Multiplex editing using CAS enzymes

Abstract

Described herein are methods, compositions, and systems for multiplex editing using Cas enzymes, or editing of T cells or related cells using Cas enzymes.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 163,510, entitled "MULTIPLEX EDITING WITH CAS ENZYMES," filed March 19, 2021, U.S. Provisional Patent Application No. 63 / 186,506, entitled "MULTIPLEX EDITING WITH CAS ENZYMES," filed May 10, 2021, and U.S. Provisional Patent Application No. 63 / 241,916, entitled "MULTIPLEX EDITING WITH CAS ENZYMES," filed September 8, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 17, 2022, is named 55921-719-601-SL.txt and is 70,612 bytes in size. [Background technology]

[0003] Cas enzymes, along with their associated clustered regularly interspaced short palindromic repeats (CRISPR)-guided ribonucleic acid (RNA), appear to be widespread components of the immune system of prokaryotes (about 45% of bacteria and about 84% of archaea), helping to protect these microorganisms from non-self nucleic acids, such as infectious viruses and plasmids, by CRISPR-RNA-guided nucleic acid cleavage. While deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse and contain a wide variety of nucleic acid-interacting domains. Although CRISPR DNA elements have been observed as early as 1987, the programmable endonuclease cleavage capabilities of CRISPR / Cas complexes have only been recognized relatively recently, leading to the use of recombinant CRISPR / Cas systems in a variety of DNA engineering and gene editing applications. Summary of the Invention

[0004] In some aspects, the disclosure provides a method of editing two or more loci in a cell, the method comprising contacting the cell with (a) a Class 2, Type II Cas endonuclease complex comprising (i) a Class 2, Type II Cas endonuclease and (ii) a first engineered guide RNA comprising an RNA sequence configured to bind to the Class 2, Type II Cas endonuclease and a spacer sequence configured to hybridize to a first set of one or more target loci; and (b) a Class 2, Type V Cas endonuclease complex comprising (i) a Class 2, Type V Cas endonuclease and (ii) a second engineered guide RNA comprising an RNA sequence configured to bind to the Class 2, Type V Cas endonuclease and a spacer sequence configured to hybridize to a second set of one or more target loci. In some embodiments, the Class 2, Type II Cas endonuclease is not a Cas9 endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease is a Cas12a endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1 or 4, or a variant thereof. In some embodiments, the Class 2, Type V Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:7 or a variant thereof.In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 3, 6, or 9. In some embodiments, the method edits the genomic sequence of the first locus with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency and / or the second locus with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease is introduced at a concentration of 200 pmol or less, 100 pmol or less, 50 pmol or less, 25 pmol or less, 5 pmol or less, or 1 pmol or less. In some embodiments, the off-target sites in the cells are disrupted at a frequency of less than 0.2% as determined by genome-wide off-target double-stranded break analysis. In some embodiments, the off-target sites in the cells are disrupted at a frequency of less than 0.01% as determined by genome-wide off-target double-stranded break analysis. In some embodiments, the first set of one or more target loci or the second set of one or more target loci comprises T cell receptor (TCR) loci. In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci or the spacer sequence configured to hybridize to the second set of one or more target loci has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 10-15, a complement thereof, or a reverse complement thereof. In some embodiments, the first set of one or more target loci or the second set of one or more target loci comprises the albumin (ALB) locus.In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci or the spacer sequence configured to hybridize to the second set of one or more target loci has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 17-19, a complement thereof, or a reverse complement thereof. In some embodiments, the first set of one or more target loci or the second set of one or more target loci comprises a Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) locus. In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci or the spacer sequence configured to hybridize to the second set of one or more target loci has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 16, 20, 21, or 22, its complement, or its reverse complement. In some embodiments, the method further comprises introducing into the cell a donor DNA sequence comprising an open reading frame encoding a heterologous engineered T cell receptor molecule, a first homology arm comprising a DNA sequence located on a first side of the first set of one or more target loci, and a second homology arm comprising a DNA sequence located on a second side of the first set of one or more target loci. In some embodiments, the edit comprises the insertion of an indel, a premature stop codon, a missense codon, a frameshift mutation, an adenine deamination, a cytosine deamination, or any combination thereof.

[0005] In some aspects, the disclosure provides a method of generating a glucocorticoid-resistant engineered T cell, comprising: administering to a T cell, or a precursor thereof, (a) an RNA-guided endonuclease complex that targets a T cell receptor (TCR) locus, the RNA-guided endonuclease complex comprising: (i) a first RNA-guided endonuclease or a DNA encoding the first RNA-guided endonuclease; and (ii) a first engineered guide RNA, the first engineered guide RNA comprising an RNA sequence configured to form a complex with the first RNA-guided endonuclease and a first spacer sequence configured to hybridize to at least a portion of the TCR locus. In some embodiments, the method includes introducing an RNA-guided endonuclease complex targeting a T cell receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) locus, and (b) an RNA-guided endonuclease complex targeting a T cell receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) locus, the RNA-guided endonuclease complex comprising: (i) a second RNA-guided endonuclease; and (ii) a second engineered guide RNA, the second engineered guide RNA comprising an RNA sequence configured to form a complex with the second RNA-guided endonuclease, and a second spacer sequence configured to hybridize to at least a portion of the NR3C1 locus. In some embodiments, the at least a portion of the TCR locus is within the T cell locus. In some embodiments, the method further comprises introducing into the cell (b) a donor DNA sequence comprising an open reading frame encoding a heterologous engineered T cell receptor molecule, a first homologous arm comprising a DNA sequence located on a first side of the target sequence within the TCR locus, and a second homologous arm comprising a DNA sequence located on a second side of the second target sequence. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease comprises a class 2, type II, or class 2, type V Cas endonuclease.In some embodiments, the first RNA-guided endonuclease comprises the class 2, type II Cas endonuclease and the second RNA-guided endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the second RNA-guided endonuclease comprises the class 2, type II Cas endonuclease and the first RNA-guided endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the heterologous engineered T cell receptor is a CAR molecule. In some embodiments, the at least a portion of the T cell receptor locus is a T cell receptor alpha constant (TRAC) locus or a T cell receptor beta constant (TRBC) locus. In some embodiments, the homology arm comprises an intronic or exonic region within the TCR locus proximal to the at least a portion of the T cell receptor locus. In some embodiments, the at least a portion of the T cell receptor locus is the first or third exon of TRAC. In some embodiments, the method disrupts genomic sequences at the TCR locus and the NR3C1 locus with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the efficiency is determined by flow cytometry on proteins expressed from the TCR locus and the NR3C1 locus. In some embodiments, the at least a portion of the NR3C1 locus is exon 2 or exon 3. In some embodiments, the method produces cells positive for a CAR molecule and negative for NR3C1 with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the method further comprises simultaneously introducing (a)-(c) into said T cells or precursors thereof.In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1, 4, or 7, or a variant thereof. In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 6, or 9, its complement, or its reverse complement. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease is present at a concentration of 100 pmol or less, 50 pmol or less, 25 pmol or less, 5 pmol or less, or 1 pmol or less. In some embodiments, the T cell or the precursor thereof comprises a T cell, a hematopoietic stem cell (HSC), or a peripheral blood mononuclear cell (PBMC). In some embodiments, the second spacer sequence comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 16, 20, 21, or 22, a complement thereof, or a reverse complement thereof. In some embodiments, the first or second spacer sequence comprises at least about 19-24 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, or at least about 24 nucleotides. In some embodiments, the donor DNA sequence is delivered in a viral vector. In some embodiments, the viral vector is an AAV vector or an AAV-6 vector.

[0006] In some aspects, the disclosure provides a population of glucocorticoid-resistant T cells or precursors thereof comprising (a) a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of any one of SEQ ID NOs: 10-15 in a TCR locus. In some embodiments, the T cells or precursors thereof further comprise (b) an NR3C1 locus comprising an indel. In some embodiments, the heterologous sequence is an indel. In some embodiments, the heterologous sequence comprises an open reading frame comprising a nucleotide sequence encoding a heterologous T cell receptor or CAR molecule. In some embodiments, the NR3C1 locus comprises an indel within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of any one of SEQ ID NOs: 16, 20, 21, or 22. In some embodiments, less than 0.2% of the cells have an indel at an off-target locus as determined by genome-wide off-target double-stranded break analysis. In some embodiments, less than 0.01% of the cells have an indel at an off-target locus as determined by genome-wide off-target double stranded break analysis, hi some embodiments, the population of cells is substantially free of chromosomal translocations.

[0007] In some aspects, the disclosure provides a method of editing two or more genetic loci in a cell, the method comprising contacting the cell with (a) a first Cas endonuclease complex comprising (i) a first Cas endonuclease and (ii) one or more engineered guide RNAs comprising an RNA sequence configured to bind to a class 2, type II Cas endonuclease and a spacer sequence configured to hybridize to a first target sequence; and (b) a second Cas endonuclease complex comprising (i) a second Cas endonuclease and (ii) one or more engineered guide RNAs comprising an RNA sequence configured to bind to a class 2, type II Cas endonuclease and a spacer sequence configured to hybridize to a second target sequence. In some embodiments, the method further comprises introducing into the cell (c) a first donor DNA sequence comprising an open reading frame encoding a first transgene, a 5' homology arm comprising a DNA sequence located 5' to the first target sequence, and a 3' homology arm comprising a DNA sequence located 3' to the first target sequence, and (d) a second donor DNA sequence comprising an open reading frame encoding a second transgene, a 5' homology arm comprising a DNA sequence located 5' to the second target sequence, and a 3' homology arm comprising a DNA sequence located 3' to the second target sequence. In some embodiments, the first transgene and the second transgene are different. In some embodiments, the first target sequence or the second target sequence is a target sequence within a T cell receptor locus, a TRAC, a TRBC, a NR3C1, or an AAVS1 locus, or any combination thereof. In some embodiments, the first or second transgene is the alpha, beta, alpha-D3, or beta-D3 isoform of GR, a CAR molecule, a truncated low affinity nerve growth factor receptor (tLNGFR) sequence, a truncated form of the epidermal growth factor receptor (tEGFR), a GFP coding sequence, or any combination thereof.In some embodiments, the 5' homology arm comprising a DNA sequence located 5' to the first target sequence or the 5' homology arm comprising a DNA sequence located 5' to the second target sequence comprises SEQ ID NO: 42 or 23, or a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the 3' homology arm comprising a DNA sequence located 5' to the first target sequence or the 3' homology arm comprising a DNA sequence located 5' to the second target sequence comprises SEQ ID NO: 43 or 24, or a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the first or the second class 2, type II Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 6, or 9, its complement, or its reverse complement. In some embodiments, the spacer sequence configured to hybridize to the first target sequence or the spacer sequence configured to hybridize to the second target sequence has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 16, 20, 21, 22, or 41, or its complement, its complement, or its reverse complement.In some embodiments, the first or second endonuclease comprises a class 2, type II Cas endonuclease, or a class 2, type V Cas endonuclease, or any combination thereof.

[0008] In some aspects, the disclosure provides an isolated nucleic acid comprising any one of SEQ ID NOs:63-65, or a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0009] In some aspects, the present disclosure provides an isolated nucleic acid comprising any of the nucleic acid sequences described herein, a complement thereof, or a reverse complement thereof. In some embodiments, the isolated nucleic acid is a guide RNA.

[0010] In some aspects, the disclosure provides a cell comprising any of the nucleic acids described herein. In some embodiments, the cell is a T cell or a precursor thereof. In some embodiments, the T cell or a precursor thereof comprises a T cell, a hematopoietic stem cell (HSC), or a peripheral blood mononuclear cell (PBMC).

[0011] In some embodiments, the present disclosure provides a vector comprising any of the nucleic acids described herein.In some embodiments, the vector is an adeno-associated virus (AAV) vector.In some embodiments, the AAV vector is an AAV-6 serotype vector.

[0012] In some aspects, the disclosure provides a vector comprising a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 23, 24, 42, or 43. In some embodiments, the vector further comprises a transgene flanked by sequences having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 23, 24, 42, or 43. In some embodiments, the transgene comprises the alpha, beta, alpha-D3, or beta-D3 isoform of GR, a CAR molecule, a truncated low affinity nerve growth factor receptor (tLNGFR) sequence, a truncated form of epidermal growth factor receptor (tEGFR), a GFP coding sequence, or any combination thereof. In some embodiments, the vector further comprises a tEGFR coding sequence of SEQ ID NO: 63, or a variant having 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the vector comprises a tLNGFR coding sequence of SEQ ID NO: 64, or a variant having 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the vector further comprises a MND promoter of SEQ ID NO: 63, or a variant having 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the vector further comprises an MSCV promoter of SEQ ID NO:64, or a variant having 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0013] In some aspects, the disclosure provides a method of editing two or more loci in a cell, the method comprising contacting or introducing into the cell (a) a Class 2, Type II Cas endonuclease complex comprising, or comprising polynucleotides encoding, (i) a Class 2, Type II Cas endonuclease and (ii) one or more engineered guide RNAs, the one or more engineered guide RNAs comprising an RNA sequence configured to bind to the Class 2, Type II Cas endonuclease and a spacer sequence configured to hybridize to a first set of one or more target loci. In some embodiments, the method comprises contacting or introducing into the cell (b) a Class 2, Type V Cas endonuclease complex comprising: (i) a Class 2, Type V Cas endonuclease; and (ii) one or more engineered guide RNAs, the one or more engineered guide RNAs comprising an RNA sequence configured to bind to the Class 2, Type V Cas endonuclease and a spacer sequence configured to hybridize to a second set of one or more target loci. In some embodiments, the Class 2, Type II Cas endonuclease is not a Cas9 endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease is a Cas12a endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1 or 4, or a variant thereof. In some embodiments, the Class 2, Type V Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 7, or a variant thereof.In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 90%, or 95% to any one of SEQ ID NOs: 3, 6, or 9, or a complement thereof. In some embodiments, the method edits the genomic sequence of the first locus with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency and / or the second locus with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease is introduced at a concentration of 200 pmol or less, 100 pmol or less, 50 pmol or less, 25 pmol or less, 5 pmol or less, or 1 pmol or less. In some embodiments, the off-target site in the cell is disrupted at a frequency of less than 0.2% as determined by genome-wide off-target double-stranded break analysis. In some embodiments, the off-target site in the cell is disrupted at a frequency of less than 0.01% as determined by genome-wide off-target double-stranded break analysis.In some embodiments, genome-wide off-target double strand break analysis is performed using an HTGTS assay (high-throughput, genome-wide translocation sequencing; see, e.g., Chiarle et al. Cell. 2011 Sep 30;147(1):107-19. doi:10.1016 / j.cell.2011.07.049, expressly incorporated by reference for all purposes), a LAM-HTGTS assay (linear amplification mediated high-throughput genome-wide sequencing; see, e.g., Hu et al. Nat Protoc. 2016.11(5):853-71. doi:10.1038 / nprot.2016.043, expressly incorporated by reference for all purposes), or a Digenome-Seq assay (in vitro Cas-digested whole genome sequencing; see, e.g., Kim et al. Nat Methods. 2015.12(3):237-43. doi:10.1038 / nmeth.3284 (expressly incorporated herein by reference for all purposes). In some embodiments, the first set of one or more target loci or the second set of one or more target loci comprise T cell receptor (TCR) loci. In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci or the spacer sequence configured to hybridize to the second set of one or more target loci has at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 10-15, or a complement thereof. In some embodiments, the first set of one or more target loci or the second set of one or more target loci comprise a nuclear receptor subfamily 3 group C member 1 (NR3C1) locus.In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci or the spacer sequence configured to hybridize to the second set of one or more target loci has at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 16, 20, 21, or 22, or a complement thereof. In some embodiments, the method further comprises introducing into the cell a donor DNA sequence comprising an open reading frame encoding a heterologous engineered T cell receptor molecule, a first homologous arm comprising a DNA sequence located on a first side of the first set of one or more target loci, and a second homologous arm comprising a DNA sequence located on a second side of the first set of one or more target loci. In some embodiments, the edit comprises the insertion of an indel, a premature stop codon, a missense codon, a frameshift mutation, an adenine deamination, a cytosine deamination, or any combination thereof.

[0014] In some aspects, the disclosure provides a method of generating glucocorticoid-resistant engineered T cells, the method comprising: injecting into a T cell, or a precursor thereof, (a) an RNA-guided endonuclease complex that targets a T cell receptor (TCR) locus, the RNA-guided endonuclease complex comprising, or a polynucleotide encoding, (i) a first RNA-guided endonuclease or a DNA encoding the first RNA-guided endonuclease; and (ii) a first engineered guide RNA, the first engineered guide RNA comprising an RNA sequence configured to form a complex with the first RNA-guided endonuclease and a first spacer sequence configured to hybridize to at least a portion of the TCR locus. In some embodiments, the method includes introducing an RNA-guided endonuclease complex targeting a T cell receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) locus, and (b) an RNA-guided endonuclease complex targeting a T cell receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) locus, the RNA-guided endonuclease complex comprising, or a polynucleotide encoding, (i) a second RNA-guided endonuclease and (ii) a second engineered guide RNA, the second engineered guide RNA comprising an RNA sequence configured to form a complex with the second RNA-guided endonuclease and a second spacer sequence configured to hybridize to at least a portion of the NR3C1 locus. In some embodiments, the at least a portion of the TCR locus is within the T cell locus. In some embodiments, the method further comprises introducing into the cell (b) a donor DNA sequence comprising an open reading frame encoding a heterologous engineered T cell receptor molecule, a first homologous arm comprising a DNA sequence located on a first side of the target sequence within the TCR locus, and a second homologous arm comprising a DNA sequence located on a second side of the second target sequence. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease comprises a class 2, type II, or class 2, type V Cas endonuclease.In some embodiments, the first RNA-guided endonuclease comprises the class 2, type II Cas endonuclease and the second RNA-guided endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the second RNA-guided endonuclease comprises the class 2, type II Cas endonuclease and the first RNA-guided endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the heterologous engineered T cell receptor is a CAR molecule. In some embodiments, the at least a portion of the T cell receptor locus is a T cell receptor alpha constant (TRAC) locus or a T cell receptor beta constant (TRBC) locus. In some embodiments, the homology arm comprises an intronic or exonic region within the TCR locus proximal to the at least a portion of the T cell receptor locus. In some embodiments, the at least a portion of the T cell receptor locus is the first or third exon of TRAC. In some embodiments, the method disrupts genomic sequences at the TCR locus and the NR3C1 locus with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the efficiency is determined by flow cytometry on proteins expressed from the TCR locus and the NR3C1 locus. In some embodiments, the at least a portion of the NR3C1 locus is exon 2 or exon 3. In some embodiments, the method produces cells positive for a CAR molecule and negative for NR3C1 with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the method comprises introducing (a)-(c) simultaneously into said T cells or precursors thereof.In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1, 4, or 7. In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 3, 6, or 9, or a complement thereof. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease is present at a concentration of 100 pmol or less, 50 pmol or less, 25 pmol or less, 5 pmol or less, or 1 pmol or less. In some embodiments, the T cell or the precursor thereof comprises a T cell, a hematopoietic stem cell (HSC), or a peripheral blood mononuclear cell (PBMC). In some embodiments, the second spacer sequence comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 16, 20, 21, or 22, or a complement thereof. In some embodiments, the first or the second spacer sequence comprises at least about 19-24 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, or at least about 24 nucleotides. In some embodiments, the donor DNA sequence is delivered in a viral vector. In some embodiments, the viral vector is an AAV vector or an AAV-6 vector.

[0015] In some aspects, the disclosure provides a population of T cells comprising (a) a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of any one of SEQ ID NOs: 10-15 within a TCR locus, or a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of SEQ ID NO: 42. In some embodiments, the population of T cells further comprises (b) an NR3C1 locus comprising an indel. In some embodiments, the indel in the NR3C1 locus confers glucocorticoid resistance to the T cell. The heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of the heterologous sequence is an indel. In some embodiments, the heterologous sequence comprises an open reading frame comprising a nucleotide sequence encoding a heterologous T cell receptor or CAR molecule. In some embodiments, the NR3C1 locus comprises an indel within 100, 75, 50, 25, or 10 nucleotides of the hybridization region of any one of SEQ ID NOs: 16, 20, 21, or 22. In some embodiments, less than 0.2% have indels at off-target loci as determined by genome-wide off-target double-stranded break analysis. In some embodiments, less than 0.01% have indels at off-target loci as determined by genome-wide off-target double-stranded break analysis. In some embodiments, the population of cells is substantially free of chromosomal translocations.

[0016] In some aspects, the disclosure provides a method of editing two or more genetic loci in a cell, the method comprising contacting the cell with (a) a first Cas endonuclease complex that comprises, or comprises polynucleotides encoding, (i) a first Cas endonuclease and (ii) one or more engineered guide RNAs, the one or more engineered guide RNAs comprising an RNA sequence configured to bind to a class 2, type II Cas endonuclease and a spacer sequence configured to hybridize to a first target sequence; and (b) a second Cas endonuclease complex that comprises (i) a second Cas endonuclease and (ii) one or more engineered guide RNAs, the one or more engineered guide RNAs comprising an RNA sequence configured to bind to a class 2, type II Cas endonuclease and a spacer sequence configured to hybridize to a second target sequence. In some embodiments, the method further comprises introducing into the cell: (c) a first donor DNA sequence comprising an open reading frame encoding a first transgene, a 5' homology arm comprising a DNA sequence located 5' to the first target sequence, and a 3' homology arm comprising a DNA sequence located 3' to the first target sequence; and (d) a second donor DNA sequence comprising an open reading frame encoding a second transgene, a 5' homology arm comprising a DNA sequence located 5' to the second target sequence, and a 3' homology arm comprising a DNA sequence located 3' to the second target sequence. In some embodiments, the second transgenes are different. In some embodiments, the first target sequence or the second target sequence is a target sequence within a T cell receptor locus, a TRAC, TRBC, NR3C1, or AAVS1 locus, or any combination thereof. In some embodiments, the first or second transgene is an alpha, beta, alpha-D3, or beta-D3 isoform of GR, a CAR molecule, or any combination thereof.In some embodiments, the 5' homology arm comprising a DNA sequence located 5' of the first target sequence or the 5' homology arm comprising a DNA sequence located 5' of the second target sequence comprises SEQ ID NO: 42 or 23. In some embodiments, the 3' homology arm comprising a DNA sequence located 5' of the first target sequence or the 3' homology arm comprising a DNA sequence located 5' of the second target sequence comprises SEQ ID NO: 43 or 24. In some embodiments, the first or the second class 2, type II Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1 or 4, or a variant thereof. In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 3, 6, or 9, or a complement thereof. In some embodiments, the spacer sequence configured to hybridize to the first target sequence or the spacer sequence configured to hybridize to the second target sequence has at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 16, 20, 21, 22, or 41, or a complement thereof. In some embodiments, the first or the second endonuclease comprises a class 2, type II Cas endonuclease, or a class 2, type V Cas endonuclease, or any combination thereof.

[0017] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0018] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0019] The novel features of the invention 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.

[0020] [Figure 1] FIG. 1 shows a scheme for producing allogeneic CAR-T cells using the Cas endonucleases described herein in combination with an AAV vector delivering the CAR-T donor sequence. [Diagram 2] 1 shows the results of the experiment in Example 1 testing indel formation in TRAC by MG3-6, MG3-8, and MG29-1 RNPs containing guide RNAs targeting TRAC, along with a Cas9 control. The left panel shows the % indel formation measured by next generation sequencing (NGS), while the right panel shows the cell phenotype (TCR+ or TCR-) assessed by flow cytometry. [Diagram 3] Figure 1 shows the results of the experiment of Example 1, which tests targeted CAR-T integration using the RNP nuclease complex described herein, which targets TRAC in combination with an AAV donor vector containing CAR-T sequences.Flow cytometry plots are shown showing TCR expression status (TCR- or TCR+, x-axis) along with the expression of CAR antigen binding domain (y-axis).Mg3-6, MG3-8, and MG29-1 all showed similar results. [Figure 4]As described in Example 2, multiplex editing of two loci (one of which is TRAC) is shown using a combination of MG3-6 and MG29-1 RNP complexes. [Diagram 5] 1 shows multiplex editing of three loci (one of which is TRAC) using a combination of MG3-6 and MG29-1 RNP complexes as described in Example 2. [Figure 6] 1 shows the design of a PCR experiment as in Example 3 to test for integration of the GR transgene into the AAVS1 locus (A) or agarose gel pictures (B and C) showing the results of an experiment in which the AAVS1 and TRAC loci were targeted simultaneously using different Cas enzymes with exposure to separate donor DNA targeting each site as in Example 3. (B) shows amplification of the GR transgene from either conditions in which T cells were exposed to a GR transgene-carrying AAV construct (lanes 2-5), a GR transgene AAV construct / SpCas9 targeting AAVS1 / MG3-6 / CAR transgene AAV targeting TRAC (lanes 6-9), or a GR AAV construct / SpCas9 targeting AAVS1 alone at a multiplicity of infection (MOI) of 25K (lanes 10-13). (C) shows amplification of the GR transgene from T cells exposed to either assay controls (mock transfection or Cas complexes without transgene; lanes 2-4), GR AAV construct at an MOI of 50K / SpCas9 targeting AAVS1 alone (lanes 5-8), or GR AAV construct at an MOI of 100K / SpCas9 targeting AAVS1 alone at a multiplicity of infection (MOI) of 50K (lanes 9-12). Results show the GR transgene integrated within the AAVS1 locus with similar efficiency, regardless of whether an additional TRAC locus was targeted. [Figure 7]Figure 1 shows flow cytometry plots showing the results of an experiment as in Example 3, in which the AAVS1 and TRAC loci were targeted simultaneously using different Cas enzymes with exposure to separate donor DNA targeting each site. Individual plots (A-D) are shown in which AAV carrying each GR transgene was introduced into T cells with AAVS1-targeted SpCas9 complexes, TRAC-targeted MG3-6 complexes, and CAR-bearing AAV. Results show that TCR knockout and CAR integration were similarly efficient for all GR transgenes, and were high (51.31% to 61.1% efficiency) despite simultaneous targeting of the AAVS1 locus. [Figure 8] As in Example 4, the results of a genome-wide off-target double-stranded break analysis assay were performed to assess the off-target specificity of MG3-6, MG3-8, and MG29-1 endonucleases along with SpCas9 ("Cas9"). [Figure 9] FIG. 1 is a diagram of the assembly of delta, gamma, and epsilon chains to make an active complete TCR. [Figure 10] 1 shows multiplex TRAC / TRBC editing in primary T cells as assessed by the percentage of sequences at the targeted locus that contain indels, as described in Example 5. The results show frequent disruption at both sites when both sites are targeted simultaneously. [Figure 11]Figure 1 shows gene editing results by flow cytometry for single gene knockout experiments described in Example 6. Bar graphs are shown showing the percentage of analyzed cells containing each of the four phenotypes (TCR-B2M-DKO, TCR-B2M+, TCR+B2M-, and TCR+B2M+) that evaluate TCR and B2M knockout. The graphs show that (a) all TCR targeting conditions efficiently result in TCR knockout, with MG3-6 TRAC6 and MG3-6 TRBC E2 sgRNA resulting in the most efficient TCR knockout, and (b) all B2M targeting conditions result in B2M knockout, with B2M H1 and B2M D2 resulting in the most efficient B2M knockout. [Figure 12] Figure 1 shows gene editing results by flow cytometry for the double gene knockout experiment described in Example 7 using the B2M and TRAC conditions of Example 6, but in combination. Bar graphs are shown showing the percentage of analyzed cells containing each of the four phenotypes evaluating the knockout of TCR and B2M (TCR-B2M-DKO, TCR-B2M+, TCR+B2M-, and TCR+B2M+). The graphs show that the most efficient double targeting conditions were A4, B4, and C4, which involved MG3-6 TRAC6 conditions with MG29-1 B2M H1, D2, or A3 conditions. The most efficient double targeting condition appeared to be B4, which used MG3-6 TRAC6 sgRNA and MG29-1 B2M D2 sgRNA. [Figure 13] 13 shows gene editing results by flow cytometry for the triple gene knockout experiment described in Example 8 using B2M, TRAC, and TRBC conditions from Example 6, but in combination. [Figure 14] 13 shows gene editing results at the DNA level for the triple gene knockout experiment described in Example 8 using B2M, TRAC, and TRBC conditions from Example 6, but in combination. [Figure 15]13 shows an analysis of gene editing outcomes as determined by next generation sequencing (NGS) for the triple gene knockout experiment described in Example 8. [Figure 16] 1 shows gene editing results at the protein level in T cells for the experiments described in Example 9. Bar graphs are shown showing the percentage of T cells positive for GFP / tEGFR, tLNGFR, double targeted integration (GFP / tLNGFR), double targeted integration (tEGFR / tLNGFR), or TCR as determined by fluorescence activated cell sorting (FACS) using the nuclease, guide, and AAV combinations described in Example 9. [Figure 17] 1 shows gene editing results at the DNA level in T cells for the AAVS1 site and the TRAC locus for the experiment described in Example 10. A bar graph of the percentage of sequences detected by next generation sequencing (Illumina MiSeq) with at least one indel detected at the AAVS1 locus (% indels) using the conditions described in Example 10 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] While various embodiments of the present invention have been shown and described herein, it will be apparent 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 used.

[0022] The practice of some of the methods disclosed herein may involve immunological, biochemical, chemical, molecular biology, microbiology, cell biology, genomics, and recombinant DNA techniques, unless otherwise indicated. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (FMA Usubel, et al. eds.), the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)) (incorporated herein in its entirety by reference).

[0023] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent the terms "comprising," "including," "having," "having," "having," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0024] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0025] As used herein, "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, single-cell eukaryotic cells, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, bryophytes, mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. agarwal), and the like). C. Agardh), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from vertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. The cells may not be derived from a naturally occurring organism (e.g., cells may be synthetically produced and sometimes referred to as artificial cells).

[0026] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may 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 may include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled, such as by using a moiety that includes an optically detectable moiety (e.g., a fluorophore). Labeling may also be performed using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif.; fluoro-conjugated deoxynucleotides, fluoro-conjugated Cy3-dCTP, fluoro-conjugated Cy5-dCTP, fluoro-conjugated fluoroX-dCTP, fluoro-conjugated Cy3-dUTP, and fluoro-conjugated Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2′-dATP available from Mannheim, Indianapolis, Ind.; and Molecular Examples of chromosomal labeling nucleotides available from Probes, Eugene, Oreg. include 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. Nucleotides may also be labeled or marked by chemical modification. The chemically modified single nucleotide may be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP). Nucleotides may include nucleotide analogs. In some embodiments, nucleotide analogs may include the structure of natural nucleotides modified at any position to change certain chemical properties of the nucleotide but retain the ability of the nucleotide analog to perform its intended function (e.g., hybridization to other nucleotides in RNA or DNA). Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine: O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or otherwise appropriately modified) nucleotides, and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug.10(4):297-310. Nucleotide analogs may also include modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine: O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or otherwise appropriately modified) nucleotides, and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug.10(4):297-310. Nucleotide analogs may also include modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0027] The terms "polynucleotide", "oligonucleotide", and "nucleic acid" are generally used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in single-stranded, double-stranded, or multiple-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locuses) defined from binding 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 may be interrupted by non-nucleotide components.A polynucleotide may comprise a mixture of naturally occurring nucleotides and nucleotide analogs (eg, synthetic nucleotide analogs).

[0028] The term "transfection" or "transfected" generally refers to the introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecule may be a genetic sequence encoding a complete protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88, which is incorporated herein by reference in its entirety.

[0029] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and generally refer to a polymer of at least two amino acid residues linked by peptide bonds. The term does not refer to a particular length of the polymer, and is not intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins (e.g., domains) with or without secondary and / or tertiary structure. The term also encompasses amino acid polymers modified by any other manipulation, such as, for example, disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and unnatural amino acids, which are chemically modified to include a non-naturally occurring group or chemical moiety on the amino acid. An amino acid analog may refer to an amino acid derivative. The term "amino acid" includes both D- and L-amino acids.

[0030] As used herein, "non-natural" may generally refer to a nucleic acid or polypeptide sequence that is not present in a naturally occurring nucleic acid or protein. Non-natural may refer to an affinity tag. Non-natural may refer to a fusion. Non-natural may refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions. A non-natural sequence may exhibit and / or encode an activity (e.g., an enzyme activity, a methyltransferase activity, an acetyltransferase activity, a kinase activity, an ubiquitination activity, etc.) that may also be exhibited by the nucleic acid and / or polypeptide sequence to which the non-natural sequence is fused. A non-natural nucleic acid or polypeptide sequence may be linked to a naturally occurring nucleic acid and / or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and / or polypeptide sequence that encodes a chimeric nucleic acid or polypeptide.

[0031] As used herein, the term "promoter" generally refers to a regulatory DNA region that controls the transcription or expression of a gene and may be located adjacent to or overlapping the nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences that bind protein factors, often called transcription factors, which promote the binding of RNA polymerase to DNA, thereby resulting in gene transcription. A "basal promoter", also called a "core promoter", may generally refer to a promoter that contains all the basic elements to promote the transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters typically, but not necessarily, contain a TATA-box and / or a CAAT box.

[0032] As used herein, the term "expression" generally refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as the "gene product." If the polynucleotide is derived from genomic DNA, expression includes splicing of the mRNA in eukaryotic cells.

[0033] As used herein, "operably linked," "operably linked," "operably linked," or grammatical equivalents thereof generally refer to the juxtaposition of genetic elements, such as promoters, enhancers, polyadenylation sequences, and the like, where the elements are in a relationship that allows them to operate in an expected manner. For example, a regulatory element, which may include a promoter sequence and / or an enhancer sequence, is operably linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and the coding region so long as this functional relationship is maintained.

[0034] As used herein, a "vector" generally refers to a polymer or an association of polymers that contains or associates with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. A vector generally includes genetic elements, such as control elements, operably linked to a gene to facilitate expression of the gene in a target.

[0035] As used herein, "expression cassette" and "nucleic acid cassette" are generally used interchangeably to refer to a nucleic acid sequence or combination of elements that are operably linked for expression. In some cases, an expression cassette refers to a combination of a gene or genes with regulatory elements that are operably linked for expression.

[0036] As used herein, an "engineered" subject generally refers to a subject that has been modified by human intervention. By way of non-limiting examples, a nucleic acid may be modified by altering its sequence to a sequence that does not occur in nature, a nucleic acid may be modified by ligating to a nucleic acid with which it is not naturally associated such that the ligated product has a function not present in the original nucleic acid, an engineered nucleic acid may be synthesized in vitro with a sequence that does not occur in nature, a protein may be modified by changing its amino acid sequence to a sequence that does not occur in nature, and an engineered protein may acquire a new function or property. An "engineered" system includes at least one engineered component.

[0037] As used herein, "synthetic" and "artificial" may generally be used interchangeably to refer to proteins or domains thereof that have low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) with naturally occurring human proteins. For example, the VPR domain and the VP64 domain are synthetic transactivation domains.

[0038] As used herein, the term "Cas12a" generally refers to a family of Cas endonucleases that are class 2, type VA Cas endonucleases that (a) use relatively small guide RNAs (about 42-44 nucleotides) that are processed by the nuclease itself after transcription from the CRISPR array, and (b) cleave DNA to leave staggered cleavage sites. Further characteristics of this enzyme family can be found, for example, in Zetsche B, Heidenreich M, Mohanraju P, et al. Nat Biotechnol 2017;35:31-34, and Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cell 2015;163:759-771, which are incorporated herein by reference.

[0039] As used herein, a "guide nucleic acid" or variants thereof may generally refer to a nucleic acid that can hybridize to another nucleic acid. A guide nucleic acid may be RNA. A guide nucleic acid may be DNA. A guide nucleic acid may be programmed to bind to a sequence of a nucleic acid in a site-specific manner. The nucleic acid to be targeted, or the target nucleic acid, may comprise nucleotides. A guide nucleic acid may comprise nucleotides. A portion of a target nucleic acid may be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to a guide nucleic acid and hybridizes with the guide nucleic acid may be referred to as a complementary strand. A strand of a double-stranded target polynucleotide that is complementary to a complementary strand and therefore not complementary to the guide nucleic acid may be referred to as a non-complementary strand. A guide nucleic acid may comprise a polynucleotide strand and may be referred to as a "single guide nucleic acid". A guide nucleic acid may comprise two polynucleotide strands and may be referred to as a "double guide nucleic acid". Otherwise, the term "guide nucleic acid" may be inclusive, referring to both single and double guide nucleic acids. The guide nucleic acid may comprise a segment that may be referred to as a "nucleic acid targeting segment" or a "nucleic acid targeting sequence" or a "spacer sequence." The nucleic acid targeting segment may comprise a sub-segment that may be referred to as a "protein binding segment" or a "protein binding sequence" or a "Cas protein binding segment." The guide nucleic acid may comprise a sgRNA. The guide nucleic acid may comprise a crRNA.

[0040] The terms "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences generally refer to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are identical or have a certain percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example, BLASTP using the BLOSUM62 scoring matrix setting parameters of a word length (W) of 3, an expectation (E) of 10, and gap costs at 11, an extension of 1, and using a conditional composition score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a word length (W) of 2, an expectation (E) of 1,000,000, and PAM30 scoring setting gap costs at 9 for open gaps and 1 for extended gaps for sequences shorter than 30 residues (default parameters for BLASTP are available in BLAST at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using Smith-Waterman homology search algorithm parameters of 2 matches, -1 mismatches, and -1 gaps; MUSCLE using default parameters; MAFFT using parameters of 2 retrees and 1000 maximum repeats; Novafold using default parameters; HMMER hmmalign using default parameters.

[0041] As used herein, the term "chimeric antigen receptor", "CAR", or "CAR molecule" generally refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined herein. In some embodiments, the stimulatory molecule is a zeta chain associated with the signaling domain of the T cell receptor complex or NKG2D. In some embodiments, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0042] The term "signaling domain" generally refers to a functional portion of a protein that acts by transmitting a signal within the cell and regulates cellular activity through a defined signaling pathway by generating second messengers or functioning as an effector by responding to such messengers.

[0043] As used herein, the term "antibody" generally refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds, for example, non-covalently, reversibly, and in a specific manner, to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. An antibody may be a tetramer of immunoglobulin molecules. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, and chimeric antibodies. The antibody may be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0044] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen-binding domain, e.g., an antigen-determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single chain or "scFv" antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the variable regions of the light and heavy chains are linked contiguously via a short flexible polypeptide linker and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless specified, an scFv may have the VL and VH variable regions in any order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0045] The portion of the CAR composition that includes an antibody or antibody fragment thereof can exist in various forms, where the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, single domain antibody fragments (sdAbs), single chain antibodies (scFvs), and humanized antibodies (Harlow et al., 1999, In:Using Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In:Antibodies:A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment. In some embodiments, the CAR comprises an antibody fragment that includes an scFv.

[0046] The present disclosure includes any variant of the enzymes described herein that have one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without destroying the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by replacing amino acids with similar hydrophobicity, polarity, and R chain length. Additionally or alternatively, by comparing the aligned sequences of homologous proteins from different species, conservative substitutions can be identified by finding amino acid residues (e.g., non-conserved residues) that are mutated between species without changing the basic function of the encoded protein. Such conservatively substituted variants may include variants having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the endonuclease protein sequences described herein. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants may include sequences with substitutions such that the activity of one or more critical active site residues or guide RNA binding residues of the endonuclease is not destroyed. In some embodiments, a functional variant of any of the proteins described herein lacks at least one substitution of a conserved or functional residue that is characteristic of a Cas endonuclease, hi some embodiments, a functional variant of any of the proteins described herein lacks all substitutions of a conserved or functional residue that is characteristic of a Cas endonuclease.

[0047] The disclosure also includes variants (e.g., reduced activity variants) of any of the enzymes described herein that have substitutions of one or more catalytic residues to reduce or eliminate activity of the enzyme. In some embodiments, reduced activity variants of the proteins described herein include disruptive substitutions of at least one, at least two, or all three RuvC catalytic residues.

[0048] Conservative substitution tables providing functionally similar amino acids are available in a variety of references (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W), 7) Serine (S), Threonine (T), and 8) Cysteine ​​(C), Methionine (M).

[0049] overview Discovery of new Cas enzymes with unique functionality and structure may further disrupt deoxyribonucleic acid (DNA) editing technologies, endowing them with the potential to improve speed, specificity, functionality, and ease of use. Compared to the predicted prevalence of clustered regularly interspaced short palindromic repeats (CRISPR) systems in microbes and the net diversity of microbial species, a relatively small number of functionally characterized CRISPR / Cas enzymes exist in the literature. This is in part because the vast number of microbial species are not easily cultured under laboratory conditions. Metagenomic sequencing from natural environmental niches containing large numbers of microbial species could dramatically increase the number of documented new CRISPR / Cas systems, endowing them with the potential to expedite the discovery of new oligonucleotide editing functions. A fruitful recent example of such an approach is illustrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0050] CRISPR / Cas systems are RNA-directed nuclease complexes that have been described to function as adaptive immune systems in microorganisms. In their natural context, CRISPR / Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally contain two parts: (i) an array of short repetitive sequences (30-40 bp) separated by equally short spacer sequences that encode RNA-based targeting elements; and (ii) an ORF encoding a Cas that encodes a nuclease polypeptide directed by the RNA-based targeting element flanked by accessory proteins / enzymes. Efficient nuclease targeting of a specific target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide; and (ii) the presence of a protospacer adjacent motif (PAM) sequence within a defined vicinity of the target seed (PAM is usually a sequence that is not commonly represented in the host genome). Depending on the exact function and composition of the system, CRISPR-Cas systems are commonly organized into two classes, five types, and 16 subtypes based on shared functional characteristics and evolutionary similarities (see Figure 1).

[0051] Class I CRISPR-Cas systems have large, multi-subunit effector complexes and include types I, III, and IV. Class II CRISPR-Cas systems generally have single polypeptide multi-domain nuclease effectors and include types II, V, and VI.

[0052] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, processing of CRISPR arrays into mature crRNA does not require the presence of special endonuclease subunits, but rather a small transcoding crRNA (tracrRNA) with a region complementary to the array repeat sequence, which interacts with both its corresponding effector nuclease (e.g., Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nuclease is specified as a DNA nuclease. Type 2 effectors generally exhibit a structure that includes a RuvC-like endonuclease domain that fits into an RNase H fold with an unrelated HNH nuclease domain inserted into the fold of the RuvC-like nuclease domain. The RuvC-like domain is involved in cleavage of the target (e.g., crRNA-complementary) DNA strand, while the HNH domain is involved in cleavage of the replacement DNA strand.

[0053] Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g., Cas12) structure similar to that of type II effectors, including a RuvC-like domain. Like type II, most (but not all) type V CRISPR systems use tracrRNA to process pre-crRNA into mature crRNA, but unlike type II systems that require RNAse III to cleave pre-crRNA into multiple crRNAs, type V systems can cleave pre-crRNA using the effector nuclease itself. Like type II CRISPR-Cas systems, type V CRISPR-Cas systems are also identified as DNA nucleases. Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to have robust single-stranded non-specific deoxyribonuclease activity that is activated by the first crRNA-directed cleavage of the double-stranded target sequence.

[0054] CRISPR-Cas systems have emerged as a gene editing technology in recent years due to their targeting and ease of use. The most commonly used systems are class 2 type II SpCas9 and class 2 type VA Cas12a (previously Cpf1). Specifically, type VA systems have become more widely used because their reported specificity in cells is higher than other nucleases and they have fewer or no off-target effects. VA systems are also advantageous in that the guide RNAs are small (42-44 nucleotides compared to about 100 nt for SpCas9) and are processed by the nuclease itself after transcription from the CRISPR array, simplifying multiplexed applications involving multiple gene editing. Furthermore, VA systems have staggered cut sites, which may facilitate directed repair pathways such as microhomology-dependent targeted integration (MITI).

[0055] The most commonly used type VA enzymes require a 5' protospacer adjacent motif (PAM) next to the selected target site: 5'-TTTV-3' for Lachnospiraceae bacteria ND2006 LbCas12a and Acidaminococcus species AsCas12a; and 5'-TTV-3' for Francisella novicida FnCas12a. Recent surveys of orthologs have revealed proteins with less restrictive PAM sequences that are also active in mammalian cell culture, such as YTV, YYN, or TTN. However, these enzymes do not fully encompass the biodiversity and targetability of VA and may not represent all possible activities and PAM sequence requirements. Here, we have pulled thousands of genomic fragments from multiple metagenomes for type VA nucleases. The documented diversity of VA enzymes may be expanded, and novel systems may be developed into highly targetable, compact, and precise gene editing agents.

[0056] Exemplary embodiments In some aspects, the disclosure provides a method of editing two or more loci in a cell, the method comprising contacting or introducing to the cell (a) a Class 2, Type II Cas endonuclease complex comprising (i) a Class 2, Type II Cas endonuclease and (ii) one or more engineered guide RNAs comprising an RNA sequence configured to bind to the Class 2, Type II Cas endonuclease and a spacer sequence configured to hybridize to a first set of one or more target loci. In some embodiments, the method comprises contacting or introducing to the cell (b) a Class 2, Type V Cas endonuclease complex comprising (i) a Class 2, Type V Cas endonuclease and (ii) one or more engineered guide RNAs comprising an RNA sequence configured to bind to the Class 2, Type V Cas endonuclease and a spacer sequence configured to hybridize to a second set of one or more target loci. In some embodiments, the Cas endonuclease is contacted in the form of a ribonucleoprotein (RNP) particle (e.g., in the case of lipid-based or electroporation / nucleofection-based transfection). In some embodiments, the Cas endonuclease is introduced in the form of a sequence encoding the endonuclease or an associated guide RNA (e.g., in the case of a vector or in vitro transcribed mRNA). In some embodiments, the edit comprises the insertion of an indel, a premature stop codon, a missense codon, a frameshift mutation, an adenine deamination, a cytosine deamination, or any combination thereof.

[0057] The Cas endonuclease may be a specific Cas endonuclease, may be introduced under specific parameters, or may be introduced in a manner to achieve a specific target metric. In some embodiments, the Class 2, Type II Cas endonuclease is not a Cas9 endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease is a Cas12a endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1 or 4, or a variant thereof. In some embodiments, the Class 2, Type V Cas endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 7 or a variant thereof. In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 3, 6, or 9. In some embodiments, the method edits the genomic sequence of the first locus with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, and / or the second locus with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease is introduced at a concentration of 200 pmol or less, 100 pmol or less, 50 pmol or less, 25 pmol or less, 5 pmol or less, or 1 pmol or less. In some embodiments, the off-target sites are disrupted at a frequency of less than 0.2% as determined by genome-wide off-target double-stranded break analysis.In some embodiments, the off-target sites are disrupted at a frequency of less than 0.01% as determined by genome-wide off-target double-stranded break analysis. In some embodiments, genome-wide off-target double strand break analysis is performed using an HTGTS assay (high-throughput, genome-wide translocation sequencing; see, e.g., Chiarle et al. Cell. 2011 Sep 30;147(1):107-19. doi:10.1016 / j.cell.2011.07.049, expressly incorporated by reference for all purposes), a LAM-HTGTS assay (linear amplification mediated high-throughput genome-wide sequencing; see, e.g., Hu et al. Nat Protoc. 2016.11(5):853-71. doi:10.1038 / nprot.2016.043, expressly incorporated by reference for all purposes), or a Digenome-Seq assay (in vitro Cas-digested whole genome sequencing; see, e.g., Kim et al. Nat Methods. 2015.12(3):237-43. doi:10.1038 / nmeth.3284 (expressly incorporated by reference herein for all purposes).

[0058] Targeted loci can include any locus. Targeted loci can be loci of particular therapeutic interest, such as T cell receptor (TCR) loci (including the constant region of the TCR locus, which is a conserved subtype of T cells, such as TRAC and TRBC), glucocorticoid receptor loci (also known as GR loci), loci encoding other nuclear hormone receptors (e.g., estrogen receptor, progesterone receptor, or androgen receptor loci), or loci encoding particular oncogenes or tumor suppressors. In some embodiments, the first set of one or more target loci or the second set of one or more target loci include T cell receptor (TCR) loci. In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci or the spacer sequence configured to hybridize to the second set of one or more target loci has at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 10-15. In some embodiments, the first set of one or more target loci or the second set of one or more target loci comprise a Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) locus. In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci or the spacer sequence configured to hybridize to the second set of one or more target loci has at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 16, 20, 21, or 22.

[0059] Any of the editing methods used herein can be used in conjunction with a donor nucleic acid molecule to introduce a transgene, for example, by homologous recombination at one of the sites targeted by a Cas enzyme or Cas complex. In some embodiments, the method further comprises introducing into the cell a donor DNA sequence comprising an open reading frame encoding a transgenic version of an endogenous gene, a first homologous arm comprising a DNA sequence located on a first side of the target sequence within the locus of the endogenous gene, and a second homologous arm comprising a DNA sequence located on a second side of the second target sequence. In some cases, the transgene may be a CAR-T molecule. In some embodiments, the method further comprises introducing into the cell a donor DNA sequence comprising an open reading frame encoding a heterologous engineered T cell receptor molecule, a first homologous arm comprising a DNA sequence located on a first side of the target sequence within the TCR locus, and a second homologous arm comprising a DNA sequence located on a second side of the second target sequence.

[0060] In some aspects, the disclosure provides a method of generating a glucocorticoid-resistant engineered T cell, the method comprising introducing into a T cell, or a precursor thereof, (a) an RNA-guided endonuclease complex targeting a T cell receptor (TCR) locus, the RNA-guided endonuclease complex comprising: (i) a first RNA-guided endonuclease, or DNA encoding the first RNA-guided endonuclease; and (ii) a first engineered guide RNA, the first engineered guide RNA comprising an RNA sequence configured to form a complex with the first RNA-guided endonuclease, and a first spacer sequence configured to hybridize to at least a portion of the TCR locus. In some embodiments, the method further comprises introducing into said T cell or said precursor thereof (b) an RNA-guided endonuclease complex targeting a T cell receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) locus, the RNA-guided endonuclease complex comprising (i) a second RNA-guided endonuclease and (ii) a second engineered guide RNA, the second engineered guide RNA comprising an RNA sequence configured to form a complex with the second RNA-guided endonuclease and a second spacer sequence configured to hybridize to at least a portion of the NR3C1 locus. In some embodiments, the at least a portion of the TCR locus is within the T cell locus. In some embodiments, the method further comprises introducing into the cell (b) a donor DNA sequence comprising an open reading frame encoding a heterologous engineered T cell receptor molecule, a first homologous arm comprising a DNA sequence located on a first side of the target sequence, within the TCR locus, and a second homologous arm comprising a DNA sequence located on a second side of the second target sequence.

[0061] A type II or type V endonuclease may comprise a particular Cas endonuclease. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease comprises a class 2, type II, or class 2, type V Cas endonuclease. In some embodiments, the first RNA-guided endonuclease comprises the class 2, type II Cas endonuclease and the second RNA-guided endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the second RNA-guided endonuclease comprises the class 2, type II Cas endonuclease and the first RNA-guided endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease comprises a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1, 4, or 7. In some embodiments, the first engineered guide RNA or the second engineered guide RNA comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 3, 6, or 9. In some embodiments, the first RNA-guided endonuclease or the second RNA-guided endonuclease is present at a concentration of 100 pmol or less, 50 pmol or less, 25 pmol or less, 5 pmol or less, or 1 pmol or less.

[0062] Any of the editing methods used herein can be used in conjunction with a donor nucleic acid molecule to introduce a transgene, for example, by homologous recombination at one of the sites targeted by a Cas enzyme or Cas complex. In some embodiments, the heterologous engineered T cell receptor is a CAR molecule. In some embodiments, the at least a portion of the T cell receptor locus is a T cell receptor alpha constant (TRAC) locus or a T cell receptor beta constant (TRBC) locus. In some embodiments, the at least a portion of the T cell receptor locus is a TRAV locus or a TRAJ locus. In some embodiments, the at least a portion of the T cell receptor locus is a TRBV locus or a TRBJ locus. In some embodiments, the homology arms include an intronic or exonic region within a TCR locus proximal to the at least a portion of the T cell receptor locus. In some embodiments, the at least a portion of the T cell receptor locus is the first or third exon of TRAC. In some embodiments, the method disrupts genomic sequences at the TCR locus and the NR3C1 locus with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the efficiency is determined by flow cytometry on protein expressed from the TCR locus or the NR3C1 locus. In some embodiments, the at least a portion of the NR3C1 locus is exon 2 or exon 3. In some embodiments, the method produces cells positive for the CAR molecule and negative for NR3C1 with at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more efficiency. In some embodiments, the method comprises simultaneously introducing (a)-(c) into said T cell or said precursor thereof. In some embodiments, said T cell or said precursor thereof comprises a T cell, a hematopoietic stem cell (HSC), or a peripheral blood mononuclear cell (PBMC).In some embodiments, the second spacer sequence comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 16, 20, 21, or 22. In some embodiments, the first or second spacer sequence comprises at least about 19-24 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, or at least about 24 nucleotides.

[0063] The donor sequence used in conjunction with the methods described herein may be provided in various forms in the method. In some embodiments, the donor sequence is provided in the form of a nucleic acid molecule (e.g., single-stranded or double-stranded DNA, or RNA). In some embodiments, the donor sequence is provided in a vector (e.g., a plasmid, a YACmid, a BACmid, a phagemid, or a viral vector). In the case of a viral vector, the viral vector can include an AAV virus having a specific serotype. In some embodiments, the donor DNA sequence is delivered in a viral vector. In some embodiments, the viral vector is an AAV vector or an AAV-6 vector.

[0064] In some aspects, the disclosure provides a population of glucocorticoid-resistant CAR-T cells comprising (a) a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of any one of SEQ ID NOs: 10-15 in a TCR locus. In some embodiments, the population further comprises (b) an NR3C1 locus comprising an indel. In some embodiments, the heterologous sequence is an indel. In some embodiments, the heterologous sequence comprises an open reading frame comprising a nucleotide sequence encoding a heterologous T cell receptor or CAR molecule. In some embodiments, the NR3C1 locus comprises an indel within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of any one of SEQ ID NOs: 16, 20, 21, or 22. In some embodiments, less than 0.2% of the cells of the population have an indel at an off-target locus as determined by genome-wide off-target double-stranded break analysis. In some embodiments, less than 0.01% of cells in the population have indels at off-target loci as determined by genome-wide off-target double-stranded break analysis. In some embodiments, genome-wide off-target double strand break analysis is performed using an HTGTS assay (high-throughput, genome-wide translocation sequencing; see, e.g., Chiarle et al. Cell. 2011 Sep 30;147(1):107-19. doi:10.1016 / j.cell.2011.07.049, expressly incorporated by reference for all purposes), a LAM-HTGTS assay (linear amplification mediated high-throughput genome-wide sequencing; see, e.g., Hu et al. Nat Protoc. 2016.11(5):853-71. doi:10.1038 / nprot.2016.043, expressly incorporated by reference for all purposes), or a Digenome-Seq assay (in vitro Cas-digested whole genome sequencing; see, e.g., Kim et al. Nat Methods. 2015.12(3):237-43. doi:10.1038 / nmeth.3284 (expressly incorporated by reference herein for all purposes).In some embodiments, the population of cells is substantially free of chromosomal translocations.

[0065] In some aspects, the disclosure provides a cell produced by any of the methods described herein.

[0066] In some aspects, the disclosure provides protein or nucleotide sequences provided in Table 1 below.

[0067] [Table 1-1]

[0068] [Table 1-2]

[0069] [Table 1-3]

[0070] [Table 1-4]

[0071] [Table 1-5]

[0072] [Table 1-6]

[0073] [Table 1-7]

[0074] [Table 1-8]

[0075]

Table 1-9

[0076]

Table 1-10

[0077]

Table 1-11

[0078]

Table 1-12

[0079]

Table 1-13

[0080]

Table 1-14

[0081]

Table 1-15

[0082]

Table 1-16

[0083]

Table 1-17

[0084]

Table 1-18

[0085] [Table 1-19]

[0086] [Table 1-20]

[0087] [Table 1-21]

[0088] [Table 1-22]

[0089] In some cases, any of the endonucleases described herein may include variants having one or more nuclear localization sequences (NLSs). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. The NLS may be added to the N-terminus or C-terminus of any one of SEQ ID NOs: 25-40, or a variant having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 25-40. In some cases, the NLS may comprise a sequence substantially identical to any one of SEQ ID NOs:25-40.

[0090] [Table 2]

[0091] In some cases, any of the endonuclease methods described herein can further comprise introducing a single-stranded or double-stranded DNA repair template into the cell. In some cases, the engineered nuclease system further comprises a single-stranded DNA repair template. In some cases, the engineered nuclease system further comprises a double-stranded DNA repair template. In some cases, the single-stranded or double-stranded DNA repair template can comprise, in a 5' to 3' direction, a first homologous arm comprising a sequence of at least 20 nucleotides 5' to the target deoxyribonucleic acid, a synthetic DNA sequence of at least 10 nucleotides, and a second homologous arm comprising a sequence of at least 20 nucleotides 3' to the target sequence.

[0092] In some cases, the first homology arm comprises a sequence of at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, or at least 1000 nucleotides. In some cases, the second homology arm comprises a sequence of at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, or at least 1000 nucleotides.

[0093] In some cases, the first and second homologous arms are homologous to a prokaryotic genomic sequence. In some cases, the first and second homologous arms are homologous to a bacterial genomic sequence. In some cases, the first and second homologous arms are homologous to a fungal genomic sequence. In some cases, the first and second homologous arms are homologous to a eukaryotic genomic sequence.

[0094] In some cases, any of the endonuclease methods described herein can further comprise introducing a DNA repair template into the cell. The DNA repair template can comprise a double-stranded DNA segment. The double-stranded DNA segment can be flanked by one single-stranded DNA segment. The double-stranded DNA segment can be flanked by two single-stranded DNA segments. In some cases, the single-stranded DNA segment is conjugated to the 5' end of the double-stranded DNA segment. In some cases, the single-stranded DNA segment is conjugated to the 3' end of the double-stranded DNA segment.

[0095] In some cases, the single stranded DNA segment has a length of 1-15 nucleotide bases. In some cases, the single stranded DNA segment has a length of 4-10 nucleotide bases. In some cases, the single stranded DNA segment has a length of 4 nucleotide bases. In some cases, the single stranded DNA segment has a length of 5 nucleotide bases. In some cases, the single stranded DNA segment has a length of 6 nucleotide bases. In some cases, the single stranded DNA segment has a length of 7 nucleotide bases. In some cases, the single stranded DNA segment has a length of 8 nucleotide bases. In some cases, the single stranded DNA segment has a length of 9 nucleotide bases. In some cases, the single stranded DNA segment has a length of 10 nucleotide bases.

[0096] In some cases, the single-stranded DNA segment has a nucleotide sequence that is complementary to a sequence within the spacer sequence. In some cases, the double-stranded DNA sequence comprises a barcode, an open reading frame, an enhancer, a promoter, a protein coding sequence, an miRNA coding sequence, an RNA coding sequence, or a transgene.

[0097] In some cases, sequence identity as described herein may be determined by the BLASTP, CLUSTALW, MUSCLE, or MAFFT algorithms, or the CLUSTALW algorithm using Smith-Waterman homology search algorithm parameters. Sequence identity may be determined by the BLASTP homology search algorithm using the BLOSUM62 scoring matrix setting parameters of word length (W) of 3, expectation (E) of 10, and gap costs at presence of 11, extension of 1, and using a conditional composition score matrix adjustment.

[0098] The disclosed systems or methods may be used for a variety of applications, such as, for example, binding to nucleic acid molecules (e.g., sequence-specific binding), nucleic acid editing (e.g., gene editing), etc. Such systems or methods may be used, for example, to address (e.g., remove or replace) genetically inherited mutations that may cause disease in a subject, to inactivate genes to confirm their function in cells, as diagnostic tools to detect disease-causing genetic elements (e.g., via cleavage of reverse-transcribed viral RNA or amplified DNA sequences that code for disease-causing mutations), as inactivated enzymes combined with probes to target specific nucleotide sequences (e.g., sequences that code for antibiotic resistance in bacteria), to inactivate viruses by targeting viral genomes or to prevent them from infecting host cells, to add genes or modify metabolic pathways to engineer organisms to produce valuable small molecules, macromolecules, or secondary metabolites, to establish gene drive elements for evolutionary selection, and as biosensors to detect cellular perturbations by exogenous small molecules and nucleotides. EXAMPLES

[0099] Example 1 - Editing at the TCAR locus A workflow was developed for producing CAR-T cells (and other T cell-like cells carrying heterologous T cell receptors) using the nucleases described herein (Figure 1). Thus, nuclease complexes for targeting T cell receptor loci (e.g., the TRAC locus) were developed. Spacer sequences (SEQ ID NOs: 10-15) were developed for targeting the TRAC gene in combination with the class 2 type II endonuclease MG3-6 (SEQ ID NO: 1), or SpCas9, or the class 2, type V endonuclease MG29-1 (SEQ ID NO: 7), and introduced into the corresponding sgRNA of each enzyme (see Table 1). RNP complexes containing each TRAC-targeting sgRNA were assembled and nucleofected into human T cells (200,000 T cells using a Lonza 4-D Nucleofector with program EO-115 and P3 buffer) that had been cultured for 4 days after purification from PBMCs by negative selection using (Stemcell Technologies Human T cell Isolation Kit #17951) and activation with CD2 / 3 / 28 beads (Miltenyi T cell Activation / Expansion Kit #130-091-441). Cells were analyzed by next-generation sequencing (NGS) for indel formation in the TRAC gene (Figure 2, left) and by flow cytometry for TCR expression together with mock-transfected T cells (Figure 2, right). Analysis by both NGS and flow cytometry showed that MG3-6 and MG29-1 were as good or better than SpCas9 at inducing indel formation or disruption of T cell receptor expression in transfected T cells.

[0100] Next, we tested the editing ability using the RNP complex described above to promote targeted integration of the CAR-T molecule into the TRAC locus. We developed an AAV-6 vector containing a nucleotide sequence that includes a CAR-T molecule flanked by 5' and 3' homology arms (e.g., SEQ ID NOs: 23-24) that target the TRAC gene. TRAC targeting using MG29-1 RNP as described above was performed, but then 100,000 vector genomes (vg) of the AAV-6 vector were added to the T cells after transfection. The cells were analyzed for TCR receptors and for expression of the CAR antigen-binding domain using flow cytometry (Figure 3). Flow cytometry showed that approximately 60% of the T cells treated with the AAV-endonuclease combination expressed the CAR antigen-binding domain. Similar results were obtained in experiments combining the AAV-6 / CAR-T integration construct with MG3-6 and MG3-8 RNPs targeting TRAC.

[0101] Example 2 - Multiplex editing in TCR-like cells In combination with the modification of the TRAC locus (e.g., CAR-T integration), it may be advantageous to modify other genes. Thus, the ability of the nuclease complex described herein to target TRAC and additional loci was determined. One such locus is the NR3C1 (also known as GR, or glucocorticoid receptor) locus, which may be advantageous to destroy to render CAR-T cells unresponsive to glucocorticoid drugs (e.g., in cancer patients who are concurrently treated with glucocorticoids, or in cancer patients with autoimmune disorders that require glucocorticoid maintenance). Three MG29-1 compatible targeting sequences (target BD; SEQ ID NO: 20, 21, 22) were designed to target the NR3C1 gene and incorporated into the MG29-1 guide RNA. RNP complexes containing MG29-1 with these guide RNAs were assembled. T cells were treated by nucleofection with various combinations of MG29-1 / NR3C1 gRNA RNP complexes and MG3-6 / TRAC RNPs as described above (Figure 4). After nucleofection, cells were analyzed using NGS to evaluate indel formation at each locus. Results showed that while different guide RNAs had different efficiencies of targeting NR3C1 (see "MG29-1 GR-13", "MG29-1 GR-28", "MG29-1 GR-29"), the combination of MG3-6 complexes targeting TRAC and MG29-1 complexes targeting NR3C1 was able to efficiently induce indel formation in both genes (see the three conditions at the far right of Figure 4).

[0102] After establishing the ability to target two different loci using the nuclease complexes described herein, the ability to target three loci (e.g., selected from TRAC, locus B-29 / SEQ ID NO:16, locus C-87 / SEQ ID NO:17, locus C-74 / SEQ ID NO:18, or locus C-83 / SEQ ID NO:19) was evaluated by nucleofecting T cells with RNPs corresponding to each of the loci alone and in combination of the three, as described above, and assessing indel formation by NGS (see FIG. 5). The results of this experiment showed that indels at all three loci were produced in significant amounts, even in conditions where three different RNPs targeting different loci were combined.

[0103] Example 3 - Multiplex editing with multiple gene replacement After establishing the ability to edit multiple loci and integrate a transgene at at least one locus, the ability to simultaneously edit two or more loci and integrate genes at both loci simultaneously was tested by editing two different loci in T cells and providing two different donor DNA templates targeting the two different loci. The AAVS1 (safe harbor) locus and the TRAC locus described above were selected as target sites. Primary T cells (2×10 5) were nucleofected with a combination of (a) SpCas9 (12 pmol) and a compatible sgRNA targeting the AAVS1 locus (60 pmol, SEQ ID NO: 41 shows the spacer sequence), and (b) MG3-6 (52 pmol) and a compatible TRAC3-6 6 sgRNA (60 pmol, SEQ ID NO: 10). After nucleofection, cells were incubated with two different AAV-6 vectors: (a) one carrying a transgene containing each of the four different isoforms of GR (GR-alpha, GR-beta, GR-alphaD3, and GR-betaD3) flanked by 5' and 3' homology arms targeting the AAVS1 locus (SEQ ID NOs: 42 and 43), and (b) one carrying a CAR flanked by 5' and 3' homology arms targeting the TRAC locus (SEQ ID NOs: 23-24), each at a multiplicity of infection (MOI) of 50,000. After 4 days of incubation, T cells were analyzed by (a) PCR for the presence of the GR transgene at the AAVS1 locus (see FIG. 6 for PCR design and results) or (b) flow cytometry for the CAR antigen-binding domain and T cell receptor to assess the integration of the CAR at the TRAC locus (FIG. 7). Data from PCR and flow cytometry experiments showed that both transgenes (GR and CAR) could be inserted simultaneously without significant loss of performance, with PCR for the GR transgene under dual AAVS1 / TCR targeting conditions (middle four lanes, FIG. 6B) showing similar integration results as AAVS1 targeting alone (last four lanes, FIG. 7B or middle four lanes, FIG. 7C), whereas flow cytometry for TCR (FIGS. 7A, 7B, 7C, 7D) showed high integration of the CAR and loss of the TCR, even when AAVS1 was targeted simultaneously.

[0104] Example 4 - Specificity analysis by genome-wide off-target double-strand break analysis The target specificity of MG3-6, MG3-8, and MG29-1 was evaluated through genome-wide off-target double-strand break analysis with SpCas9 ("Cas9"). The results are shown in Figure 8. The results showed that MG3-6, MG3-8, and MG29-1 had lower levels of off-target editing than Cas9.

[0105] Example 5 - Multiplex editing in T cells Creating a recombinant TCR-based T cell product requires the introduction of new, desired alpha and beta chains of the TCR into a pool of T cells, since the a / b chains are the subunits of the TCR that confer antigen-specific recognition. These new a / b chains can then assemble with delta / gamma / epsilon chains to create an active, complete TCR (see FIG. 9). Unfortunately, in this simple case, the existing a / b chains are still expressed in the recipient cells. This introduces the undesirable possibility that the existing alpha can pair with the new beta, and the new alpha can pair with the existing beta, e.g., the new, desired TCR a / b chains do not know that they are "supposed" to pair together. Without further action, the T cells now express four different TCRs (a / b, a' / b, a / b', a' / b'), one with the engineered specificity. This creates two problems: i) it reduces the expression of new, desirable TCRs by 4-fold, ii) it poses a risk of autoimmunity since the two hybrid a / b pairs (a' / b and a / b') recognize antigens in an unpredictable and potentially autoreactive manner.

[0106] In this experiment, primary T cells grown on CD2 / 3 / 28 beads were nucleofected using a Lonza 4D electroporator and solution P3 with 200K cells per condition to deliver 104 pmol of MG3-6 protein and 128 pmol of guide RNA, or the same amount of type V enzyme MG29-1, or both MG3-6 and MG29-1. The MG3-6 guides used were MG3-6-TRAC-6 (SEQ ID NO: 44) and MG3-6-TRBC (SEQ ID NO: 45), 22 nt in length. Genomic DNA was harvested from these cells after 3 days and analyzed by NGS (see FIG. 10). The results in FIG. 10 showing the percentage of sequences at the target site with indels demonstrate that there is a double TRAC / TRBC knockout in these cells when RNPs targeting both sites are introduced into the cells simultaneously.

[0107] Example 6 - Gene editing results by flow cytometry for single gene knockouts. Primary T cells were purified from PBMCs (peripheral blood mononuclear cells) using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of RNP (100 pmol protein / 150 pmol guide RNA) into T cells (200,000) was performed using a Lonza 4D electroporator. For analysis by flow cytometry, 3 days after nucleofection, 100,000 T cells were stained with anti-CD3 and anti-B2M antibodies for 30 min at 4°C and analyzed on an Attune Nxt flow cytometer (Figure 11). FIG. 11, which shows the percentage of analyzed cells containing each of the four phenotypes assessing TCR and B2M knockout, shows that (a) all of the TCR targeting conditions efficiently resulted in TCR knockout, with MG3-6 TRAC6 and MG3-6 TRBC E2 sgRNAs resulting in the most efficient TCR knockout, and (b) all of the B2M targeting conditions resulted in B2M knockout, with B2M H1 and B2M D2 resulting in the most efficient B2M knockout.

[0108] Example 7 - Gene editing results by flow cytometry for double gene knockout. After evaluating the performance of TCR / B2M targeting conditions alone in Example 6, simultaneous double cleavage using a combination of conditions was also tested for TRAC and B2M targeting (Figure 12). Primary T cells were purified from PBMCs using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of RNP (100 pmol protein / 150 pmol guide RNA) into T cells (200,000) was performed using a Lonza 4D electroporator. For analysis by flow cytometry, 3 days after nucleofection, 100,000 T cells were stained with anti-CD3 and anti-B2M antibodies for 30 minutes at 4°C and analyzed on an Attune Nxt flow cytometer (Figure 12). Figure 12, which shows the percentage of analyzed cells containing each of the four phenotypes evaluating TCR and B2M knockout, shows that the most efficient dual targeting conditions were A4, B4, and C4, which involved MG3-6 TRAC6 conditions along with MG29-1 B2M H1, D2, or A3 conditions. The most efficient dual targeting condition appeared to be B4, which used MG3-6 TRAC6 sgRNA and MG29-1 B2M D2 sgRNA.

[0109] Example 8 - Gene editing results by flow cytometry for triple gene knockout. After evaluating the performance of TCR / B2M targeting conditions alone in Example 6 and dually in Example 7, simultaneous dual cleavage using combinations of conditions was also tested for simultaneous TRAC, TRBC, and B2M targeting. Primary T cells were purified from PBMCs using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of RNP (100 pmol protein / 150 pmol guide RNA) into T cells (200,000) was performed using a Lonza 4D electroporator. For analysis by flow cytometry, 3 days after nucleofection, 100,000 T cells were stained with anti-CD3 and anti-B2M antibodies for 30 minutes at 4°C and analyzed on an Attune Nxt flow cytometer (Figure 13). Flow cytometry results show that conditions B2, E1, and F1 were the most efficient triple targeting conditions for knockout.

[0110] Cells were harvested and genomic DNA was prepared 5 days after transfection. PCR primers suitable for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences of each guide RNA. Amplicons were sequenced on an Illumina MiSeq machine and analyzed using a custom Python script to measure gene editing (Figure 14). The sequencing results here contradicted those in Figure 13, as indels may not necessarily reflect functional disruption of a gene (as measured by flow cytometry).

[0111] [Table 3]

[0112] Therefore, additional analysis was performed to verify the generation of triple knockout cells by sequencing (see FIG. 15). Data demonstrating the successful generation of triple knockout cells are shown in FIG. 15. The data in the "Editing" column is taken from FIG. 14, and the data in the "Wild Type" column is 100% minus the percentage of editing. The minimum (Min.) frequency of double and triple knockouts is calculated assuming minimal overlap between editing events in individual cells. Therefore, the minimum double knockout frequency between TRBC and B2M is 100% minus the percentage of wild type cells for TRBC and the percentage of wild type cells for B2M. Therefore, the minimum triple knockout frequency is 100% minus the percentage of cells that may not contain a double knockout minus the percentage of wild type cells for TRAC. The high editing frequency observed excludes the possibility that all of the editing events occurred in separate cells. Thus, the data in FIG. 15 demonstrates the successful generation of triple knockout TRAC / TRBC / B2M cells.

[0113] Example 9 - Expression of GFP and surface markers in edited T cells Primary human T cells were purified from PBMCs using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of MG3-6 mRNA (500ng / 150pmol guide), MG29-1 RNP (100pmol / 150pmol guide), and / or SpCas9 RNP (12pmol / 60pmol guide) into T cells (200,000) was performed using a Lonza 4D electroporator. After nucleofection, cells were immediately harvested in medium containing AAV-6 (50,000 MOI). The AAV vectors used included (a) an AAV vector delivering an MSCV promoter-driven truncated low affinity nerve growth factor receptor (tLNGFR) coding sequence flanked by homology arms corresponding to the cleavage sites in MG3-6-TRAC-6 (SEQ ID NO: 64) or MG29-1-TRAC-35 (SEQ ID NO: 65), and (b) an AAV vector delivering an MND promoter-driven polycistronic construct encoding GFP along with a truncated form of the epidermal growth factor receptor (tEGFR) flanked by homology arms corresponding to the cleavage sites in Mali et al. AAVS1 T2 (SEQ ID NO: 63). Four days after transfection, 100,000 cells were stained for viability (Live / Dead Fixable Aqua Cell Stain Kit; ThermoFisher Scientific) and expression of tLNGFR (CD271) (VioBlue REAfinity™, clone REA844; Miltenyi Biotech), tEGFR (Cetuximab Biosimilar, AlexaFluor® 647, clone Hu1; R&D Systems), and TCR a / b (Brilliant Violet 785, clone IP26; BioLegend). Cells were stained for 30 minutes at 4° C. and data were acquired on an Attune NxT flow cytometer. Cells expressing tLNGFR, GFP, tEGFR, and / or TCR a / b were gated on single live cells ( FIG. 16 ).

[0114] Example 10 - Indel analysis at the AAVS1 site in edited T cells Primary T cells were purified from PBMCs using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of MG3-6 mRNA (500ng / 150pmol guide), MG29-1 RNP (100pmol / 150pmol guide), and / or SpCas9 RNP (12pmol / 60pmol guide) into T cells (200,000) was performed using a Lonza 4D electroporator. After nucleofection, cells were immediately harvested in medium containing AAV-6 (50,000 MOI). The AAV vectors used included an MSCV promoter-driven truncated low affinity nerve growth factor receptor (tLNGFR) coding sequence flanked by homology arms corresponding to the cleavage sites in MG3-6-TRAC-6 or MG29-1-TRAC-35, an MND promoter-driven polycistronic construct encoding GFP, and a truncated form of epidermal growth factor receptor (tEGFR) flanked by homology arms corresponding to the cleavage sites in Mali et al. AAVS1 T2. Cells were harvested and genomic DNA was prepared 4 days after transfection. PCR primers suitable for use in NGS-based DNA sequencing were generated, optimized, and used to amplify regions containing the target sites of the different AAVS1 site-specific RNA guides used in these experiments. Amplicons were sequenced on an Illumina MiSeq machine and analyzed using a proprietary Python script to measure gene editing (Figure 17). The results showed that the most efficient dual targeting conditions for TRAC and AAVS1 were those involving MG29-1 with sgRNA F3 and MG3-6 with sgRNA TRAC3-6 #6.

[0115] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing description, the description and explanation of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it will be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. It is therefore contemplated that the present invention will encompass any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention and its methods and structures within the scope of these claims and their equivalents encompassed thereby.

Claims

1. 1. A method for editing two or more genetic loci in a cell, comprising administering to the cell: (a) a class 2, type II Cas endonuclease or a nucleic acid encoding the class 2, type II Cas endonuclease; (b) a first engineered guide ribonucleic acid or a nucleic acid encoding said first engineered guide ribonucleic acid, said first engineered guide ribonucleic acid comprising: (i) a first ribonucleic acid sequence configured to bind to the class 2, type II Cas endonuclease; and (ii) a first engineered guide ribonucleic acid or a nucleic acid encoding said first engineered guide ribonucleic acid, the first spacer sequence being configured to hybridize to a first set of one or more target loci; (c) a class 2, type V Cas endonuclease or a nucleic acid encoding the class 2, type V Cas endonuclease; (d) a second engineered guide ribonucleic acid or a nucleic acid encoding said second engineered guide ribonucleic acid, said second engineered guide ribonucleic acid comprising: (i) a second ribonucleic acid sequence configured to bind to the Class 2, Type V Cas endonuclease; and (ii) introducing a second engineered guide ribonucleic acid, or a nucleic acid encoding said second engineered guide ribonucleic acid, comprising a second spacer sequence configured to hybridize to a second set of one or more target loci.

2. The method of claim 1, wherein the class 2, type V Cas endonuclease is a Cas12a endonuclease.

3. The method described in claim 1 or 2, wherein the class 2, type V Cas endonuclease comprises a sequence having at least 75% sequence identity to SEQ ID NO:

7.

4. The method of claim 1, wherein the class 2, type V Cas endonuclease comprises the sequence of SEQ ID NO:

7.

5. A method described in any one of claims 1 to 4, wherein the second manipulated guide guide ribonucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO:

9.

6. A method described in any one of claims 1 to 5, wherein the second manipulated guide guide ribonucleic acid comprises the sequence of SEQ ID NO:

9.

7. The method of claim 1, wherein the Class 2, Type II Cas endonuclease comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 75% sequence identity to SEQ ID NO:

62.

8. The method of claim 1, wherein the class 2, type II Cas endonuclease comprises a polypeptide sequence encoded by SEQ ID NO:

62.

9. The method of claim 1, wherein the first manipulated guide ribonucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO:

3.

10. The method of claim 1, wherein the first manipulated guide ribonucleic acid comprises the sequence of SEQ ID NO:

3.

11. The method of any one of claims 1 to 10, wherein the first set of one or more target loci or the second set of one or more target loci include a T cell receptor (TCR) locus, an albumin (ALB) locus, or a nuclear receptor subfamily 3 group C member 1 (NR3C1) locus.

12. The method of any one of claims 1 to 11, wherein the first spacer sequence configured to hybridize to the first set of one or more target loci, or the second spacer sequence configured to hybridize to the second set of one or more target loci, has at least 80% sequence identity to any one of SEQ ID NOs: 10 to 22, or their complements.

13. A method according to any one of claims 1 to 12, wherein the editing comprises the insertion of an indel, a premature stop codon, a missense codon, a frameshift mutation, adenine deamination, cytosine deamination, or any combination thereof.

14. A method for producing engineered T cells resistant to glucocorticoids, comprising administering to a T cell or a precursor thereof: (a) a first ribonucleic acid-guided endonuclease or a nucleic acid encoding said first ribonucleic acid-guided endonuclease; (b) a first engineered guide ribonucleic acid or a nucleic acid encoding said first engineered guide ribonucleic acid, said first engineered guide ribonucleic acid comprising: (i) a first ribonucleic acid sequence configured to form a complex with the first ribonucleic acid guide endonuclease; and (ii) a first engineered guide ribonucleic acid or a nucleic acid encoding said first engineered guide ribonucleic acid, comprising a first spacer sequence configured to hybridize to at least a portion of a T cell receptor (TCR) locus; (c) a second ribonucleic acid-guided endonuclease or a nucleic acid encoding the second ribonucleic acid-guided endonuclease; (d) a second engineered guide ribonucleic acid or a nucleic acid encoding said second engineered guide ribonucleic acid, said second engineered guide ribonucleic acid comprising: (i) a second ribonucleic acid sequence configured to form a complex with the second ribonucleic acid guide endonuclease; and (ii) introducing a second engineered guide ribonucleic acid, or a nucleic acid encoding the second engineered guide ribonucleic acid, comprising a second spacer sequence configured to hybridize to at least a portion of the T cell receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) locus.

15. The method of claim 14, wherein the first ribonucleic acid-guided endonuclease is a class 2, type II Cas endonuclease and the second ribonucleic acid-guided endonuclease is a class 2, type V Cas endonuclease. (a) a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of a hybridization region of any one of SEQ ID NOs: 10-15 within a TCR locus; and (b) the NR3C1 locus containing an indel; A population of glucocorticoid-resistant T cells comprising:

17. The population of glucocorticoid-resistant T cells described in claim 16, wherein the heterologous sequence is an indel.

18. An isolated nucleic acid comprising any one of SEQ ID NOs:63-65, or a sequence having at least 75% sequence identity thereto.

19. A cell comprising the isolated nucleic acid described in claim 18.

20. The cell described in claim 19, wherein the cell is a T cell or a precursor thereof.

21. A vector comprising the sequence of the isolated nucleic acid described in claim 18.

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