any one of claims 2-6 or the nuclease system of claim 24, wherein the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer-adjacent motif (PAM) sequence.

26. The composition or the nuclease system of claim 25, wherein the PAM sequence is a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G.

27. The composition or the nuclease system of claim 25, wherein the PAM sequence is a non- canonical PAM sequence.

28. The composition or the nuclease system of claim 27, wherein the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG.

29. The composition or the nuclease system of claim 27, wherein the PAM sequence (or the non-canonical PAM sequence) comprises or is one or more nucleic acid sequence selected from Table 11.

30. The composition or nuclease system of any one of the preceding claims, wherein the programmable nuclease is a Cas nuclease.

31. The composition or nuclease system of any one of the preceding claims, wherein the programmable nuclease comprises or consists of an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

1.

32. A guide RNA comprising: (i) a region that binds to a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1; and (ii) a guide sequence that binds to a target sequence of a target nucleic acid adjacent to a protospacer-adjacent motif (PAM) sequence (or a non-canonical PAM sequence) that is different than a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G.

33. A guide RNA comprising: a region that binds to a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1; and a guide sequence that binds to a target sequence of a target gene, wherein the guide sequence comprises a spacer sequence that is at least partially complementary to the target sequence, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, and wherein the seed sequence is 100% complementary to a corresponding nucleotide sequenceof the target sequence.

34. A guide RNA comprising: a region that binds to a programmable nuclease comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1; and a guide sequence that binds to a target sequence of a target allele comprising a single nucleotide polymorphism (SNP), wherein the guide sequence comprises a spacer sequence that is at least partially complementary to the target sequence, wherein the spacer sequence comprises a seed sequence from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’-end of the spacer sequence, wherein the seed sequence is 100% complementary to a corresponding nucleotide sequence of the target sequence, and wherein the corresponding nucleotide sequence comprises the SNP.

35. The guide RNA of claim 34, wherein the SNP is a disease-causing SNP.

36. The guide RNA of any one of claims 33-35, wherein the spacer sequence comprises additional nucleotides at the 3’-end of the seed sequence, optionally wherein the additional nucleotides are 1-20 additional nucleotides.

37. The guide RNA of claim 36, wherein the additional nucleotides form a nucleotide sequence that is partially or 100% complementary to a corresponding nucleotide sequence of the nucleotide sequence within the one or more target genes, the target allele, or regulatory region thereof.

38. The guide RNA of any one of claims 33-37, wherein the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof is adjacent to a protospacer- adjacent motif (PAM) sequence.

39. The guide RNA of claim 38, wherein the PAM sequence is a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G.

40. The guide RNA of claim 38, wherein the PAM sequence is a non-canonical PAM sequence.

41. The guide RNA of claim 40, wherein the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of: CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG.

42. The guide RNA of claim 40, wherein the PAM sequence (or the non-canonical PAMsequence) comprises or is one or more nucleic acid sequence selected from Table 11.

43. The guide RNA of any one of claims 32-42, wherein the region that binds to the programmable nuclease comprises or consists of a polynucleotide sequence having at least about 74% sequence identity to the polynucleotide sequence of SEQ ID NO:

597.

44. The guide RNA of any one of claims 32-43, wherein said scaffold sequence has a length of at most about 110 nucleotides.

45. The guide RNA of any one of claims 32-44, wherein said scaffold sequence has a length of at most about 105 nucleotides.

46. A nucleic acid encoding the guide RNA of any one of claims 32-45.

47. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease is a Cas nuclease.

48. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease comprises or consists of an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

1.

49. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 700-848.

50. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 12 or 112-201.

51. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the amino acid sequence comprises at least one deletion from amino acid residues 2-100, as compared to the amino acid sequence of SEQ ID NO:

1.

52. The composition, nuclease system or guide RNA of claim 51, wherein the at least one deletion comprises at least one deletion from amino acid residues 31-40, 41-60, and / or 61-80 as compared to the amino acid sequence of SEQ ID NO:

1.

53. The composition, nuclease system or guide RNA of claim 51 or 52, wherein the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO:

1.

54. The composition, nuclease system or guide RNA of any one of the preceding claims,wherein the programmable nuclease has a length of about 350 amino acids to about 900 amino acids.

55. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease has a length of at most about 528 amino acids.

56. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease comprises an amino acid mutation which results in reduced nuclease activity.

57. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease is nuclease inactive.

58. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO:

1.

59. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO:

1.

60. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:

11.

61. The composition, nuclease system or guide RNA of any one of the preceding claims, wherein the programmable nuclease is coupled or fused to a gene modulator or a gene editing moiety, optionally wherein the gene editing moiety has transposase activity, or is a transposase or a functional domain or functional fragment thereof.

62. A vector comprising the guide RNA of any one of claims 32-45 or the nucleic acid of claim 46.

63. The vector of claim 62, wherein the vector is a particle or a viral vector.

64. The vector of claim 63, wherein the particle is a lipid nanoparticle (LNP).

65. The vector of claim 63, wherein the viral vector is an adeno-associated viral (AAV) vector.

66. The vector of any one of claims 62-65, further comprising the programmable nuclease, or a nucleic acid sequence encoding the programmable nuclease.

67. A system comprising the composition of any one of claims 1-6, 25-31, or 47-61, whereinthe programmable nuclease is in a complex with the non-naturally occurring guide RNA.

68. A vector comprising the composition of any one of claims 1-6, 25-31, or 47-61, the nuclease system of any one of claims 7-31 or 47-61, or the system of claim 67.

69. The vector of claim 68, wherein the vector is a particle or a viral vector.

70. The vector of claim 69, wherein the particle is a lipid nanoparticle (LNP).

71. The vector of claim 69, wherein the viral vector is an adeno-associated viral (AAV) vector.

72. A method of modulating one or more target genes in a cell, the method comprising contacting the cell with the composition of any one of claims 1, 28-31, or 47-61, the system of claim 67, or the vector of any one of claims 68-71, wherein the programmable nuclease and the guide RNA form a complex and modulate the one or more target genes in the cell.

73. A method of modulating one or more target genes or a target allele in a cell, the method comprising: contacting the cell with the composition of any one of claims 2-6, 25-31, or 47-61, the system of claim 67, or the vector of any one of claims 62-66 or 68-71, wherein the programmable nuclease and the non-naturally occurring guide RNA form a complex and modulate the one or more target genes or the target allele in the cell.

74. A method of modulating one or more target genes or a target allele in a cell, the method comprising: contacting the cell with the nuclease system of any one of claims 7-31 or 47-61 or the vector of any one of claims 68-71, wherein the programmable nuclease and the non-naturally occurring guide RNA form a complex and modulate one or more target genes or the target allele in the cell associated with the one or more disease conditions, wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced immunogenicity compared to that of a control system that does not comprise the programmable nuclease, and wherein the nuclease system, upon administering to a subject, is configured to exhibit reduced toxicity compared to that of a control system that does not comprise the programmable nuclease.

75. The method of claim 73 or 74, wherein the method results in enhanced binding of the programmable nuclease to the nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof, relative to a method that uses a guide RNA with a seed sequence that is partially complementary to a corresponding nucleotide sequence within the oneor more target genes, the target allele, or regulatory regions thereof.

76. The method of any one of claims 73-75, wherein the method results in enhanced modulation of the one or more target genes or the target allele relative to a method that uses a guide RNA with a seed sequence that is partially complementary to a corresponding nucleotide sequence within the one or more target genes, the target allele, or regulatory regions thereof.

77. The method of any one of claims 72-76, wherein the cell is a eukaryotic cell.

78. The method of claim 77, wherein the eukaryotic cell is a mammalian cell.

79. The method of claim 78, wherein the mammalian cell is a human cell.

80. A method of preventing or treating a genetic disorder in a subject, the method comprising administering to the subject the composition of any one of claims 1, 28-31, or 47-61, the system of claim 67, or the vector of any one of claims 68-71, wherein the programmable nuclease and the guide RNA form a complex and modulate one or more target gene associated with the genetic disorder.

81. The method of claim 80, wherein the composition, the system, or the vector are administered in an amount sufficient to modulate the one or more target gene associated with the genetic disorder.

82. The method of claim 80 or 81, wherein the subject is human.

83. The method of any one of claims 80-82, wherein the genetic disorder is selected from the group consisting of: X-linked severe combined immune deficiency, sickle cell anemia, thalassemia, hemophilia, neoplasia, cancer, age-related macular degeneration, schizophrenia, trinucleotide repeat disorders, fragile X syndrome, prion-related disorders, amyotrophic lateral sclerosis, drug addiction, autism, Alzheimer’s disease, Parkinson’s disease, cystic fibrosis, blood and coagulation disease or disorders, inflammation, facioscapulohumeral muscular dystrophy, retinitis pigmentosa, Leber congenital amaurosis, glaucoma, immune-related diseases or disorders, metabolic diseases and disorders, liver diseases and disorders, kidney diseases and disorders, muscular / skeletal diseases and disorders, neurological and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and ocular diseases and disorders.

84. A computer-implemented method for identifying guide RNA (gRNA) spacer sequences, the method comprising: (a) identifying an optimal target region of a target gene, wherein the optimal target regioncomprises a target nucleotide sequence that can be bound by a Cas protein-gRNA complex to modulate the target gene; (b) identifying one or more protospacer adjacent motif (PAM) sequences present within the optimal target region, wherein the PAM sequence is specific for the Cas protein; (c) extracting a candidate spacer sequence that is immediately 3’ to the PAM sequence identified in (b); and (d) identifying one or more potential off-target binding sites for the candidate spacer sequence based on a pre-defined rule; and (e) generating an association between a candidate spacer sequence extracted in (c) and the one or more potential off-target binding sites identified in (d).

85. The computer-implemented method of claim 84, wherein the optimal target region is an optimal target region for activating the target gene.

86. The computer-implemented method of claim 85, wherein the optimal target region is -1000 bp to +200 bp from the transcription start site (TSS) of the target gene.

87. The computer-implemented method of claim 84, wherein the optimal target region is an optimal target region for suppressing the target gene.

88. The computer-implemented method of claim 86, wherein the optimal target region is -200 bp to +1000 bp from the TSS of the target gene.

89. The computer-implemented method of any one of claims 84-88, wherein the Cas protein is Cas12f or a variant thereof.

90. The computer-implemented method of any one of claims 84-88, wherein the Cas protein comprises or consists of an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1000.

91. The computer-implemented method of any one of claims 84-88, wherein the Cas protein comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 1092- 1240.

92. The computer-implemented method of any one of claims 84-88, wherein the Cas protein comprises or consists of an amino acid sequence according to any one of SEQ ID NOS: 1000- 1091.

93. The computer-implemented method of any one of claims 84-88, wherein the Cas protein comprises or consists of at least one deletion from amino acid residues 2-100, as compared to theamino acid sequence of SEQ ID NO: 1000.

94. The computer-implemented method of claim 93, wherein the at least one deletion comprises at least one deletion from amino acid residues 31-40, 41-60, and / or 61-80, as compared to the amino acid sequence of SEQ ID NO: 1000.

95. The computer-implemented method of claim 93 or 94, wherein the at least one deletion is a deletion of at least 10 amino acid residues, as compared to the amino acid sequence of SEQ ID NO: 1000.

96. The computer-implemented method of any one of claims 84-95, wherein the Cas protein has a length of about 350 amino acids to about 900 amino acids.

97. The computer-implemented method of any one of claims 84-96, wherein the Cas protein has a length of at most about 528 amino acids.

98. The computer-implemented method of any one of claims 84-97, wherein the Cas protein comprises an amino acid mutation which results in reduced nuclease activity.

99. The computer-implemented method of any one of claims 84-98, wherein the Cas protein is nuclease inactive.

100. The computer-implemented method of any one of claims 84-99, wherein the Cas protein comprises an amino acid substitution at D326 or D510, as compared to the amino acid sequence of SEQ ID NO: 1000.

101. The computer-implemented method of any one of claims 84-100, wherein the Cas protein comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R, and E528R, as compared to the amino acid sequence of SEQ ID NO: 1000.

102. The computer-implemented method of any one of claims 84-101, wherein the Cas protein comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1010.

103. The computer-implemented method of any one of claims 84-102, wherein the PAM sequence specific for the Cas protein is a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G.

104. The computer-implemented method of any one of claims 84-103, wherein the PAM sequence specific for the Cas protein is a non-canonical PAM sequence.

105. The computer-implemented method of claim 104, wherein the non-canonical PAM sequence comprises or is one or more nucleic acid sequence selected from the group consisting of:CCCC, TTAT, GTCC, TTTC, TCAT, ACCC, TCTG, TTAG, GTTA, ACTC, GCCA, TTTT, TTGG, GTTG, ATTG, TTAA, CTTA, TTGA, GCTC, GCCC, TTCG, CTTG, TTCA, TCTA, TTTA, and TTTG.

106. The computer-implemented method of claim 104, wherein the PAM sequence comprises or is one or more nucleic acid sequence selected from Table 11.

107. The computer-implemented method of any one of claims 84-106, wherein the candidate spacer sequence is from 10-30 nucleotides in length.

108. The computer-implemented method of any one of claims 84-107, wherein the identifying one or more potential off-target binding sites of (d) comprises mapping the candidate spacer sequence to a reference genome to identify at least one candidate off-target binding site.

109. The computer-implemented method of any one of claims 84-108, wherein the pre-defined rule identifies a candidate off-target binding site as a potential off-target binding site when the candidate off-target binding site has a PAM sequence specific for the Cas protein within 5 nucleotides upstream of the 5’ end of the candidate off-target binding site.

110. The computer-implemented method of any one of claims 84-109, wherein the pre-defined rule eliminates a candidate off-target binding site as a potential off-target binding site when the candidate off-target binding site has one or more nucleotide mismatches within a seed sequence of the candidate spacer sequence.

111. The computer-implemented method of claim 110, wherein the seed sequence is from nucleotide positions 1-6, nucleotide positions 1-7, or nucleotide positions 1-8 from the 5’ end of the candidate spacer sequence.

112. The computer-implemented method of claim 110 or 111, wherein the pre-defined rule eliminates a candidate off-target binding site as a potential off-target binding site when the candidate off-target binding site has more than three nucleotide mismatches within the candidate spacer sequence in a region outside of the seed sequence.

113. The computer-implemented method of any one of claims 84-112, wherein the generating an associating of (e) comprises outputting a list or table of candidate spacer sequences associated with corresponding potential off-target binding sites.

114. The computer-implemented method of claim 113, further comprising associating each candidate spacer sequence with an indication of whether the candidate spacer sequence binds to the optimal target region for activating the target gene, or binds to the optimal target region forsuppressing the target gene.

115. A computer-implemented system comprising at least one processor and instructions executable by the at least one processor to provide an application for implementing the computer- implemented method of any one of claims 84-114.

116. A non-transitory computer-readable storage media encoded with instructions executable by one or more processors for implementing the computer-implemented method of any one of claims 84-114.

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