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, filed Mar. 19, 2021, entitled "MULTIPLEX EDITING WITH CAS ENZYMES"; U.S. Provisional Patent Application No. 63 / 186,506, filed May 10, 2021, entitled "MULTIPLEX EDITING WITH CAS ENZYMES"; and U.S. Provisional Patent Application No. 63 / 241,916, filed Sep. 8, 2021, entitled "MULTIPLEX EDITING WITH CAS ENZYMES", each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a sequence listing that was electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on Mar. 17, 2022 is named 55921-719-601-SL.txt and is 70,612 bytes in size.

Background Art

[0003] Cas enzymes, along with their associated clustered and regularly arranged short palindromic sequence repeats (CRISPR) guided ribonucleic acid (RNA), appear to be a widespread component of the prokaryotic immune system (approximately 45% of bacteria and 84% of archaea), helping to protect these microorganisms from non-self nucleic acids such as infectious viruses and plasmids through CRISPR-RNA guided nucleic acid cleavage. While the deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements can be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse and contain a wide variety of nucleic acid interaction domains. Although CRISPR DNA elements were observed as early as 1987, the programmable endonuclease cleavage capability of the CRISPR / Cas complex has only been recognized relatively recently, leading to the use of recombinant CRISPR / Cas systems in a wide range of DNA manipulation and gene editing applications. [Overview of the project]

[0004] In certain embodiments, the disclosure provides a method for editing two or more loci in a cell, the method comprising contacting the cell with a class 2, type II Cas endonuclease complex comprising: (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: 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 second engineered guide RNA comprising: a second engineered guide RNA comprising: 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 contains 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 with any one of sequence numbers 1 or 4, or its variants. In some embodiments, the class 2, type V Cas endonuclease contains a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to SEQ ID NO: 7 or its variants.In some embodiments, the first manipulated guide RNA or the second manipulated guide RNA contains a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of sequence numbers 3, 6, or 9. In some embodiments, the method edits the genomic sequences of the first locus and / or the second locus having at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher efficiency. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease is introduced at concentrations 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 intracellular off-target sites are disrupted at a frequency of less than 0.2% when determined by genome-wide off-target double-strand break analysis. In some embodiments, the intracellular off-target sites are disrupted at a frequency of less than 0.01% when determined by genome-wide off-target double-strand break analysis. In some embodiments, the first set of one or more target loci, or the second set of one or more target loci, includes a T cell receptor (TCR) locus. In some embodiments, the spacer sequence configured to hybridize to a first set of one or more target loci, or to a 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 with any one of sequence numbers 10-15, its complement, or its reverse complement. In some embodiments, the first set of one or more target loci, or the second set of one or more target loci, includes an albumin (ALB) locus.In some embodiments, the spacer sequence configured to hybridize to the first set of one or more target loci, or 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 with any one of sequence numbers 17-19, its complement, or its reverse complement. In some embodiments, the first set of one or more target loci, or the second set of one or more target loci, includes the nuclear receptor subfamily 3 group C member 1 (NR3C1) locus. In some embodiments, the spacer sequence configured to hybridize to a first set of one or more target loci, or to a 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 with any one of SEQ ID NOs. 16, 20, 21, or 22, its complement, or its reverse complement. In some embodiments, the method further includes 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 DNA sequences located on the first side of the first set of one or more target loci, and a second homologous arm comprising DNA sequences located on the second side of the first set of one or more target loci. In some embodiments, editing includes indel insertion, immature stop codon, missense codon, frameshift mutation, adenine deamination, cytosine deamination, or any combination thereof.

[0005] In certain embodiments, the present disclosure provides a method for producing glucocorticoid-resistant engineered T cells, the method comprising: (a) an RNA guide endonuclease complex targeting a T cell receptor (TCR) locus, comprising (i) a first RNA guide endonuclease or DNA encoding the first RNA guide endonuclease; and (ii) a first engineered guide RNA comprising an RNA sequence configured to form a complex with the first RNA guide endonuclease and a first spacer sequence configured to hybridize to at least a portion of the TCR locus, thereby targeting the TCR locus. (b) an RNA guide endonuclease complex that targets the T cell receptor NR3C1 locus, comprising (i) a second RNA guide endonuclease, and (ii) a second engineered guide RNA comprising an RNA sequence configured to form a complex with the second RNA guide endonuclease, and a second spacer sequence configured to hybridize to at least a portion of the NR3C1 locus. In some embodiments, at least a portion of the TCR locus is located within the T cell locus. In some embodiments, the method further includes introducing into the cells (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 the first side of the target sequence within the TCR locus, and a second homologous arm comprising a DNA sequence located on the second side of the second target sequence. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease comprises a class 2, type II, or class 2, type V Cas endonuclease.In some embodiments, the first RNA guide endonuclease comprises the class 2, type II Cas endonuclease, and the second RNA guide endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the second RNA guide endonuclease comprises the class 2, type II Cas endonuclease, and the first RNA guide 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, at least a portion of the T cell receptor locus is the T cell receptor alpha stationary (TRAC) locus or the T cell receptor beta stationary (TRBC) locus. In some embodiments, the homologous arm comprises an intronic or exonic region within the TCR locus proximal to at least a portion of the T cell receptor locus. In some embodiments, at least a portion of the T cell receptor locus is the first or third exon of TRAC. In some embodiments, the method disrupts the genomic sequences of the TCR locus and the NR3C1 locus with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, this efficiency is determined by flow cytometry of the proteins expressed from the TCR locus and the NR3C1 locus. In some embodiments, at least a portion of the NR3C1 locus is exon 2 or exon 3. In some embodiments, the method produces cells that are positive for CAR molecules and negative for NR3C1 with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, the method further includes simultaneously introducing (a) to (c) into the T cell or its precursor.In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease contains 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 with any one of SEQ ID NOs: 3, 6, or 9, its complement, or its reverse complement. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease is present at concentrations 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 its precursor includes a T cell, a hematopoietic stem cell (HSC), or a peripheral blood mononuclear cell (PBMC). In some embodiments, the second spacer sequence includes a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of sequence numbers 16, 20, 21, or 22, its complement, or its reverse complement. In some embodiments, the first or second spacer sequence contains at least about 19 to 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 embodiments, the disclosure provides a population of glucocorticoid-resistant T cells or their precursors, comprising (a) a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides in the hybridization region of any one of SEQ ID NOs: 10-15 within the TCR locus. In some embodiments, the T cells or their precursors further comprise (b) an NR3C1 locus containing an indel. In some embodiments, the heterologous sequence is an indel. In some embodiments, the heterologous sequence comprises an open reading frame containing 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 in the 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-strand break analysis. In some embodiments, less than 0.01% of the cells have indels at off-target loci, as determined by genome-wide off-target double-strand break analysis. In some embodiments, the population of cells is substantially free of chromosomal translocations.

[0007] In certain embodiments, the disclosure provides a method for editing two or more gene loci in a cell, the method comprising contacting the cell with a first Cas endonucleases complex comprising (a) a first Cas endonucleases complex comprising (i) a first Cas endonucleases and (ii) one or more manipulated guide RNAs comprising an RNA sequence configured to bind to class 2, type II Cas endonucleases and a spacer sequence configured to hybridize to a first target sequence; and (b) a second Cas endonucleases complex comprising (i) a second Cas endonucleases and (ii) one or more manipulated guide RNAs comprising an RNA sequence configured to bind to class 2, type II Cas endonucleases and a spacer sequence configured to hybridize to a second target sequence. In some embodiments, the method further includes introducing into the cells a first donor DNA sequence comprising (c) an open reading frame encoding a first transgene, a 5' homologous arm containing a DNA sequence located 5' to the first target sequence, and a 3' homologous arm containing 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' homologous arm containing a DNA sequence located 5' to the second target sequence, and a 3' homologous arm containing 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, 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, a truncated low-affinity nerve growth factor receptor (tLNGFR) sequence, a truncated epidermal growth factor receptor (tEGFR), a GFP coding sequence, or any combination thereof.In some embodiments, the 5' homologous arm, which includes a DNA sequence located 5' to the first target sequence, or the 5' homologous arm, which includes a DNA sequence located 5' to the second target sequence, includes sequence number 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' homologous arm, which includes a DNA sequence located 5' to the first target sequence, or the 3' homologous arm, which includes a DNA sequence located 5' to the second target sequence, includes sequence number 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 second class 2, type II Cas endonuclease includes 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. In some embodiments, the first manipulated guide RNA or the second manipulated guide RNA contains a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of sequence numbers 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 with any one of sequence numbers 16, 20, 21, 22, or 41, or its complement, its complement, or its reverse complement.In some embodiments, the first or second endonuclease includes a class 2, type II Cas endonuclease, a class 2, type V Cas endonuclease, or any combination thereof.

[0008] In some embodiments, the Disclosure provides isolated nucleic acids comprising any one of the sequences SEQ ID NOs. 63 to 65, or 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 thereto.

[0009] In some embodiments, the disclosure provides isolated nucleic acids comprising any of the nucleic acid sequences described herein, their complements, or their reverse complements. In some embodiments, the isolated nucleic acid is a guide RNA.

[0010] In some embodiments, the Disclosure provides cells comprising any of the nucleic acids described herein. In some embodiments, such cells are T cells or their precursors. In some embodiments, such T cells or their precursors include T cells, hematopoietic stem cells (HSCs), or peripheral blood mononuclear cells (PBMCs).

[0011] In some embodiments, this disclosure provides a vector comprising one 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 embodiments, 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 with any one of SEQ ID NOs. In some embodiments, the vector further comprises an adjacent transgene to the 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 with any one of SEQ ID NOs. In some embodiments, the transgene includes an 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 epidermal growth factor receptor (tEGFR), a GFP coding sequence, or any combination thereof. In some embodiments, the vector further includes the 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 includes the 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 includes the 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 the MSCV promoter of SEQ ID NO: 64, or variants having 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0013] In certain embodiments, the disclosure provides a method for editing two or more loci in a cell, the method comprising contacting or introducing a class 2, type II Cas endonuclease complex into a cell, the class 2, type II Cas endonuclease complex comprising (a) a class 2, type II 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 set of one or more target loci. In some embodiments, the method involves contacting or introducing a class 2, type V Cas endonuclease complex into the cells, comprising (b)(i) a class 2, type V Cas endonuclease and (ii) one or more engineered guide RNAs comprising an RNA sequence configured to bind to a 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 contains a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with any one of sequence numbers 1 or 4, or its variants. In some embodiments, the class 2, type V Cas endonuclease contains a sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with any one of sequence numbers 7, or its variants.In some embodiments, the first manipulated guide RNA or the second manipulated guide RNA includes a sequence having at least 80%, 85%, 90%, or 95% of any one of sequence numbers 3, 6, or 9, or its complement. In some embodiments, the method edits the genomic sequences of the first locus and / or the second locus having at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher efficiencies. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease is introduced at concentrations 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 within the cell are disrupted at a frequency of less than 0.2% when determined by genome-wide off-target double-strand break analysis. In some embodiments, the off-target sites within the cell are disrupted at a frequency of less than 0.01% when determined by genome-wide off-target double-strand break analysis.In some embodiments, genome-wide off-target double-strand break analysis is performed using HTGTS assays (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 herein for all purposes by reference)), LAM-HTGTS assays (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 herein for all purposes by reference)), or Digenome-Seq assays (in vitro Cas-digestion whole-genome sequencing; see, e.g., Kim et al. Nat This includes 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, comprises a T cell receptor (TCR) 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 with any one of sequence numbers 10-15 or its complement. 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 a first set of one or more target loci, or to a second set of one or more target loci, has at least 80%, 85%, 90%, or 95% sequence identity with any one of SEQ ID NOs. 16, 20, 21, or 22, or its complement. In some embodiments, the method further includes 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 DNA sequences located on the first side of the first set of one or more target loci, and a second homologous arm comprising DNA sequences located on the second side of the first set of one or more target loci. In some embodiments, the editing includes indel insertion, immature stop codons, missense codons, frameshift mutations, adenine deamination, cytosine deamination, or any combination thereof.

[0014] In certain embodiments, the Disclosure provides a method for producing glucocorticoid-resistant engineered T cells, the method comprising: a T cell or its precursor comprising an RNA guide endonuclease complex targeting a T cell receptor (TCR) locus, comprising (i) a first RNA guide endonuclease or DNA encoding the first RNA guide endonuclease, and (ii) a first engineered guide RNA comprising an RNA sequence configured to form a complex with the first RNA guide endonuclease and a first spacer sequence configured to hybridize to at least a portion of the TCR locus, or comprising a polynucleotide encoding the TCR locus. (b) an RNA guide endonuclease complex that targets the T cell receptor NR3C1 locus, comprising (i) a second RNA guide endonuclease, and (ii) a second engineered guide RNA comprising an RNA sequence configured to form a complex with the second RNA guide endonuclease, and a second spacer sequence configured to hybridize to at least a portion of the NR3C1 locus, or a polynucleotide encoding such a second engineered guide RNA. In some embodiments, at least a portion of the TCR locus is located within the T cell locus. In some embodiments, the method further includes introducing into the cells (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 the first side of the target sequence within the TCR locus, and a second homologous arm comprising a DNA sequence located on the second side of the second target sequence. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease comprises a class 2, type II, or class 2, type V Cas endonuclease.In some embodiments, the first RNA guide endonuclease comprises the class 2, type II Cas endonuclease, and the second RNA guide endonuclease comprises the class 2, type V Cas endonuclease. In some embodiments, the second RNA guide endonuclease comprises the class 2, type II Cas endonuclease, and the first RNA guide 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, at least a portion of the T cell receptor locus is the T cell receptor alpha stationary (TRAC) locus or the T cell receptor beta stationary (TRBC) locus. In some embodiments, the homologous arm comprises an intronic or exonic region within the TCR locus proximal to at least a portion of the T cell receptor locus. In some embodiments, at least a portion of the T cell receptor locus is the first or third exon of TRAC. In some embodiments, the method disrupts the genomic sequences of the TCR locus and the NR3C1 locus with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, this efficiency is determined by flow cytometry of the proteins expressed from the TCR locus and the NR3C1 locus. In some embodiments, at least a portion of the NR3C1 locus is exon 2 or exon 3. In some embodiments, the method produces cells that are positive for CAR molecules and negative for NR3C1 with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, the method includes simultaneously introducing (a) to (c) into the T cell or its precursor.In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease contains 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 with any one of SEQ ID NOs: 1, 4, or 7. In some embodiments, the first manipulated guide RNA or the second manipulated guide RNA contains a sequence having at least 80%, 85%, 90%, or 95% sequence identity with any one of SEQ ID NOs: 3, 6, or 9, or its complement. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease is present at concentrations 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 its precursor comprises a T cell, a hematopoietic stem cell (HSC), or a peripheral blood mononuclear cell (PBMC). In some embodiments, the second spacer sequence comprises one of sequence numbers 16, 20, 21, or 22, or a sequence having at least 80%, 85%, 90%, or 95% sequence identity with its complement. In some embodiments, the first or second spacer sequence comprises at least about 19 to 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 embodiments, the Disclosure provides a population of T cells comprising (a) a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of the hybridization region of any one of SEQ ID NOs: 10-15 within the TCR locus, or a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of the hybridization region of SEQ ID NO: 42. In some embodiments, the population of T cells further comprises (b) an NR3C1 locus containing an indel. In some embodiments, the indel in the NR3C1 locus confers glucocorticoid resistance to the T cells. The heterologous sequence within 100, 75, 50, 25, or 10 nucleotides of the hybridization region of the heterologous sequence is an indel. In some embodiments, the heterologous sequence comprises an open reading frame containing a nucleotide sequence encoding a heterologous T cell receptor or CAR molecule. In some embodiments, the NR3C1 locus contains 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 an indel at an off-target locus, as determined by genome-wide off-target double-strand break analysis. In some embodiments, less than 0.01% have an indel at an off-target locus, as determined by genome-wide off-target double-strand break analysis. In some embodiments, the cell population is substantially free of chromosomal translocations.

[0016] In certain embodiments, the disclosure provides a method for editing two or more loci in a cell, the method comprising contacting the cell with a first Cas endonuclease complex comprising (a) a first Cas endonuclease complex comprising (i) a first Cas endonuclease and (ii) one or more manipulated 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, or a polynucleotide comprising (b) a second Cas endonuclease complex comprising (i) a second Cas endonuclease and (ii) one or more manipulated 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 includes introducing into the cells a first donor DNA sequence comprising (c) an open reading frame encoding a first transgene, a 5' homologous arm containing a DNA sequence located 5' to the first target sequence, and a 3' homologous arm containing 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' homologous arm containing a DNA sequence located 5' to the second target sequence, and a 3' homologous arm containing a DNA sequence located 3' to the second target sequence. In some embodiments, the second transgene is different. In some embodiments, the first or second target sequence is a target sequence within a T cell receptor locus, 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' homologous arm, which includes a DNA sequence located 5' to the first target sequence, or the 5' homologous arm, which includes a DNA sequence located 5' to the second target sequence, includes SEQ ID NOs. 42 or 23. In some embodiments, the 3' homologous arm, which includes a DNA sequence located 5' to the first target sequence, or the 3' homologous arm, which includes a DNA sequence located 5' to the second target sequence, includes SEQ ID NOs. 43 or 24. In some embodiments, the first or second class 2, type II Cas endonuclease includes 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 with respect to any one of SEQ ID NOs. 1 or 4, or a variant thereof. In some embodiments, the first manipulated guide RNA or the second manipulated guide RNA includes a sequence having at least 80%, 85%, 90%, or 95% sequence identity with any one of SEQ ID NOs: 3, 6, or 9, or its 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%, 90%, or 95% sequence identity with any one of SEQ ID NOs: 16, 20, 21, 22, or 41, or its complement. In some embodiments, the first or second endonuclease includes a class 2, type II Cas endonuclease, a class 2, type V Cas endonuclease, or any combination thereof.

[0017] Further aspects and advantages of the Disclosure will be readily apparent to those skilled in the art from the following detailed description, and only exemplary embodiments of the Disclosure are shown and described herein. As will be understood, the Disclosure can be made into other different embodiments, and some of its details can be modified in various obvious ways without departing from the Disclosure. Accordingly, the drawings and description should be considered illustrative in nature and not as limitations.

[0018] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as to be incorporated by reference. [Brief explanation of the drawing]

[0019] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings.

[0020] [Figure 1] This document presents a scheme for producing allogeneic CAR-T cells using the Cas endonuclease described herein, in combination with an AAV vector that delivers a CAR-T donor sequence. [Figure 2] The results of the experiment in Example 1, which tested indel formation in TRAC using MG3-6, MG3-8, and MG29-1 RNPs containing guide RNAs that target TRAC, along with a Cas9 control, are shown. The left panel shows the percentage of indel formation measured by next-generation sequencing (NGS), while the right panel shows the cell phenotype (TCR+ or TCR-) evaluated by flow cytometry. [Figure 3] The results of Example 1, which tests targeted CAR-T incorporation using the RNP nuclease complex described herein, which targets TRAC in combination with an AAV donor vector containing a CAR-T sequence, are shown. Flow cytometry plots showing TCR expression status (TCR- or TCR+, x-axis) along with CAR antigen-binding domain expression (y-axis) are shown. Similar results were obtained for all three: MG3-6, MG3-8, and MG29-1. [Figure 4]As described in Example 2, we demonstrate multiplex editing of two loci (one of which is TRAC) using a combination of the MG3-6 and MG29-1 RNP complex. [Figure 5] As described in Example 2, we demonstrate multiplex editing of three loci (one of which is TRAC) using a combination of the MG3-6 and MG29-1 RNP complex. [Figure 6] As shown in Example 3, the design of a PCR experiment to test the integration of the GR transgene into the AAVS1 locus (A) or agarose gel images (B and C) shows the results of an experiment in which the AAVS1 and TRAC loci were simultaneously targeted using different Cas enzymes along with exposure to separate donor DNAs targeting each site. (B) shows the amplification of the GR transgene from any of the following conditions in which T cells were exposed to a GR transgene-carrying AAV construct (lanes 2-5), a GR transgene AAV construct / MG3-6 / CAR transgene AAV targeting AAVS1 / TRAC (lanes 6-9), or a GR AAV construct / SpCas9 targeting AAVS1 alone at a multiple of infection (MOI) of 25K (lanes 10-13). (C) shows amplification of the GR transgene from T cells exposed to one of the following conditions: assay control (pseudotransfection or Cas complex without transgene; lanes 2-4), SpCas9 targeting GR AAV construct / AAVS1 alone at a 50K MOI (lanes 5-8), or SpCas9 targeting GR AAV construct / AAVS1 alone at a 100K MOI / 50K multiple infection degree (MOI) (lanes 9-12). The results show GR transgenes integrated into the AAVS1 locus with similar efficiency, regardless of whether additional TRAC loci were targeted. [Figure 7]As in Example 3, a flow cytometry plot is shown that depicts the results of an experiment such as in Example 3, where the AAVS1 and TRAC loci were simultaneously targeted using different Cas enzymes along with exposure to separate donor DNAs that target each site. Individual plots (A - D) are shown where AAVs carrying each GR transgene were introduced into T cells together with the AAVS1-targeting SpCas9 complex, the TRAC-targeting MG3-6 complex, and the CAR-carrying AAV. The results show that TCR knockout and CAR integration were equally efficient with all GR transgenes, and were high (51.31% - 61.1% efficiency) despite the simultaneous targeting of the AAVS1 locus. [Figure 8] As in Example 4, the results of a genome-wide off-target double-strand break assay performed to evaluate the off-target specificity of the MG3-6, MG3-8, and MG29-1 endonucleases together with SpCas9 (「Cas9」) are shown. [Figure 9] Diagram of the assembly of the delta, gamma, and epsilon chains that make up an active full TCR. [Figure 10] Multiplex TRAC / TRBC editing in primary T cells as described in Example 5, when evaluated by the proportion of sequences at the target locus containing indels, is shown. The results show high-frequency disruption at both sites when both sites are simultaneously targeted. [Figure 11]Flow cytometry results for the single gene knockout experiments described in Example 6 are shown, demonstrating gene editing results. A bar graph showing the percentage of analyzed cells containing each of four phenotypes (TCR-B2M-DKO, TCR-B2M+, TCR+B2M-, and TCR+B2M+) that were evaluated for knockout of TCR and B2M is shown. This graph shows that (a) all TCR targeting conditions resulted in efficient TCR knockout, and that MG3-6 TRAC6 and MG3-6 TRBC E2 sgRNAs resulted in the most efficient TCR knockout, and (b) all B2M targeting conditions resulted in B2M knockout, and that B2M H1 and B2M D2 resulted in the most efficient B2M knockout. [Figure 12] Flow cytometry results for the double gene knockout experiments described in Example 7 are shown, demonstrating gene editing results. These experiments used the B2M and TRAC conditions of Example 6 and were used in combination. A bar graph showing the percentage of analyzed cells containing each of four phenotypes (TCR-B2M-DKO, TCR-B2M+, TCR+B2M-, and TCR+B2M+) that were evaluated for knockout of TCR and B2M is shown. The graph shows that the most efficient dual targeting conditions were A4, B4, and C4, which involved the MG3-6 TRAC6 condition in combination with the MG29-1 B2M H1, D2, or A3 conditions. The most efficient dual targeting condition appeared to be B4, which used the MG3-6 TRAC6 sgRNA and the MG29-1 B2M D2 sgRNA. [Figure 13] Flow cytometry results for the triple gene knockout experiments described in Example 8 are shown, demonstrating gene editing results. These experiments used the B2M, TRAC, and TRBC conditions from Example 6 and were used in combination. [Figure 14] DNA-level gene editing results for the triple gene knockout experiments described in Example 8 are shown, demonstrating gene editing results. These experiments used the B2M, TRAC, and TRBC conditions from Example 6 and were used in combination. [Figure 15]This section shows the analysis of gene editing results determined by next-generation sequencing (NGS) for the triple gene knockout experiment described in Example 8. [Figure 16] The results of gene editing at the protein level in T cells for the experiment described in Example 9 are shown. The bar graph shows the percentage (%) of T cells positive for GFP / tEGFR, tLNGFR, dual-target incorporation (GFP / tLNGFR), dual-target incorporation (tEGFR / tLNGFR), or TCR, as determined by fluorescence-activated cell sorting (FACS) using the nuclease, guide, and AAV combination described in Example 9. [Figure 17] The results of DNA-level gene editing at the AAVS1 site and TRAC locus in T cells for the experiment described in Example 10 are shown. A bar graph shows the percentage of sequences (indels) detected by next-generation sequencing (Illumina MiSeq) with at least one indel detected at the AAVS1 locus using the conditions described in Example 10. [Modes for carrying out the invention]

[0021] Various embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Numerous variations, modifications, and substitutions can occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0022] The practices of some of the methods disclosed herein, unless otherwise indicated, utilize techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (FMAusubel, et al. eds.), the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor 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 (RIFreshney, ed. (2010)) (fully incorporated herein by reference).

[0023] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context otherwise explicitly indicates. Furthermore, to the extent that the terms "including," "including," "having," "having," or their variants are used in either the Detailed Description and / or the Claims, such terms are intended to be inclusive in a manner similar to that of the term "including."

[0024] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which in part depends on how the value is measured or determined, i.e., the limits of the measuring system. For example, “about” may mean within one or more standard deviations according to the practice of the art. Alternatively, “about” may mean 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 can be the basic structure, function, and / or biological unit of a living organism. A cell may originate from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, single-cell eukaryotic cells, protozoan cells, plant-derived cells (e.g., 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, clubmosses, hornworts, bryophytes, moss-derived cells), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens) Examples of cells include those derived from C. agardh, seaweed (e.g., kelp), fungal cells (e.g., yeast cells, mushroom-derived cells), animal cells, cells derived from vertebrates (e.g., fruit flies, knidarians, echinoderm, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells derived from mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Cells may not always originate from naturally occurring organisms (for example, cells may be synthetically produced and are sometimes called artificial cells).

[0026] As used herein, the term “nucleotide” generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may also be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, e.g., dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Examples of such derivatives include [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 also refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled, for example, by using a portion containing an optically detectable moiety (e.g., a fluorophore). Labeling may also be performed using quantum dots. Examples of detectable labels include radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Fluorescent labeling of nucleotides includes, but is 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; fluoro-linked deoxynucleotides available from Amersham, Arlington Heights, Il.: fluoro-linked Cy3-dCTP, fluoro-linked Cy5-dCTP, fluoro-linked fluoro-X-dCTP, fluoro-linked Cy3-dUTP, and fluoro-linked Cy5-dUTP; Boehringer Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-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 chromosome-labeled nucleotides available from Probes, Eugene, and 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. A chemically modified single nucleotide can be 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). The nucleotides may also include nucleotide analogs. In some embodiments, the nucleotide analogs may include structures of native nucleotides that are modified at arbitrary positions to alter 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 5th position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6th position, e.g., 6-(2-amino)propyluridine; and for adenosine and / or guanosine, the 8th position, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. 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, as well as 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 are described in U.S. Patents No. 5,858,988 and No. 6,291,438.Examples of nucleotide positions that can be derivatized include the 5th position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6th position, e.g., 6-(2-amino)propyluridine; and for adenosine and / or guanosine, the 8th position, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. 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, as well as 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 are described in U.S. Patents No. 5,858,988 and No. 6,291,438.

[0027] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are generally used interchangeably to refer to polymeric forms of nucleotides of any length, in single-stranded, double-stranded, or multi-stranded form, of either deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides may be exogenous or endogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any function. Polynucleotides may contain one or more analogues (e.g., modified backbone, sugar, or nucleic acid base). If present, modifications to the nucleotide structure may be introduced before or after the assembly of the polymer. Some non-exclusive examples of analogs include 5-bromouracil, peptide nucleic acids, heteronucleotides, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein bound to sugar), thiol-containing nucleotides, biotin-bound nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosin, and waiosin. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined by 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, non-cellular polynucleotides including non-cellular DNA (cfDNA) and non-cellular RNA (cfRNA), nucleic acid probes, and primers. The sequence of a nucleotide may be interrupted by non-nucleotide components.Polynucleotides may include mixtures of naturally occurring nucleotides and nucleotide analogs (e.g., synthetic nucleotide analogs).

[0028] The term “transfection” or “transfected” generally refers to the introduction of nucleic acids into cells by non-viral or virus-based methods. Nucleic acid molecules may be complete proteins or gene sequences encoding functional portions thereof. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1–18.88 (fully incorporated herein by reference).

[0029] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and generally refer to polymers of at least two amino acid residues linked by peptide bonds. The terms do not imply a specific length of polymer and are not intended to imply or distinguish whether peptides are produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, polymers may be interrupted by non-amino acids. The terms include full-length proteins and amino acid chains of any length, including proteins with or without secondary and / or tertiary structure (e.g., domains). The terms also encompass amino acid polymers modified by any other operations, such as disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, oxidation, and conjugation with labeling components. Where used herein, the terms “amino acid” and “multiple 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 both natural and unnatural amino acids, which are chemically modified to include groups or chemical moieties that do not naturally exist on the amino acid. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0030] As used herein, “unnatural” generally refers to nucleic acid or polypeptide sequences that are not present in natural nucleic acids or proteins. “Unnatural” may also refer to affinity tags. “Unnatural” may also refer to fusions. “Unnatural” may also refer to naturally occurring nucleic acid or polypeptide sequences, including mutations, insertions, and / or deletions. A nonnatural sequence may exhibit and / or encode activities (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that may also be exhibited by the nucleic acid and / or polypeptide sequence to which the nonnatural sequence is fused. A nonnatural nucleic acid or polypeptide sequence may be ligated to a naturally occurring nucleic acid and / or polypeptide sequence (or a variant thereof) by genetic engineering to produce a chimeric nucleic acid or polypeptide sequence encoding 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 with a nucleotide or region of nucleotides on which RNA transcription is initiated. A promoter may often contain a specific DNA sequence that binds to a protein factor called a transcription factor, which facilitates the binding of RNA polymerase to the DNA, thereby resulting in gene transcription. A “basic promoter,” also called a “core promoter,” may generally refer to a promoter containing all the basic elements necessary to facilitate the transcriptional expression of a operably linked polynucleotide. Eukaryotic basic promoters typically, but not necessarily, contain a TATA-box and / or 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 (for example, into mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene products.” When the polynucleotide is derived from genomic DNA, expression includes the splicing of mRNA in eukaryotic cells.

[0033] As used herein, “operably linked,” “operably linked,” “operably linked,” or its grammatical equivalents generally refer to the parallelization of gene elements, such as promoters, enhancers, polyadenylation sequences, etc., where the elements are related in a way that enables them to operate in the expected manner. For example, regulatory elements, which may include promoter sequences and / or enhancer sequences, are operably linked to a coding region if the regulatory elements help initiate transcription of the coding sequence. Intervening residues may be present between the regulatory elements and the coding region, as long as this functional relationship is maintained.

[0034] As used herein, “vector” generally refers to a macromolecule or association of macromolecules that contains or associates with polynucleotides and can be used to mediate the delivery of polynucleotides to cells. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. Vectors generally include gene elements, such as regulatory elements, that are operably linked to a gene to promote the expression of the gene in the target.

[0035] As used herein, “expression cassette” and “nucleic acid cassette” are generally used interchangeably to refer to a combination of nucleic acid sequences or elements that are operablely linked for expression. In some cases, an expression cassette refers to a combination of a gene or multiple genes and a regulatory element that is operablely linked for expression.

[0036] As used herein, the term “engineered” generally indicates that the subject has been modified by human intervention. In non-limiting examples, nucleic acids may be modified by altering their sequence to a sequence that does not occur naturally, nucleic acids may be modified by ligating them to nucleic acids that are not naturally associated such that the ligated product has a function not present in the original nucleic acid, engineered nucleic acids may be synthesized in vitro using sequences that do not occur naturally, proteins may be modified by altering their amino acid sequence to a sequence that does not occur naturally, and engineered proteins may acquire new functions or properties. An “engineered” system includes at least one engineered component.

[0037] As used herein, “synthetic” and “artificial” can generally be used interchangeably to refer to proteins or domains having low sequence identity with naturally occurring human proteins (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). 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, which (a) use a relatively small guide RNA (approximately 42-44 nucleotides) that is processed by the nuclease itself after transcription from a CRISPR array, and (b) cleave DNA in such a way that it leaves alternating 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, “guide nucleic acid” or its variants may generally refer to a nucleic acid that can hybridize to another nucleic acid. The guide nucleic acid may be RNA. The guide nucleic acid may be DNA. The guide nucleic acid may be programmed to bind site-specifically to a nucleic acid sequence. The nucleic acid to be targeted, or the target nucleic acid, may contain nucleotides. The guide nucleic acid may contain nucleotides. Part of the target nucleic acid may be complementary to part of the guide nucleic acid. A double-stranded target polynucleotide chain that is complementary to the guide nucleic acid and hybridizes with it may be called a complementary chain. A double-stranded target polynucleotide chain that is complementary to the complementary chain and therefore not complementary to the guide nucleic acid may be called a non-complementary chain. The guide nucleic acid may contain a polynucleotide chain and may also be called a “single guide nucleic acid”. The guide nucleic acid may contain two polynucleotide chains and may also be called a “double guide nucleic acid”. Otherwise, the term “guide nucleic acid” may be comprehensive, referring to both single guide nucleic acids and double guide nucleic acids. The guide nucleic acid may include a segment that may be called a "nucleic acid targeting segment," "nucleic acid targeting sequence," or "spacer sequence." The nucleic acid targeting segment may include a subsegment that may be called a "protein binding segment," "protein binding sequence," or "Cas protein binding segment." The guide nucleic acid may include sgRNA. The guide nucleic acid may include crRNA.

[0040] In the context of two or more nucleic acid or polypeptide sequences, the terms “sequence identity” or “percent identity” generally refer to two (e.g., in pairwise alignment) or more (e.g., in multiple sequence alignments) sequences that are identical or have a certain percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence across 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 a BLOSUM62 scoring matrix with parameters of word length (W) x 3, expected value (E) x 10, and gap cost set by presence x 11 and extension x 1, and conditional composition score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of word length (W) x 2, expected value (E) x 1,000,000, and PAM30 scoring set gap cost of 9 for open gaps and 1 for extended gaps for sequences shorter than 30 residues (default parameters for BLASTP exist in BLAST, available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using Smith-Waterman homology search algorithm parameters of match x 2, mismatch x 1, and gap x 1; MUSCLE using default parameters; MAFFT using parameters of retrieval x 2 and maximum repeats x 1,000; Novafold using default parameters; and HMMER hmmalign using default parameters.

[0041] As used herein, the terms “chimeric antigen receptor,” “CAR,” or “CAR molecule” generally refer to a recombinant polypeptide construct comprising a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”), which comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a functional signaling domain derived from a stimulating molecule as defined herein. In some embodiments, the stimulating molecule is a zeta chain associated with the signaling domain of a 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 co-stimulatory molecule as defined below. In some embodiments, the co-stimulatory 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 stimulating 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 co-stimulatory molecule and a functional signaling domain derived from a stimulating 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 two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulating 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 stimulating 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, the leader sequence being optionally cleaved from the antigen recognition domain, e.g., scFv) during cell 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 modulates cellular activity through defined signaling pathways by acting by transmitting information within the cell, by generating a second messenger, or by functioning as an effector in response to such a messenger.

[0043] As used herein, the term “antibody” generally refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen, for example, non-covalently, reversibly, and in a particular manner. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). Each VH and VL consists 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 region of the antibody can mediate the binding of immunoglobulins 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, camel 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 its recombinant variant, and refers to the antigen-determining variable region of the intact antibody that is sufficient to confer an antigen-binding domain, such as recognition of the antibody fragment and specific binding 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(VL or VH), camel VHH domains, and multispecific antibodies formed from antibody fragments. The term “scFv” refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked via a short mobile polypeptide linker and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless otherwise specified, scFv may have VL and VH variable regions in either order, for example, with respect to the N-terminus and C-terminus of the polypeptide, and scFv may include a VL-linker-VH or a VH-linker-VL.

[0045] The portion of the CAR composition containing an antibody or an antibody fragment may exist in various forms, where the antigen-binding domain is expressed as part of a continuous polypeptide chain, for example, containing a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), and a humanized antibody (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 present invention contains an antibody fragment. In some embodiments, the CAR contains an antibody fragment containing an scFv.

[0046] Any variant of the enzymes described herein having one or more conserved amino acid substitutions is included in this disclosure. Such conserved substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R-chain length to each other. In addition, or alternatively, by comparing the aligned sequences of homologous proteins from different species, conserved substitutions can be identified by finding interspecies mutated amino acid residues (e.g., non-conserved residues) without altering the fundamental 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%, and 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 important active site residues or guide RNA binding residues of the endonuclease is not disrupted. In some embodiments, any functional variant of the proteins described herein lacks at least one substitution of a conserved or functional residue characteristic of Cas endonucleases. In some embodiments, any functional variant of the proteins described herein lacks all substitutions of conserved or functional residues characteristic of Cas endonucleases.

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

[0048] Tables of conserved substitutions resulting in functionally similar amino acids are available from various 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 conserved substitutions with each other: 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 The discovery of novel Cas enzymes with unique functionalities and structures could further disrupt deoxyribonucleic acid (DNA) editing technologies, potentially improving their speed, specificity, functionality, and ease of use. Relatively few functionally characterized CRISPR / Cas enzymes exist in the literature, compared to the predicted prevalence of clustered and regularly arranged short palindromic sequence repeat (CRISPR) systems in microorganisms and the net diversity of microbial species. This is partly because, under laboratory conditions, a vast number of microbial species are not readily cultured. Metagenomic sequencing from natural environmental niches containing numerous microbial species could dramatically increase the number of documented novel CRISPR / Cas systems, potentially accelerating the discovery of new oligonucleotide editing functions. A fruitful recent example of such an approach is the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0050] The CRISPR / Cas system is an RNA-directed nuclease complex described as functioning as an adaptive immune system in microorganisms. In their natural context, the CRISPR / Cas system arises in a CRISPR (clustered and regularly arranged short palindromic sequence repeats) operon or locus, which generally consists of two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences encoding an RNA-based targeting element; and (ii) an ORF encoding a Cas that encodes a nuclease polypeptide directed by the RNA-based targeting element, aligned with an accessory protein / enzyme. Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and a crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined neighborhood of the target seed (PAMs are typically sequences not commonly represented in the host genome). Depending on the exact function and configuration of the system, CRISPR-Cas systems are generally structured into two classes, five types, and sixteen 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 their components. In Type II CRISPR-Cas systems, processing the CRISPR array into mature crRNA does not require the presence of a special endonuclease subunit, but rather a small transcoding crRNA (tracrRNA) having a region complementary to the array repeat sequence. The tracrRNA interacts with both its corresponding effector nuclease (e.g., Cas9) and the repeat sequence to form a precursor dsRNA structure, which is then cleaved by endogenous RNAse III to produce a mature effector enzyme loaded with both tracrRNA and crRNA. The Cas II nuclease is identified as a DNA nuclease. Type II effectors generally exhibit a structure containing 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 cleaving target (e.g., crRNA-complementary) DNA strands, while the HNH domain is involved in cleaving substitution DNA strands.

[0053] Type V CRISPR-Cas systems are characterized by nuclease effector structures (e.g., Cas12) that are similar in structure to type II effectors, including a RuvC-like domain. Similar to type II, most (but not all) type V CRISPR systems use tracrRNA to process precrRNA into mature crRNA; however, unlike type II systems which require RNAse III to cleave precrRNA into multiple crRNAs, type V systems can cleave precrRNA using the effector nuclease itself. As with 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 possess robust single-strand nonspecific deoxyribonuclease activity, activated by the first crRNA-directed cleavage of a double-stranded target sequence.

[0054] CRISPR-Cas systems have emerged as a gene editing technology in recent years due to their targeting capabilities and ease of use. The most commonly used systems are Class 2 Type II SpCas9 and Class 2 Type VA Cas12a (formerly Cpf1). Specifically, Type VA systems are becoming 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 also have the advantage of having small guide RNAs (42-44 nucleotides compared to about 100 nt for SpCas9), which are processed by the nuclease itself after transcription from the CRISPR array, simplifying multiplexing applications involving multiple gene editing. Furthermore, VA systems have staggered cleavage sites, which can facilitate directed repair pathways such as microhomology-dependent targeted integration (MITI).

[0055] The most commonly used type VA enzymes require a 5' protospacer adjacency motif (PAM) adjacent to the selected target site: 5'-TTTV-3' for Lachnospiraceae bacterium ND2006 LbCas12a and Acidaminococcus species AsCas12a; and 5'-TTV-3' for Francisella novicida FnCas12a. Recent investigations of orthologues have revealed proteins with less restrictive PAM sequences that are also active in mammalian cell cultures, 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 activity and PAM sequence requirements. Here, we have drawn thousands of genomic fragments from numerous metagenomics for type VA nucleases. The diversity of documented VA enzymes may be expanding, and novel systems may be developing into highly targetable, compact, and precise gene-editing agents.

[0056] Exemplary Embodiments In some embodiments, the disclosure provides a method for editing two or more loci in a cell, the method comprising contacting or introducing a class 2, type II Cas endonuclease complex into the cell, the class 2, type II Cas endonuclease complex comprising (a) a class 2, type II 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 set of one or more target loci. In some embodiments, the method comprises contacting or introducing a class 2, type V Cas endonuclease complex into the cell, the class 2, type V Cas endonuclease complex comprising (b) a class 2, type V Cas endonuclease and (ii) one or more engineered guide RNAs comprising an RNA sequence configured to bind to a 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 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 associated guide RNA (e.g., in a vector or in vitro transcribed mRNA). In some embodiments, editing includes indel insertion, immature stop codon, missense codon, frameshift mutation, adenine deamination, cytosine deamination, or any combination thereof.

[0057] A Cas endonuclease may be a specific Cas endonuclease, may be introduced under specific parameters, or may be introduced in a manner that achieves specific target metrics. 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 contains 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 with any one of sequence numbers 1 or 4, or its variants. In some embodiments, the class 2, type V Cas endonuclease contains 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 with SEQ ID NOs: 3, 6, or 9. In some embodiments, the first manipulated guide RNA or the second manipulated guide RNA contains a sequence having at least 80%, 85%, 90%, or 95% sequence identity with any one of SEQ ID NOs: 3, 6, or 9. In some embodiments, the method edits the genome sequence of the first locus having an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher, and / or the second locus having an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, the first RNA guide endonuclease or the second RNA guide 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, off-target sites are disrupted at a frequency of less than 0.2% when determined by genome-wide off-target double-strand break analysis.In some embodiments, off-target sites are disrupted at a frequency of less than 0.01% when determined by genome-wide off-target double-strand break analysis. In some embodiments, genome-wide off-target double-strand break analysis is performed using HTGTS assays (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 herein for all purposes by reference)), LAM-HTGTS assays (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 herein for all purposes by reference)), or Digenome-Seq assays (in vitro Cas-digestion whole-genome sequencing; see, e.g., Kim et al. Nat This includes Methods.2015.12(3):237-43.doi:10.1038 / nmeth.3284 (expressly incorporated herein for all purposes by reference).

[0058] The targeted locus may include any locus. The targeted locus may be a specific therapeutically interesting locus, such as a T cell receptor (TCR) locus (including the constant region of the TCR locus, which is a plurality of conserved subtypes of T cells such as TRAC and TRBC), a glucocorticoid receptor locus (also known as the GR locus), a locus encoding another nuclear hormone receptor (e.g., estrogen receptor, progesterone receptor, or androgen receptor locus), or a locus encoding a specific oncogene or tumor suppressor. In some embodiments, the first set of one or more targeted loci, or the second set of one or more targeted loci, includes a T cell receptor (TCR) locus. In some embodiments, the spacer sequence configured to hybridize to the first set of one or more targeted loci, or the spacer sequence configured to hybridize to the second set of one or more targeted loci, has at least 80%, 85%, 90%, or 95% sequence identity to any one of sequence numbers 10-15. In some embodiments, the first set of one or more target loci, or the second set of one or more target loci, includes the 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 with any one of sequence numbers 16, 20, 21, or 22.

[0059] One 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 includes introducing into the cell a donor DNA sequence containing an open reading frame encoding a transgenic form of an endogenous gene, a first homologous arm containing a DNA sequence located on the first side of the target sequence within the locus of the endogenous gene, and a second homologous arm containing a DNA sequence located on the second side of the second target sequence. In some cases, the transgene may be a CAR-T molecule. In some embodiments, the method further includes introducing into the cell a donor DNA sequence containing an open reading frame encoding a heterologous engineered T cell receptor molecule, a first homologous arm containing a DNA sequence located on the first side of the target sequence within the TCR locus, and a second homologous arm containing a DNA sequence located on the second side of the second target sequence.

[0060] In certain embodiments, the Disclosure provides a method for producing glucocorticoid-resistant engineered T cells, the method comprising introducing a T cell receptor (TCR) locus-targeting RNA guide endonuclease complex into a T cell or its precursor, the complex comprising: (a) an RNA guide endonuclease complex targeting a T cell receptor (TCR) locus, comprising (i) a first RNA guide endonuclease or DNA encoding the first RNA guide endonuclease; and (ii) a first engineered guide RNA comprising an RNA sequence configured to form a complex with the first RNA guide endonuclease and a first spacer sequence configured to hybridize to at least a portion of the TCR locus. In some embodiments, the method further includes introducing into the T cell or its precursor an RNA guide endonuclease complex that targets the T cell receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) locus, comprising: (i) a second RNA guide endonuclease; and (ii) a second engineered guide RNA comprising an RNA sequence configured to form a complex with the second RNA guide endonuclease, and a second spacer sequence configured to hybridize to at least a portion of the NR3C1 locus. In some embodiments, at least a portion of the TCR locus is located within the T cell locus. In some embodiments, the method further includes introducing into the cells (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 the first side of the target sequence within the TCR locus, and a second homologous arm comprising a DNA sequence located on the second side of the second target sequence.

[0061] Type II or type V endonucleases may include specific Cas endonucleases. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease includes a class 2, type II, or class 2, type V Cas endonuclease. In some embodiments, the first RNA guide endonuclease includes the class 2, type II Cas endonuclease, and the second RNA guide endonuclease includes the class 2, type V Cas endonuclease. In some embodiments, the second RNA guide endonuclease includes the class 2, type II Cas endonuclease, and the first RNA guide endonuclease includes the class 2, type V Cas endonuclease. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease contains 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 with any one of SEQ ID NOs: 1, 4, or 7. In some embodiments, the first manipulated guide RNA or the second manipulated guide RNA contains a sequence having at least 80%, 85%, 90%, or 95% sequence identity with any one of SEQ ID NOs: 3, 6, or 9. In some embodiments, the first RNA guide endonuclease or the second RNA guide endonuclease is present at concentrations of 100 pmol or less, 50 pmol or less, 25 pmol or less, 5 pmol or less, or 1 pmol or less.

[0062] One 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, at least a portion of the T cell receptor locus is the T cell receptor alpha stationary (TRAC) locus or the T cell receptor beta stationary (TRBC) locus. In some embodiments, at least a portion of the T cell receptor locus is the TRAV locus or the TRAJ locus. In some embodiments, at least a portion of the T cell receptor locus is the TRBV locus or the TRBJ locus. In some embodiments, the homologous arm includes an intronic or exonic region within a TCR locus proximal to at least a portion of the T cell receptor locus. In some embodiments, at least a portion of the T cell receptor locus is the first or third exon of TRAC. In some embodiments, the method disrupts the genomic sequences of the TCR locus and the NR3C1 locus with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, this efficiency is determined by flow cytometry of the protein expressed from the TCR locus or the NR3C1 locus. In some embodiments, at least a portion of the NR3C1 locus is exon 2 or exon 3. In some embodiments, the method produces cells that are positive for CAR molecules and negative for NR3C1 with an efficiency of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, the method includes simultaneously introducing (a) to (c) into the T cell or its precursor. In some embodiments, the T cell or its precursor includes a T cell, a hematopoietic stem cell (HSC), or a peripheral blood mononuclear cell (PBMC).In some embodiments, the second spacer sequence includes a sequence having at least 80%, 85%, 90%, or 95% sequence identity with any one of sequence numbers 16, 20, 21, or 22. In some embodiments, the first or second spacer sequence includes at least about 19 to 24 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, or at least about 24 nucleotides.

[0063] Donor sequences used in conjunction with the methods described herein may be provided in various forms in those methods. 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., plasmid, YACmid, BACmid, phagemid, or viral vector). In the case of a viral vector, the viral vector may contain an AAV virus having a specific serotype. In some embodiments, the donor DNA sequence is delivered in the viral vector. In some embodiments, the viral vector is an AAV vector or an AAV-6 vector.

[0064] In some embodiments, the disclosure provides a population of glucocorticoid-resistant CAR-T cells comprising (a) a heterologous sequence within 100, 75, 50, 25, or 10 nucleotides in the hybridization region of any one of SEQ ID NOs: 10-15 within the 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 in the hybridization region of any one of SEQ ID NOs: 16, 20, 21, or 22. In some embodiments, less than 0.2% of the cells in the population have an indel at an off-target locus, as determined by genome-wide off-target double-strand break analysis. In some embodiments, less than 0.01% of the cells in the population have indels at off-target loci, as determined by genome-wide off-target double-strand break analysis. In some embodiments, genome-wide off-target double-strand break analysis is performed using HTGTS assays (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 herein for all purposes by reference)), LAM-HTGTS assays (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 herein for all purposes by reference)), or Digenome-Seq assays (in vitro Cas-digestion whole-genome sequencing; see, e.g., Kim et al. Nat This includes Methods.2015.12(3):237-43.doi:10.1038 / nmeth.3284 (expressly incorporated herein for all purposes by reference).In some embodiments, the population of cells is substantially free of chromosomal translocations.

[0065] In some embodiments, this disclosure provides cells produced by any of the methods described herein.

[0066] In some embodiments, the disclosure provides protein sequences 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 a variant having one or more nuclear localization sequences (NLSs). The NLSs may be located proximal to the N-terminus or C-terminus of the endonuclease. The NLSs may be added to the N-terminus or C-terminus of any one of SEQ ID NOs. 25-40, or to 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. In some cases, the NLS may contain sequences that are substantially identical to any one of sequence numbers 25-40.

[0090] [Table 2]

[0091] In some cases, any of the endonuclease methods described herein may further include introducing a single-stranded or double-stranded DNA repair template into cells. In some cases, the engineered nuclease system further includes a single-stranded DNA repair template. In some cases, the engineered nuclease system further includes a double-stranded DNA repair template. In some cases, the single-stranded or double-stranded DNA repair template may include a first homologous arm in the 5' to 3' direction, comprising a sequence of at least 20 nucleotides 5' relative 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' relative to the target sequence.

[0092] In some cases, the first homologous arm includes 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 homologous arm includes 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 prokaryotic genome sequences. In some cases, the first and second homologous arms are homologous to bacterial genome sequences. In some cases, the first and second homologous arms are homologous to fungal genome sequences. In some cases, the first and second homologous arms are homologous to eukaryotic genome sequences.

[0094] In some cases, any of the endonuclease methods described herein may further include introducing a DNA repair template into cells. The DNA repair template may include a double-stranded DNA segment. A double-stranded DNA segment may have one single-stranded DNA segment adjacent to it. A double-stranded DNA segment may have two single-stranded DNA segments adjacent to it. 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, a single-stranded DNA segment has a length of 1 to 15 nucleotides. In some cases, a single-stranded DNA segment has a length of 4 to 10 nucleotides. In some cases, a single-stranded DNA segment has a length of 4 nucleotides. In some cases, a single-stranded DNA segment has a length of 5 nucleotides. In some cases, a single-stranded DNA segment has a length of 6 nucleotides. In some cases, a single-stranded DNA segment has a length of 7 nucleotides. In some cases, a single-stranded DNA segment has a length of 8 nucleotides. In some cases, a single-stranded DNA segment has a length of 9 nucleotides. In some cases, a single-stranded DNA segment has a length of 10 nucleotides.

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

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

[0098] The systems or methods of this disclosure may be used for a variety of applications, such as binding to nucleic acid molecules (e.g., sequence-specific binding) and nucleic acid editing (e.g., gene editing). Such systems or methods may be used, for example, to address genetically inherited mutations that may cause disease in a subject (e.g., removal or substitution), to inactivate genes to confirm their function in cells, as a diagnostic tool to detect disease-causing genetic elements (e.g., via cleavage of reverse-transcribed viral RNA or amplified DNA sequences encoding disease-causing mutations), as an inactivated enzyme combined with a probe to target specific nucleotide sequences (e.g., sequences encoding 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 manipulate organisms to produce valuable small molecules, macromolecules or secondary metabolites, to establish gene-driven elements for evolutionary selection, and as a biosensor to detect cellular perturbations by foreign small molecules and nucleotides. [Examples]

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

[0100] Next, we tested the editing capability of the above RNP complex to promote targeted incorporation of CAR-T molecules into the TRAC gene locus. We developed an AAV-6 vector containing a nucleotide sequence with CAR-T molecules flanked by 5' and 3' homologous arms (e.g., SEQ ID NOs. 23-24) that target the TRAC gene. We performed TRAC targeting using the above MG29-1 RNP, and then, after transfection, added 100,000 vector genomes (vg) of the AAV-6 vector to T cells. The cells were analyzed using flow cytometry for TCR receptor expression and CAR antigen-binding domain expression (Figure 3). Flow cytometry showed that approximately 60% of 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 incorporation construct with MG3-6 and MG3-8 RNPs that target TRAC.

[0101] Example 2 - Multiplex editing in TCR-like cells Modification of other genes in combination with modification of the TRAC locus (e.g., CAR-T integration) may be advantageous. Therefore, 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 for disrupting CAR-T cells to confer nonresponsiveness to glucocorticoid agents (e.g., in cancer patients being concurrently treated with glucocorticoids, or in cancer patients with autoimmune disorders requiring glucocorticoid maintenance). Three MG29-1 compatible targeting sequences (target BDs; SEQ ID NOs. 20, 21, and 22) were designed to target the NR3C1 gene and incorporated into MG29-1 guide RNAs. RNP complexes containing MG29-1 with these guide RNAs were assembled. T cells were treated with nucleofection using various combinations of the MG29-1 / NR3C1 gRNA RNP complex and MG3-6 / TRAC RNP (Figure 4). After nucleofection, cells were analyzed using NGS to evaluate indel formation at each locus. The results showed that different guide RNAs had different efficiencies in targeting NR3C1 (see "MG29-1 GR-13", "MG29-1 GR-28", and "MG29-1 GR-29"), while combinations of the MG3-6 complex targeting TRAC and the MG29-1 complex targeting NR3C1 efficiently induced indel formation in both genes (see the three conditions on 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 / sequence number 16, locus C-87 / sequence number 17, locus C-74 / sequence number 18, or locus C-83 / sequence number 19) was evaluated by nucleofecting T cells with the RNPs corresponding to each locus individually and in combination of the three, as described above, and evaluating indel formation by NGS (see Figure 5). The results of this experiment showed that indels were produced in considerable quantities at all three loci, even under conditions of combining three different RNPs targeting different loci.

[0103] Example 3 - Multiplex editing with multiple gene substitutions After establishing the ability to edit multiple loci and integrate a transgene into at least one locus, the ability to simultaneously edit two or more loci and integrate genes into both loci was tested by editing two different loci within T cells and providing two different donor DNA templates targeting two different loci. The AAVS1 (Safe Harbor) locus and the TRAC locus were selected as target sites. Primary T cells (2 × 10⁶) prepared as described in Examples 1 and 2 were used. 5The cells were nucleofected with (a) SpCas9 (12 pmol) and compatible sgRNA targeting the AAVS1 locus (60 pmol, SEQ ID NO: 41 shows a spacer sequence), and (b) MG3-6 (52 pmol) and compatible TRAC3-6 6 sgRNA (60 pmol, SEQ ID NO: 10). After nucleofection, the 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-alpha D3, and GR-beta D3) with 5' and 3' homologous arms targeting the AAVS1 locus (SEQ ID NOs: 42 and 43) adjacent to each other, and (b) one carrying CAR with 5' and 3' homologous arms targeting the TRAC locus (SEQ ID NOs: 23-24) adjacent to each other, at a multiple infection degree (MOI) of 50,000. After a 4-day incubation, T cells were analyzed by (a) PCR for the presence of the GR transgene at the AAVS1 locus (see Figure 6 for PCR design and results), or (b) flow cytometry for the CAR antigen-binding domain and T cell receptor to assess CAR integration at the TRAC locus (Figure 7). Data from PCR and flow cytometry experiments showed that both transgenes (GR and CAR) could be inserted simultaneously without significant loss of performance. PCR for the GR transgene under dual AAVS1 / TCR targeting conditions (center four lanes, Figure 6B) showed similar integration results to AAVS1 targeting alone (last four lanes, Figure 7B or center four lanes, Figure 7C), while flow cytometry for TCR (Figures 7A, 7B, 7C, 7D) showed high CAR integration and TCR loss, even when AAVS1 was simultaneously targeted.

[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 along with SpCas9 ("Cas9") via genome-wide off-target double-strand break analysis. The results are shown in Figure 8. The results indicated that MG3-6, MG3-8, and MG29-1 exhibited lower levels of off-target editing than Cas9.

[0105] Example 5 - Multiplex editing in T cells The production of recombinant TCR-based T cell products requires introducing novel, desirable alpha and beta chains of the TCR into the T cell pool, since the a / b chains are the subunits of the TCR that give antigen-specific recognition. These novel a / b chains can then assemble with the delta / gamma / epsilon chain to form an active, complete TCR (see Figure 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 novel beta, and the novel alpha can pair with the existing beta, for example, the novel, desirable TCR a / b chains do not know that they "are supposed to" pair together. Without further action, the T cells here express four different TCRs (a / b, a' / b, a / b', a' / b'), one of which has the manipulated specificity. This presents two problems: i) it reduces the expression of new, desirable TCRs by fourfold, and ii) the two hybrid a / b pairs (a' / b and a / b') recognize the antigen in an unpredictable, potentially autoreactive manner, thus posing an autoimmune risk.

[0106] In this experiment, primary T cells grown with CD2 / 3 / 28 beads were nucleofected using a Lonza 4D electroporator and solution P3, with 200K cells per condition, and delivered either 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), both 22 nt in length. Genomic DNA was collected from these cells after 3 days and analyzed by NGS (see Figure 10). The results in Figure 10, showing the sequence percentage at indel-containing target sites, demonstrate that there is dual 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 knockout. 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 200,000 T cells with RNP (100 pmol protein / 150 pmol guide RNA) was performed using a Lonza 4D electroporator. For flow cytometry analysis, 100,000 T cells were stained with anti-CD3 and anti-B2M antibodies at 4°C for 30 minutes three days after nucleofection and analyzed using an Attune Nxt flow cytometer (Figure 11). Figure 11, showing the percentage of analyzed cells containing each of the four phenotypes used to evaluate TCR and B2M knockout, indicates that (a) all TCR targeting conditions efficiently resulted in TCR knockout, with MG3-6 TRAC6 and MG3-6 TRBC E2 sgRNA yielding the most efficient TCR knockout, and (b) all B2M targeting conditions resulted in B2M knockout, with B2M H1 and B2M D2 yielding the most efficient B2M knockout.

[0108] Example 7 - Gene editing results by flow cytometry for dual gene knockout. In Example 6, after evaluating the performance of TCR / B2M targeting conditions individually, simultaneous double cleavage using combinations 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 200,000 T cells with RNP (100 pmol protein / 150 pmol guide RNA) was performed using a Lonza 4D electroporator. For flow cytometry analysis, 100,000 T cells were stained with anti-CD3 and anti-B2M antibodies at 4°C for 30 minutes three days after nucleofection and analyzed using an Attune Nxt flow cytometer (Figure 12). Figure 12, showing the percentage of analyzed cells containing each of the four phenotypes used to evaluate TCR and B2M knockout, indicates that the most efficient dual-targeting conditions were A4, B4, and C4, with MG3-6 TRAC6 conditions along with MG29-1 B2M H1, D2, or A3 conditions. The most efficient dual-targeting condition appeared to be B4, using 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 individually in Example 6 and in 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 T cells (200,000) with RNP (100 pmol protein / 150 pmol guide RNA) was performed using a Lonza 4D electroporator. For flow cytometry analysis, 100,000 T cells were stained with anti-CD3 and anti-B2M antibodies at 4°C for 30 minutes three days after nucleofection and analyzed using an Attune Nxt flow cytometer (Figure 13). Flow cytometry results indicate 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 created and optimized and used to amplify individual target sequences of each guide RNA. The 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 genes (as measured by flow cytometry).

[0111] [Table 3]

[0112] Therefore, additional analyses were performed to verify the generation of triple knockout cells by sequencing (see Figure 15). Figure 15 shows data demonstrating the successful generation of triple knockout cells. The data in the "Edited" column are taken from Figure 14, and the data in the "Wild-Type" column are 100% minus the percentage of editing. The minimum (Min.) frequencies of double and triple knockout are calculated assuming the minimum 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 wild-type cells for TRAC from the percentage of cells that may not contain double knockout. The observed high editing frequencies rule out the possibility that all editing events occurred in separate cells. Therefore, the data in Figure 15 demonstrates the successful generation of triple knockout TRAC / TRBC / B2M cells.

[0113] Example 9 - Expression of GFP markers 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 200,000 T cells with MG3-6 mRNA (500 ng / 150 pmol guide), MG29-1 RNP (100 pmol / 150 pmol guide), and / or SpCas9 RNP (12 pmol / 60 pmol guide) was performed using a Lonza 4D electroporator. After nucleofection, cells were immediately harvested in a medium containing AAV-6 (50,000 MOI). The AAV vectors used included (a) an AAV vector that delivers an MSCV promoter-driven truncated low-affinity nerve growth factor receptor (tLNGFR) coding sequence with homologous arms adjacent to the cleavage sites of MG3-6-TRAC-6 (SEQ ID NO: 64) or MG29-1-TRAC-35 (SEQ ID NO: 65), and (b) an AAV vector that delivers a MND promoter-driven polycistronic construct encoding GFP along with a truncated epidermal growth factor receptor (tEGFR) with homologous arms adjacent to the cleavage site of 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 at 4°C for 30 minutes, and data were acquired using an Attune NxT flow cytometer. Cells expressing tLNGFR, GFP, tEGFR, and / or TCR a / b were gated on single live cells (Figure 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 200,000 T cells with MG3-6 mRNA (500 ng / 150 pmol guide), MG29-1 RNP (100 pmol / 150 pmol guide), and / or SpCas9 RNP (12 pmol / 60 pmol guide) was performed using a Lonza 4D electroporator. After nucleofection, cells were immediately harvested in a 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 homologous arms corresponding to the cleavage sites of MG3-6-TRAC-6 or MG29-1-TRAC-35, a MND promoter-driven polycistronic construct encoding GFP, and a truncated epidermal growth factor receptor (tEGFR) flanked by homologous arms corresponding to the cleavage site of 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 and optimized and used to amplify regions containing the target sites of different AAVS1 site-specific RNA guides used in these experiments. The amplicons were sequenced on an Illumina MiSeq machine and analyzed using a custom 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] Preferred embodiments of the present invention are shown and described herein, but 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 any specific examples provided herein. The present invention is described with reference to the foregoing description, but the description and explanation of embodiments herein are not intended to be construed as limiting. Numerous variations, alterations, and substitutions will arise herein without departing from the present invention to those skilled in the art. Furthermore, it will be understood that all aspects of the present invention are not limited to any specific descriptions, configurations, or relative proportions described herein, depending on various 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. Accordingly, the present invention is intended to encompass any such alternatives, modifications, alterations, or equivalents. The following claims are intended to define the scope of the present invention and its methods and structures within these claims and their equivalents that are encompassed therein.

Claims

1. A modified nuclease system for editing two or more gene loci within a cell, wherein the modified nuclease system is (a) Class 2, type II Cas endonuclease or nucleic acid encoding the Class 2, type II Cas endonuclease, (b) A first manipulated guide ribonucleic acid or a nucleic acid encoding the first manipulated guide ribonucleic acid, wherein the first manipulated guide ribonucleic acid is (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 the first engineered guide ribonucleic acid, comprising a first spacer sequence configured to hybridize to a first set of one or more target gene loci, (c) Class 2, type V Cas endonuclease or nucleic acid encoding the Class 2, type V Cas endonuclease, (d) A second manipulated guide ribonucleic acid or a nucleic acid encoding the second manipulated guide ribonucleic acid, wherein the second manipulated guide ribonucleic acid is (i) a second ribonucleic acid sequence configured to bind to the class 2, type V Cas endonuclease, and (ii) 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 a second set of one or more target gene loci, An engineered nuclease system in which the class 2, type V Cas endonuclease comprises a sequence having at least 90% sequence identity with respect to sequence number 7.

2. The manipulated nuclease system according to claim 1, wherein the class 2, type V Cas endonuclease comprises a sequence having at least 95% sequence identity with respect to sequence number 7.

3. The operated nuclease system according to claim 1 or 2, wherein the class 2, type V Cas endonuclease comprises the sequence of sequence number 7.

4. The manipulated nuclease system according to any one of claims 1 to 3, wherein the second manipulated guide ribonucleic acid comprises a sequence having at least 90% sequence identity with respect to SEQ ID NO:

9.

5. The manipulated nuclease system according to any one of claims 1 to 4, wherein the second manipulated guide ribonucleic acid comprises the sequence of SEQ ID NO:

9.

6. The manipulated nuclease system according to any one of claims 1 to 5, wherein the class 2, type II Cas endonuclease comprises a polypeptide sequence encoded by a polynucleotide sequence having at least 90% sequence identity with respect to SEQ ID NO:

62.

7. The operated nuclease system according to any one of claims 1 to 6, wherein the class 2, type II Cas endonuclease comprises a polypeptide sequence encoded by sequence number 62.

8. The manipulated nuclease system according to any one of claims 1 to 7, wherein the first manipulated guide ribonucleic acid comprises a sequence having at least 90% sequence identity with respect to SEQ ID NO:

3.

9. The manipulated nuclease system according to any one of claims 1 to 8, wherein the first manipulated guide ribonucleic acid comprises the sequence of SEQ ID NO:

3.

10. The manipulated nuclease system according to any one of claims 1 to 9, wherein the first set of one or more target loci, or the second set of one or more target loci, includes a T cell receptor (TCR) locus, an albumin (ALB) locus, a beta-2 microglobulin (B2M) locus, or a nuclear receptor subfamily 3 group C member 1 (NR3C1) locus.

11. The manipulated nuclease system according to any one of claims 1 to 10, wherein the first spacer sequence configured to hybridize to the first set of one or more target gene loci, or the second spacer sequence configured to hybridize to the second set of one or more target gene loci, has at least 90% sequence identity with respect to any one of sequence numbers 10 to 22 or its complement.

12. The modified nuclease system according to any one of claims 1 to 11, wherein the editing includes indel insertion, immature stop codon, missense codon, frameshift mutation, adenine deamination, cytosine deamination, or any combination thereof.

13. The manipulated nuclease system according to any one of claims 1 to 12, wherein the first set of one or more target loci or the second set of one or more target loci includes a TCR locus.

14. The manipulated nuclease system according to claim 13, wherein the first set of one or more target loci or the second set of one or more target loci comprises a T cell receptor alpha stationary (TRAC) locus or a T cell receptor beta stationary (TRBC) locus.

15. The manipulated nuclease system according to claim 14, wherein the first set of one or more target loci or the second set of one or more target loci includes the TRAC locus.

16. The manipulated nuclease system according to claim 14, wherein the first set of one or more target loci or the second set of one or more target loci includes the TRBC locus.

17. The manipulated nuclease system according to any one of claims 13 to 16, wherein the first set of one or more target loci or the second set of one or more target loci includes the B2M locus.

18. The manipulated nuclease system according to any one of claims 1 to 17, wherein the cells are T cells or their precursors.

19. The engineered nuclease system according to claim 18, further comprising a donor deoxyribonucleic acid sequence encoding an engineered T cell receptor.

20. The modified nuclease system according to claim 19, wherein the modified T cell receptor is a chimeric antigen receptor (CAR) molecule.

21. The operated nuclease system according to any one of claims 1 to 20, wherein the class 2, type V Cas endonuclease is Cas12a endonuclease.

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