RU2832314C1 - Dna-containing polynucleotides and guide sequences for crispr type v systems and methods for production and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing a cell expressing a chimeric antigen receptor (CAR), comprising bringing a first target nucleic acid containing a TRAC sequence, in a cell in contact with a first nucleic acid/protein CRISPR composition containing a CRISPR targeting molecule and a Cas12 protein, and bringing a second target nucleic acid sequence comprising a B2M sequence in the same cell into contact with a second nucleic acid/CRISPR protein composition, containing a CRISPR guide molecule and a Cas12 protein, as well as to a CAR-expressing cell obtained using said method.

EFFECT: invention is effective for producing a CAR-expressing cell for adaptive cell therapy.

20 cl, 17 dwg, 29 tbl, 14 ex

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/229,870, filed August 5, 2021, U.S. Provisional Patent Application Ser. No. 63/127,648, filed December 18, 2020, and U.S. Provisional Patent Application Ser. No. 63/093,459, filed October 19, 2020, each of which is incorporated herein by reference.

Federally Sponsored Research or Development Application

[0002] Missing.

List of sequences

[0003] This application contains a sequence listing that was filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on March 20, 2023 is named CBI039_30_SL.txt and is 203259 kilobytes in size.

Field of technology

[0004] In general, the present invention relates to clustered regularly interspaced short palindromic repeats (CRISPR) systems. In particular, the present invention relates to polynucleotides and CRISPR guides for use in CRISPR-Cas12 systems, wherein the CRISPR polynucleotides and Cas12 guides are engineered to include ribonucleotide bases and one or more deoxyribonucleotide bases. The present invention further relates to Cas12 guide/nucleoprotein complexes comprising an engineered Cas12 guide CRISPR and a CRISPR-Cas12 protein, and to the production of modified cells using such Cas12 guide/nucleoprotein complexes. The present invention also relates to compositions comprising CRISPR polynucleotides, Cas12 guides, and Cas12 guide/nucleoprotein complexes, as well as methods for making and using them. Furthermore, the present invention relates to the production and therapeutic use of cells modified using the Cas12 targeting/nucleoprotein complexes of the invention, and, for example, to the production of cells expressing a chimeric antigen receptor (CAR) for the treatment of cancer.

Prerequisites for the creation of an invention

[0005] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are found in the genomes of many prokaryotes (bacteria and archaea). These systems provide adaptive immunity to prokaryotes against foreign pathogens (e.g., viruses, bacteriophages). Thus, the CRISPR system functions as a type of immune system that helps protect prokaryotes from foreign pathogens. See, e.g., Barrangou et al. ( Science , 2007, 315:1709–1712); Makarova et al. ( Nature Reviews Microbiology , 2011, 9:467–477); Garneau et al. ( Nature , 2010, 468:67–71); Sapranauskas et al. ( Nucleic Acids Research , 2011, 39:9275-9282); Koonin et al. ( Curr. Opin. Microbiol ., 2017, 37:67-78); Shmakov et al. ( Nat. Rev. Microbiol ., 2017, 15(3):169-182); Makarova et al. ( Nat. Rev. Microbiol ., 2020, 18:67-83).

[0005] The action of immune CRISPR-Cas systems occurs in three main stages: (1) acquisition, (2) expression, and (3) interference. Acquisition involves cleavage of the genome of invading viruses and plasmids and integration of segments (called protospacers) of the genomic DNA into the host CRISPR locus. Segments that are integrated into the host genome are known as spacers, which mediate protection against subsequent attack by the same (or a sufficiently related) virus or plasmid. Expression involves transcription of the CRISPR locus and subsequent enzymatic processing to produce short mature CRISPR RNAs, each containing a single spacer sequence. Interference is induced after CRISPR RNAs bind to Cas proteins to form effector complexes, which then target complementary protospacers in foreign genetic elements, thereby inducing nucleic acid degradation.

[0007] Different CRISPR-Cas systems in their native hosts are capable of targeting DNA (class 1 type I; class 2 types II and V), targeting RNA (class 2 type VI), and co-targeting DNA and RNA (class 1 type III). See, e.g., Makarova et al. ( Nat. Rev. Microbiol. , 2015, 13:722–736); Shmakov et al. ( Nat. Rev. Microbiol ., 2017, 15:169–182); Abudayyeh et al. (Science, 2016, 353: 1–17) Makarova et al. (The CRISPR Journal , 2018, 1:325–336).

[0008] Type V systems are classified into several different subtypes, including, for example, V-A, V-B, V-C, V-D, V-E, V-F, V-G, V-H, V-I, V-J, V-K, and V-U. See, for example, Makarova et al. (Nat. Rev. Microbiol., 2020, 18:67-83) and Pausch et al. (Science, 2020, 369(6501):333-337). The V-A subtype encodes the Cas12a protein (formerly known as Cpf1). Cas12a has a RuvC-like nuclease domain that is homologous to the corresponding domain of Cas9, but lacks the HNH nuclease domain that is present in Cas9 proteins.

[0009] Type V systems have been identified in several bacteria, including Parcubacteria GWC2011_GWC2_44_17 (PbCpf1), Lachnospiraceae MC2017 (Lb3 Cpf1), Butyrivibrio proteoclasticus (BpCpf1), P eregrinibacteria GW2011_GWA_33_10 (PeCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), Porphyromonas macacae (PmCpf1), Lachnospiraceae ND2006 (LbCpf1), Porphyromonas crevioricanis (PcCpf1), Prevotella disiens (PdCpf1), Moraxella bovoculi 237 (MbCpf1), Smithella sp. SC_K08D17 (SsCpf1), Leptospira inadai (LiCpf1), Lachnospiraceae bacterium MA2020 (Lb2Cpf1), Franciscella novicida U112 (FnCpf1), Candidatus methanoplasma termitum (CMtCpf1) and Eubacterium eligens (EeCpf1).

[0010] CRISPR-Cas systems are powerful tools for site-directed genome editing by deleting, inserting, mutating, or replacing specific nucleic acid sequences. The change can be gene-specific or site-specific. Genome editing can be accomplished by using site-directed nucleases, such as Cas proteins and related polynucleotides, to cleave a target nucleic acid, thereby creating a site for modification. In some cases, the cleavage can result in a double-strand break (DSB) in the target DNA sequence. DSBs can be repaired, such as by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-based repair (HDR). HDR depends on the availability of a repair template. In some examples of such genome editing, a donor polynucleotide or portion thereof can be inserted into the break.

Description of the essence of the invention

[0011] The present invention is based on the discovery of novel polynucleotides and guide sequences for use in type V CRISPR-Cas systems, polynucleotides and guide sequences comprising ribonucleotide bases and one or more deoxyribonucleotide bases. The guide sequences disclosed herein, when complexed with a type V CRISPR-Cas protein such as Cas12a, are capable of robust editing of a target genome and reduced levels of off-target genome editing.

[0012] This genome editing process is particularly suitable for producing genetically modified cells used for therapeutic purposes. For example, using such a genome editing process, immune cells (such as T cells) can be genetically modified to express CAR. Such CAR-expressing cells can be used, for example, in adoptive immunotherapy, where CAR-expressing immune cells, such as T cells (CAR-T cells), can be administered to patients to target cells expressing a target antigen recognized by the CAR (e.g., a foreign antigen or a cancer-associated antigen).

[0013] The following non-limiting embodiments of the invention are:

[0014] [1] A CRISPR guide molecule comprising: a targeting region capable of binding to a target nucleic acid sequence; and an activating region capable of forming a nucleoprotein complex with a Cas12 protein, wherein said CRISPR guide molecule comprises ribonucleotide bases and at least one deoxyribonucleotide base.

[0015] [2] The CRISPR guide molecule of [1], wherein the CRISPR guide molecule comprises at least one deoxyribonucleotide base in the activating region, the targeting region, or both regions.

[0015] [3] The CRISPR guide molecule of [1], wherein the CRISPR guide molecule further comprises one or more base analogs selected from the group consisting of inosine, deoxyinosine, deoxyuracil, xanthosine, C3 spacer, 5-methyl-dC, 5-hydroxybutynyl-2'-deoxyuridine, 5-nitroindole, 5-methylisodeoxycytosine, isodeoxyguanosine, deoxyuridine, and isodeoxycytidine.

[0017] [4] The CRISPR guide molecule of [1], wherein the CRISPR guide molecule further comprises one or more non-basic sites.

[0018] [5] The CRISPR guide molecule of [1], wherein the CRISPR guide molecule is selected from: a CRISPR guide molecule comprising the RNA sequence UAAUUUCUACUUCUUGUAGAUGAGUCUCUCAGCUGGUACAC, wherein at least one of the bases in the sequence is replaced with a corresponding deoxyribonucleotide base, and optionally at least one of the bases in the sequence is replaced with a base analogue or a non-basic site; and a CRISPR guide molecule comprising the RNA sequence UAAUUUCUACUUCUUGUAGAUAGUAGUGGGGGUGGUGAUUCAGUGU, wherein at least one of the bases in the sequence is replaced with a corresponding deoxyribonucleotide base, and optionally at least one of the bases in the sequence is replaced with a base analogue or a non-basic site.

[0019] [6] The CRISPR guide molecule of [5], wherein the number of deoxyribonucleotide bases in the CRISPR guide molecule as a percentage of the total size of the CRISPR guide molecule is 50% or less.

[0020] [7] The CRISPR guide molecule of [6], wherein the number of deoxyribonucleotide bases in the CRISPR guide molecule as a percentage of the total size of the guide molecule is 25% or less.

[0021] [8] The CRISPR targeting molecule of [5], wherein the number of deoxyribonucleotide bases in the targeting region as a percentage of the total size of the targeting region is 25% or less.

[0022] [9] The CRISPR targeting molecule of [8], wherein the number of deoxyribonucleotide bases in the targeting region as a percentage of the total size of the targeting region is 5% or less.

[0023] [10] The CRISPR guide molecule of [5], wherein the number of deoxyribonucleotide bases in the activating region as a percentage of the total size of the activating region is 50% or less.

[0024] [11] The CRISPR guide molecule of [10], wherein the number of deoxyribonucleotide bases in the activating region as a percentage of the total size of the activating region is 25% or less.

[0025] [12] The CRISPR guide molecule of [5], wherein the CRISPR guide molecule has reduced off-target activity compared to a CRISPR guide molecule containing only RNA that binds to a target nucleic acid sequence and that is capable of forming a nucleoprotein complex with the Cas12 protein.

[0025] [13] The CRISPR guide molecule of [5], wherein one or more positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, 19, 21, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, and 40 in the sequence comprise a deoxyribonucleotide base.

[0027] [14] The CRISPR guide molecule of [13], wherein fifteen or fewer positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, 19, 21, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, and 40 in the sequence contain deoxyribonucleotide bases.

[0028] [15] The CRISPR guide molecule of [14], wherein twelve or fewer positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, 19, 21, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, and 40 in the sequence contain deoxyribonucleotide bases.

[0029] [16] The CRISPR guide molecule of [2], wherein the CRISPR guide molecule comprises an activating region comprising the RNA sequence UAAUUUCUACUUCUUGUAGAU, wherein at least one of the bases in the sequence is replaced by a corresponding deoxyribonucleotide base, and optionally at least one of the bases in the sequence is replaced by a base analogue or a non-basic site.

[0030] [17] The CRISPR guide molecule of [16], wherein one or more positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19 in the sequence comprise a deoxyribonucleotide base.

[0031] [18] The CRISPR guide molecule of [17], wherein ten or fewer positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19 in the sequence contain deoxyribonucleotide bases.

[0032] [19] The CRISPR guide molecule of [18], wherein eight or fewer positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19 in the sequence contain deoxyribonucleotide bases.

[0033] [20] The CRISPR guide molecule of [2], wherein the CRISPR guide molecule comprises a targeting region comprising the RNA sequence GAGUCUCUCAGCUGGUACAC, wherein at least one of the bases in the sequence is replaced with a corresponding deoxyribonucleotide base, and optionally, at least one of the bases in the sequence is replaced with a base analogue or a non-basic site.

[0034] [21] The CRISPR guide molecule of [20], wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence comprise a deoxyribonucleotide base.

[0035] [22] The CRISPR guide molecule of [21], wherein five or fewer positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence contain deoxyribonucleotide bases.

[0035] [23] The CRISPR guide molecule of [22], wherein three or less positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence contain deoxyribonucleotide bases.

[0037] [24] The CRISPR guide molecule of [2], wherein the CRISPR guide molecule comprises a targeting region comprising the RNA sequence AGUGGGGGUGAAUUCAGUGU, wherein at least one of the bases in the sequence is replaced with a corresponding deoxyribonucleotide base, and optionally, at least one of the bases in the sequence is replaced with a base analogue or a non-basic site.

[0038] [25] The CRISPR guide molecule of [24], wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence comprise a deoxyribonucleotide base.

[0039] [26] The CRISPR guide molecule of [25], wherein five or fewer positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence contain deoxyribonucleotide bases.

[0040] [27] The CRISPR guide molecule of [26], wherein three or less positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence contain deoxyribonucleotide bases.

[0041] [28] The CRISPR guide molecule of [5], wherein the CRISPR guide molecule comprises the sequence TAAUUUCUACUCUTGUAGAUGAGUCUCUCAGCUGGUACAC, wherein positions 2, 4, 5, 6, 8, 9, 11, 13, 16, 17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38 and 39 in the sequence comprise ribonucleotide bases, and wherein positions 1, 3, 7, 10, 12, 14, 15, 19, 21, 31 and 40 in the sequence comprise deoxyribonucleotide bases.

[0042] [29] The CRISPR guide molecule of [5], wherein the CRISPR guide molecule comprises the sequence TAAUUUCUACUCUTGUAGAUAGUGGGGGUGAUUCAGUGT, wherein positions 2, 4, 5, 6, 8, 9, 11, 13, 16, 17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38 and 39 in the sequence comprise ribonucleotide bases, and wherein positions 1, 3, 7, 10, 12, 14, 15, 19, 21, 31 and 40 in the sequence comprise deoxyribonucleotide bases.

[0043] [30] The CRISPR guide molecule of [2], wherein the activating region is 20 bases long, and wherein one or more positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19 in the 20 nucleotide activating region sequence include a deoxyribonucleotide base.

[0044] [31] The CRISPR guide molecule of [30], wherein ten or fewer positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19 in the sequence contain deoxyribonucleotide bases.

[0045] [32] The CRISPR guide molecule of [31], wherein eight or fewer positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19 in the sequence contain deoxyribonucleotide bases.

[0045] [33] The CRISPR targeting molecule of [2], wherein the targeting region is 20 bases long and wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 and 20 in the sequence of the 20 nucleotide targeting region comprise a deoxyribonucleotide base.

[0047] [34] The CRISPR guide molecule of [33], wherein five or fewer positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence contain deoxyribonucleotide bases.

[0048] [35] The CRISPR guide molecule of [34], wherein three or less positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the sequence contain deoxyribonucleotide bases.

[0049] [36] A nucleic acid/CRISPR protein composition comprising: a CRISPR guide molecule according to any one of [1]-[35]; and a Cas12 protein.

[0050] [37] The CRISPR nucleic acid/protein composition of [36], wherein the CRISPR guide molecule is in a complex with the Cas12 protein.

[0051] [38] The CRISPR nucleic acid/protein composition of [36], wherein the Cas12 protein is the Cas12a protein.

[0052] [39] The CRISPR nucleic acid/protein composition of any one of [36]-[38], wherein the Cas12 protein comprises at the C-terminus a sequence comprising a linker and an NLS that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 479-490.

[0053] [40] The CRISPR nucleic acid/protein composition of [39], wherein the sequence comprising the linker and NLS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 483, 485, 487 and 489.

[0054] [41] A cell comprising a CRISPR guide molecule according to any one of [1]-[35].

[0055] [42] The cell according to [41], additionally containing the Cas12 protein.

[0055] [43] The cell according to [42], wherein the Cas12 protein is the Cas12a protein.

[0057] [44] The cell according to [42] or [43], wherein the Cas12 protein comprises at the C-terminus a sequence that comprises a linker and an NLS, and that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 479-490.

[0058] [45] The cell of [44], wherein the sequence comprising the linker and NLS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 483, 485, 487 and 489.

[0059] [46] The cell of any one of [42]-[45], wherein the CRISPR guide molecule is in a complex with a Cas12 protein.

[0060] [47] The cell according to any one of [41]-[46], wherein the cell is a prokaryotic cell or a eukaryotic cell.

[0061] [48] The cell of [47], wherein the cell is a eukaryotic cell selected from the group consisting of a unicellular eukaryotic organism, a cell of a eukaryotic organism, a protozoan cell, a plant cell, an algal cell, a fungal cell, an animal cell, an invertebrate cell, a vertebrate cell, a mammalian cell, a stem cell, and a progenitor cell.

[0062] [49] The cell of [48], wherein the cell is a lymphocyte, a chimeric antigen receptor (CAR) T cell, a T cell receptor (TCR) cell, a TCR-engineered CAR-T cell, a tumor infiltrating lymphocyte (TIL), a CAR TIL, a dendritic cell (DC), a CAR-DC, a macrophage, a CAR-macrophage (CAR-M), a natural killer (NK) cell, or a CAR-NK cell.

[0063] [50] The cell of [49], wherein the cell is a CAR-T cell.

[0064] [51] The cell of any one of [41]-[50], further comprising a donor polynucleotide.

[0065] [52] A method for cleaving a target nucleic acid sequence, wherein said method comprises: contacting a first target nucleic acid sequence with a nucleoprotein complex comprising a catalytically active Cas12 protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule comprises a CRISPR guide molecule according to any one of [1]-[35]; wherein the targeting region of the first CRISPR guide molecule is capable of hybridizing with the first target nucleic acid sequence, and the nucleoprotein complex is capable of cleaving the first target nucleic acid sequence.

[0065] [53] The method according to [52], further comprising obtaining a donor polynucleotide.

[0067] [54] The method according to [53], wherein the target nucleic acid sequence is cleaved to form a cleavage site, and wherein the method further comprises modifying the target nucleic acid sequence.

[0068] [55] The method of [54], wherein the modification comprises introducing at least a portion of a donor polynucleotide into the cleavage site.

[0069] [56] The method according to [54], wherein the modification comprises deleting one or more nucleotides at the cleavage site.

[0070] [57] The method according to [55], wherein the target nucleic acid sequence is located in a cell.

[0071] [58] The method according to [57], wherein the cell comprises a eukaryotic cell.

[0072] [59] The method according to [58], wherein the donor polynucleotide comprises a vector for expressing a CAR.

[0073] [60] The method of [59], further comprising introducing a CAR expression vector into the cell using a viral vector.

[0074] [61] The method according to [60], wherein said administering comprises transduction.

[0075] [62] The method according to any one of [57]-[61], wherein the obtained cell comprises a lymphocyte, a CAR-T cell, a TCR cell, a CAR-T cell engineered based on a TCR, a TIL, a CAR-TIL, a dendritic cell, a CAR-DC, a macrophage, a CAR-M, an NK cell, or a CAR-NK cell.

[0075] [63] The method according to any one of [52]-[62], wherein the first target nucleic acid sequence is within a target gene encoding a protein selected from the group consisting of TRAC; TRBV; beta-2-microglobulin (B2M); PD1; PD-L1; CTLA-4; LAG-3; TIGIT; TIM3; HLA-E; HLA-A; HLA-B; HLA-C; HLA-DRA; ADAM17; BTLA; CD160; SIGLEC10; 2B4; LAIR1; CD52; CD96; VSIR; VISTA; KIR2DL1; KIR2DL2; KIR2DL3; CEACAM1; CBLB; CISH; IL-1R8; AHR; adenosine receptor 2A; GMCSF; VISTA; CII2A; and NKG2A.

[0077] [64] The method according to any one of [59]-[61], wherein the CAR expression vector encodes a CAR comprising an extracellular ligand binding domain.

[0078] [65] The method of [64], wherein the CAR expression vector further encodes a hinge region, a transmembrane region, and one or more intracellular signaling regions.

[0079] [66] The method according to [64] or [65], wherein the extracellular ligand binding domain comprises a single-chain immunoglobulin variable fragment (scFv).

[0080] [67] The method according to [66], wherein the scFv is capable of binding to a cellular target selected from the group consisting of CD37, CD38, CD47, CD73, CD4, CS1, PD-L1, NGFR, ENPP3, PSCA, CD79B, TACI, VEGFR2, B7-H3, B7-H6, B cell maturation antigen (BCMA), CD123, CD138, CD171/L1CAM, CD19, CD20, CD22, CD30, CD33, CD70, CD371, CEA, claudin 18.1, claudin 18.2, CSPG4, EFGRvIII, EpCAM, EphA2, epidermal growth factor receptor, ErbB, ErbB2 (HER2), FAP, FRα, GD2, GD3, glypican 3, IL-11Ra, IL-13Ra2, IL13 receptor alpha, Lewis antigen Y/LeY, mesothelin, MUC1, MUC16, NKG2D ligands, PD1, PSMA, ROR-1, SLAMF7, TAG72, ULBP and MICA/B, VEGF2 and WT1 proteins.

[0081] [68] The method according to [67], wherein the scFv is capable of binding to a cellular target selected from the group consisting of BCMA, CD19, CD20, CD22, CD47, CD371, ROR-1, EphA2, MUC16, glypican 3, PSCA and claudin 18.2.

[0082] [69] The method according to [68], wherein the scFv is capable of binding to BCMA.

[0083] [70] The method according to [68], wherein the scFv is capable of binding to CD371.

[0084] [71] The method according to any one of [57]-[62] and [64]-[70], wherein said method further comprises contacting a second target nucleic acid sequence in a cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule comprises a CRISPR guide molecule according to any one of [1]-[35], which is capable of binding to another target nucleic acid sequence different from the first CRISPR guide molecule, wherein the targeting region of the second CRISPR guide molecule is capable of hybridizing to the second target nucleic acid sequence, and the nucleoprotein complex is capable of cleaving the second target nucleic acid sequence.

[0085] [72] The method according to [71], wherein said first and second target nucleic acid sequences are independently located within a target gene encoding a protein selected from the group consisting of TRAC; TRBV protein; beta-2-microglobulin (B2M); PD1; PD-L1; CTLA-4; LAG-3; TIGIT; TIM3; HLA-E; HLA-A; HLA-B; HLA-C; HLA-DRA; ADAM17; BTLA; CD160; SIGLEC10; 2B4; LAIR1; CD52; CD96; VSIR; VISTA; KIR2DL1; KIR2DL2; KIR2DL3; CEACAM1; CBLB; CISH; IL-1R8; AHR; adenosine receptor 2A; GMCSF; VISTA; CII2A; and NKG2A.

[0085] [73] The method according to [71] or [72], wherein the donor polynucleotide comprises a vector for expressing a CAR, wherein the CAR comprises an extracellular ligand binding domain, and wherein the extracellular ligand binding domain comprises an scFv.

[0087] [74] The method according to [73], wherein the scFv is capable of binding to BCMA.

[0088] [75] The method according to [73], wherein the scFv is capable of binding to CD371.

[0089] [78] The method according to [72], wherein said first target nucleic acid sequence is located within a gene encoding a TRAC protein, and wherein said second target nucleic acid sequence is located within a gene encoding a PD1 protein.

[0090] [77] The method according to [72], wherein said first target nucleic acid sequence is located within a gene encoding a TRAC protein, and wherein said second target nucleic acid sequence is located within a gene encoding a B2M protein.

[0091] [78] The method according to [77], further comprising introducing into the cell a second donor polynucleotide comprising a B2M-HLA-E fusion construct, and wherein at least a portion of the second donor polynucleotide comprising a B2M-HLA-E fusion construct is introduced into a cleavage site of a second target nucleic acid sequence, wherein the B2M-HLA-E fusion construct encodes a fusion protein comprising, from the N- to the C-terminus, a B2M secretion signal, an HLA-G peptide signal sequence, a first linker sequence, a B2M sequence, a second linker sequence and an HLA-E sequence.

[0092] [79] The method according to [69] or [74], wherein the anti-BCMA scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:474; and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:475.

[0093] [80] The method according to [79], wherein the scFv further comprises a linker between VH and VL.

[0094] [81] The method according to [80], wherein the linker comprises the amino acid sequence of SEQ ID NO:476.

[0095] [82] The method according to [81], wherein said scFv comprises the amino acid sequence of SEQ ID NO:477.

[0095] [83] The method according to [64] or [73], wherein the CAR comprises: an scFv comprising a VH and a VL; a transmembrane domain; a costimulatory domain; and an activating domain.

[0097] [84] The method of [83], wherein the transmembrane domain is a transmembrane domain derived from a T cell receptor α chain, a T cell receptor β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR.

[0098] [85] The method of [84], wherein the transmembrane domain comprises a transmembrane domain derived from CD8.

[0099] [86] The method according to [83], wherein the costimulatory domain is a costimulatory domain derived from CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40, or DAP10.

[00100] [87] The method according to [86], wherein the costimulatory domain comprises a 4-1BB costimulatory domain.

[00101] [88] The method according to [83], wherein the activating domain comprises a CD3ζ activating domain.

[00102] [89] The method according to [83], wherein the transmembrane domain comprises a transmembrane domain derived from CD8, the costimulatory domain comprises a 4-1BB costimulatory domain, and the activating domain comprises a CD3ζ activating domain.

[00103] [90] The method according to [83], wherein VH comprises the amino acid sequence of SEQ ID NO:474 and VL comprises the amino acid sequence of SEQ ID NO:475.

[00104] [91] The method according to any one of [59]-[62], [64]-[70], [73]-[75] and [78]-[90], wherein the polynucleotide sequence encoding the CAR in said CAR expression vector has a leader sequence at the 5' end.

[00105] [92] The method according to [91], wherein the leader sequence comprises the nucleic acid sequence of SEQ ID NO:478.

[00105] [93] The method according to [91], wherein the CAR expression vector comprises a promoter.

[00107] [94] The method according to [93], wherein the promoter comprises a MND promoter.

[00108] [95] The method according to any of [52]-[94], wherein the Cas12 protein comprises at the C-terminus a sequence that includes a linker and an NLS, and that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 479-490.

[00109] [96] The method according to [95], wherein the sequence comprising the linker and the NLS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 483, 485, 487 and 489.

[00110] [97] A cell obtained by the method according to any of [57]-[96].

[00111] [98] A CAR-T cell produced by the method of any one of [59]-[96].

[00112] [99] The CAR-T cell of [98], wherein said CAR-T cell is an allogeneic CAR-T cell.

[00113] [100] The CAR-T cell of [98], wherein said CAR-T cell is an autologous CAR-T cell.

[00114] [101] A method for producing a CAR-T cell, comprising performing the method of any one of [59]-[96] using a T lymphocyte as the cell.

[00115] [102] A method of adoptive cell therapy comprising administering to an individual in need thereof a cell obtained by the method of any of [57]-[96].

[00115] [103] A method of adoptive cell therapy comprising administering to an individual in need thereof a CAR-T cell obtained by the method of any one of [59]-[96].

[00117] [104] A method for killing BCMA-positive cancer cells, wherein said method comprises contacting BCMA-positive cancer cells with CAR-T cells obtained by the method of any one of [69], [74] and [90].

[00118] [105] The method of [104], wherein the BCMA-positive cancer cells comprise multiple myeloma cancer cells.

[00119] [106] The method according to [105], wherein the multiple myeloma cancer cells comprise human cells.

[00120] [107] The method according to [104], wherein the contacting is intratumoral.

[00121] [108] A method for producing a CAR-expressing cell, wherein said method comprises: contacting a first target nucleic acid sequence in the cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule comprises a CRISPR guide molecule according to any one of [1]-[35], wherein the targeting region of the first CRISPR guide molecule is capable of hybridizing with the first target nucleic acid sequence, and the nucleoprotein complex is capable of cleaving the first target nucleic acid sequence; contacting a second target nucleic acid sequence in a cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule comprises a CRISPR guide molecule according to any one of [1]-[35], which is capable of binding to another target nucleic acid sequence different from the first CRISPR guide molecule, wherein the targeting region of the second CRISPR guide molecule is capable of hybridizing to the second target nucleic acid sequence, and the nucleoprotein complex is capable of cleaving the second target nucleic acid sequence; and introducing into said cell a donor polynucleotide comprising a vector for expressing a CAR, wherein at least a portion of the donor polynucleotide comprising said vector for expressing a CAR can be inserted into a cleavage site in said first target nucleic acid sequence, and wherein the CAR comprises an extracellular ligand-binding domain.

[00122] [109] The method according to [108], wherein the donor polynucleotide containing the CAR expression vector is introduced into the cell using a viral vector.

[00123] [110] The method of [108], wherein the CAR expression vector further encodes a hinge region, a transmembrane region, and one or more intracellular signaling regions.

[00124] [111] The method according to any one of [108]-[110], wherein said first target nucleic acid sequence is located within a gene encoding a TRAC protein, and wherein said second target nucleic acid sequence is located within a gene encoding a PD1 protein.

[00125] [112] The method according to any one of [108]-[110], wherein said first target nucleic acid sequence is located within a gene encoding a TRAC protein, and wherein said second target nucleic acid sequence is located within a gene encoding a B2M protein.

[00125] [113] The method according to any one of [108]-[112], wherein the extracellular ligand binding domain comprises a single chain immunoglobulin variable fragment (scFv).

[00127] [114] The method according to [113], wherein the scFv is capable of binding to BCMA.

[00128] [115] The method according to [113], wherein the scFv is capable of binding to CD371.

[00129] [116] The method according to [114], wherein the anti-BCMA scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:474 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:475.

[00130] [117] The method according to [116], wherein the scFv further comprises a linker between VH and VL.

[00131] [118] The method according to [117], wherein the linker comprises the amino acid sequence of SEQ ID NO:476.

[00132] [119] The method according to [114], wherein said scFv comprises the amino acid sequence of SEQ ID NO:477.

[00133] [120] The method according to any one of [108]-[119], further comprising introducing into the cell a second donor polynucleotide comprising a B2M-HLA-E fusion construct, wherein at least a portion of the second donor polynucleotide comprising a B2M-HLA-E fusion construct is introduced into a cleavage site of a second target nucleic acid sequence, wherein the B2M-HLA-E fusion construct encodes a fusion protein comprising, from the N- to the C-terminus, a B2M secretion signal, an HLA-G peptide signal sequence, a first linker sequence, a B2M sequence, a second linker sequence, and an HLA-E sequence.

[00134] [121] The method according to any one of [108]-[120], wherein the CAR comprises: an scFv comprising a VH and a VL; a transmembrane domain; a costimulatory domain; and an activating domain.

[00135] [122] The method of [121], wherein the transmembrane domain comprises a transmembrane domain derived from a T cell receptor α chain, a T cell receptor β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR.

[00135] [123] The method of [122], wherein the transmembrane domain comprises a transmembrane domain derived from CD8.

[00137] [124] The method of [121], wherein the costimulatory domain comprises a costimulatory domain derived from CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40, or DAP10.

[00138] [125] The method according to [124], wherein the costimulatory domain comprises a 4-1BB costimulatory domain.

[00139] [126] The method according to [121], wherein the activating domain comprises a CD3ζ activating domain.

[00140] [127] The method according to [121], wherein the transmembrane domain comprises a transmembrane domain derived from CD8, the costimulatory domain comprises a 4-1BB costimulatory domain, and the activating domain comprises a CD3ζ activating domain.

[00141] [128] The method according to any one of [108]-[127], wherein the CAR-expressing cell is a CAR-T cell.

[00142] [129] The method of [128], wherein the CAR-T cell is an allogeneic CAR-T cell.

[00143] [130] The method of [128], wherein the CAR-T cell is an autologous CAR-T cell.

[00144] [131] The method according to any of [108]-[130], wherein the Cas12 protein complexed with the first CRISPR guide molecule and/or the Cas12 protein complexed with the second CRISPR guide molecule comprises at the C-terminus a sequence that includes a linker and an NLS, and that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 479-490.

[00145] [132] The method according to [131], wherein the Cas12 protein complexed with the first CRISPR guide molecule and/or the Cas12 protein complexed with the second CRISPR guide molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 483, 485, 487 and 489.

[00145] [133] The method according to [78], wherein the second donor polynucleotide further comprises a P2A sequence at the N-terminus of the B2M-HLA-E fusion construct.

[00147] [134] The method according to [120], wherein the second donor polynucleotide further comprises a P2A sequence at the N-terminus of the B2M-HLA-E fusion construct sequence.

[00148] In some embodiments, the present invention relates to a CRISPR guide molecule comprising a targeting region capable of binding to a target nucleic acid sequence and an activating region comprising the RNA sequence UAAUUUCUACUUCUUGUAGAU, including at least one deoxyribonucleotide instead of a ribonucleotide, wherein the activating region is capable of forming a nucleoprotein complex with the Cas12 protein. In some embodiments, one or more (e.g., ten or less) positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19 of the activating region comprise a deoxyribonucleotide base. In some embodiments, the molecule comprises one or more chemical modifications selected from the group consisting of base modifications including inosine, deoxyinosine, deoxyuracil, xanthosine, C3 spacer, 5-methyl-dC, 5-hydroxybutynyl-2'-deoxyuridine, 5-nitroindole, 5-methylisodeoxycytosine, isodeoxyguanosine, deoxyuridine, isodeoxycytidine and a minor site, and backbone modifications including a phosphorothioate modification.

[00149] In some embodiments, the targeting region of the CRISPR guide sequence targets the B2M gene and comprises the RNA sequence AGUGGGGGUGAAUUCAGUGU, wherein, optionally, at least one of the bases in the sequence is replaced with a base analog or a non-basic site. In some embodiments, one or more (such as five or less) positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the targeting region comprise a deoxyribonucleotide base. In some embodiments, the targeting region is capable of hybridizing to a sequence selected from SEQ ID NOs: 51-133. In some embodiments, the CRISPR guide sequence comprises a sequence selected from SEQ ID NO: 212-231, 275-315, and 331-350. In some embodiments, the CRISPR guide sequence comprises the sequence of SEQ ID NO: 416.

[00150] In some embodiments, the targeting region of the CRISPR guide sequence targets the TRAC gene and comprises the RNA sequence GAGUCUCUCAGCUGGUACAC, wherein, optionally, at least one of the bases in the sequence is replaced with a base analog or a non-basic site. In some embodiments, one or more (such as five or less) positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20 in the targeting region comprise a deoxyribonucleotide base. In some embodiments, the targeting region is capable of hybridizing to a sequence selected from SEQ ID NOs: 15-20. In some embodiments, the CRISPR guide sequence comprises a sequence selected from SEQ ID NOs: 233-252, 317-329, 491-492, and 508. In some embodiments, the CRISPR guide molecule further comprises a chemical modification and a sequence selected from SEQ ID NOs: 512-517. In some embodiments, the CRISPR guide molecule comprises the sequence of SEQ ID NO: 415.

[00151] In some embodiments, the targeting region targets the CISH gene and is capable of hybridizing to a sequence selected from SEQ ID NOs: 157-165. In some embodiments, the CRISPR guide molecule comprises a sequence selected from SEQ ID NOs: 509 and 519-529.

[00152] In some embodiments, the targeting region targets the PDCD1 gene and is capable of hybridizing to a sequence selected from SEQ ID NO: 135-155.

[00153] In some embodiments, the targeting region targets the CBLB gene and is capable of hybridizing to a sequence selected from SEQ ID NO: 167-189. In some embodiments, the CRISPR guide molecule comprises the sequence of SEQ ID NO: 510.

[00154] In some embodiments, the present invention relates to a nucleic acid/CRISPR protein composition comprising a CRISPR guide molecule as described above and a Cas12 protein. In some embodiments, the Cas12 protein is a Cas12 protein comprising a sequence at the C-terminus that includes a linker and a nuclear localization signal (NLS) and that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 479-490.

[00155] In some embodiments, the present invention relates to a cell comprising the above-described CRISPR nucleic acid/protein composition, wherein the cell is a lymphocyte, a T cell comprising a chimeric antigen receptor (CAR), a cell comprising a T cell receptor (TCR), a CAR-T cell engineered with a TCR, a tumor-infiltrating lymphocyte (TIL), a CAR-TIL, a dendritic cell (DC), a CAR-DC, a macrophage, a CAR-macrophage (CAR-M), a natural killer (NK) cell, an induced pluripotent stem cell (iPSC), a cell differentiated from an iPSC, or a CAR-NK cell.

[00155] In some embodiments, the present invention relates to a method of producing a cell expressing a chimeric antigen receptor (CAR), wherein said method comprises: contacting a first target nucleic acid sequence comprising a TRAC sequence in a cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a first CRISPR guide molecule having a targeting region capable of binding to the first target nucleic acid sequence; and an activating region capable of forming a nucleoprotein complex with the Cas12 protein, wherein said CRISPR guide molecule comprises ribonucleotide bases and at least one deoxyribonucleotide base in the activating region, the targeting region, or both, and the nucleoprotein complex is capable of cleaving the first target nucleic acid sequence; contacting a second target nucleic acid sequence comprising a B2M sequence in the same cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a second CRISPR guide molecule having a targeting region capable of binding to the second target nucleic acid sequence; and an activating region capable of forming a nucleoprotein complex with the Cas12 protein, wherein said CRISPR guide molecule comprises ribonucleotide bases and at least one deoxyribonucleotide base in the activating region, in the targeting region, or in both regions, and the nucleoprotein complex is capable of cleaving the second target nucleic acid sequence; obtaining a first donor polynucleotide encoding a CAR comprising an scFv, a transmembrane domain, a costimulatory domain, and an activating domain, wherein the CAR is capable of inserting into a cleavage site of the first target nucleic acid sequence; obtaining a second donor polynucleotide encoding a B2M-HLA-E fusion construct comprising a B2M secretion signal, an HLA-G peptide signal sequence, a first linker sequence, a B2M sequence, a second linker sequence, an HLA-E sequence, wherein the B2M-HLA-E fusion construct is capable of inserting into a cleavage site in a second target nucleic acid sequence; cleaving the first target nucleic acid sequence and inserting at least a portion of the first donor polynucleotide into the cleavage site; and cleaving the second target nucleic acid sequence and inserting at least a portion of the second donor polynucleotide into the cleavage site. In some embodiments, the second donor polynucleotide further comprises a P2A sequence at the 5' end of the B2M-HLA-E fusion construct. In some embodiments, the first donor polynucleotide comprises SEQ ID NO: 413. In some embodiments, the second donor polynucleotide comprises SEQ ID NO: 414.

[00157] In some embodiments, the scFv in the CAR is capable of binding to a cellular target selected from the group consisting of CD37, CD38, CD47, CD73, CD4, CS1, PD-L1, NGFR, ENPP3, PSCA, CD79B, TACI, VEGFR2, B7-H3, B7-H6, B cell maturation antigen (BCMA), CD123, CD138, CD171/L1CAM, CD19, CD20, CD22, CD30, CD33, CD70, CD371, CEA, claudin 18.1, claudin 18.2, CSPG4, EFGRvIII, EpCAM, EphA2, epidermal growth factor receptor, ErbB, ErbB2 (HER2), FAP, FRα, GD2, GD3, glypican 3, IL-11Rα, IL-13Rα2, IL13 receptor alpha, Lewis antigen Y/LeY, mesothelin, MUC1, MUC16, NKG2D ligands, PD1, PSMA, ROR-1, SLAMF7, TAG72, ULBP and MICA/B, VEGF2 and WT1 proteins. In some embodiments, the scFv is capable of binding to BCMA and comprises a first variable region comprising the amino acid sequence of SEQ ID NO: 474, a second variable region comprising the amino acid sequence of SEQ ID NO: 475, and a linker located between the first and second variable regions and comprising the amino acid sequence of SEQ ID NO: 476. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 477.

[00158] In some embodiments, the transmembrane domain of the CAR is derived from a T cell receptor α chain, a T cell receptor β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR. In some embodiments, the costimulatory domain of the CAR is derived from CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40, or DAP10. In some embodiments, the CAR comprises a transmembrane domain derived from CD8, the costimulatory domain of 4-1BB, and the activation domain of CD3ζ. In some embodiments of the invention, a vector comprising a CAR sequence comprises a leader sequence having the nucleic acid sequence of SEQ ID NO: 478.

[00159] In some embodiments, the catalytically active Cas12 protein used in the method comprises a sequence at the C-terminus that includes a linker and a nuclear localization signal (NLS) and that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 479-490.

[00160] In some embodiments, the method further comprises contacting a third target nucleic acid sequence in the same cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a third CRISPR guide molecule having a targeting region capable of binding to the third target nucleic acid sequence; and an activating region capable of forming a nucleoprotein complex with the Cas12 protein, wherein said CRISPR guide molecule comprises ribonucleotide bases and at least one deoxyribonucleotide base in the activating region, in the targeting region, or in both regions, and the nucleoprotein complex is capable of cleaving the third target nucleic acid sequence; cleaving a third target nucleic acid sequence and deleting one or more nucleotides from the third target nucleic acid sequence at the cleavage site, wherein the third target nucleic acid sequence is selected from the PDCD gene, the CISH gene and the CBLB gene.

[00161] In some embodiments of the invention, the CAR-expressing cell is an allogeneic or autologous CAR T cell produced from a T lymphocyte.

[00162] In some embodiments, the present invention relates to a CAR-expressing cell obtained by the above-described method, wherein said cell is selected from a lymphocyte, a CAR-T cell, a TCR cell, a CAR-T cell engineered based on a TCR, a TIL, a CAR-TIL, a dendritic cell, a CAR-DC, a macrophage, a CAR-M, an iPSC cell, a cell differentiated from an iPSC cell, an NK cell, or a CAR-NK cell.

[00163] In some embodiments, the present invention relates to a method of performing adoptive cell therapy comprising administering to an individual in need thereof a CAR-expressing cell as described above. In some embodiments, adoptive cell therapy comprises killing BCMA-positive cancer cells, such as multiple myeloma cancer cells.

Inclusion by reference

[00164] All patents, publications, and patent applications mentioned in this specification are herein incorporated by reference as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety and for all purposes.

Brief description of the drawings

[00165] The features of the present invention are set forth in detail in the appended claims. For a better understanding of the features and advantages of the present invention, the invention is described in detail below with reference to illustrative embodiments of the invention, which set forth the principles of its disclosure, and to the accompanying drawings. The numbers are not proportional and are not to scale. The location of the pointers is approximate.

[00165] Fig. 1A, Fig. 1B, and Fig. 1C show examples of type V CRISPR-Cas12a guide RNAs.

[00167] Fig. 2 illustrates the cleavage of a target polynucleotide by the chRDNA Cas12a guide sequence/nucleoprotein complex.

[00168] Figures 3A-3I illustrate various canonical and non-canonical nucleotides for use in chRDNA Cas12 guides.

[00169] Fig. 4 illustrates the cleavage of a target polynucleotide by the chRDNA Cas12a guide sequence/nucleoprotein complex.

[00170] Fig. 5 illustrates the Cas12a cr-RNA guide.

[00171] Fig. 6 illustrates a chRDNA Cas12a guide comprising DNA bases in the activating region and a target binding sequence.

[00172] Fig. 7 illustrates a chRDNA Cas12a guide comprising DNA bases and chemically modified nucleic acids in the activating region and in the target binding sequence.

[00173] Fig. 8 illustrates the formation of the chRDNA Cas12a guide sequence/nucleoprotein complex and binding of the target polynucleotide.

[00174] Fig. 9 illustrates the production of insertions or deletions (indels) in a target polynucleotide by the chRDNA Cas12a guide sequence/nucleoprotein complex.

[00175] Fig. 10 illustrates the insertion of a donor polynucleotide sequence into a target polynucleotide via the chRDNA Cas12a guide sequence/nucleoprotein complex.

[00175] Fig. 11 illustrates a single-strand break in a target polynucleotide by the chRDNA Cas12a guide sequence/nucleoprotein complex.

[00177] Fig. 12 illustrates a tandem single-strand break in a target polynucleotide by two chRDNA Cas12a guide sequence/nucleoprotein complexes and the incorporation of a donor polynucleotide sequence into the target polynucleotide.

[00178] Figure 13 illustrates the average normalized editing rates of chRDNA Cas12a guide sequence/nucleoprotein complexes with a single DNA base in the target binding sequence.

[00179] Figure 14 illustrates the normalized editing rates of chRDNA Cas12a guide sequence/nucleoprotein complexes with a single DNA base in the activating region.

[00180] Fig. 15A and Fig. 15B illustrate the phenotypic and cytotoxic profile of CAR-T cells generated using chRDNA Cas12a guide sequence/nucleoprotein complexes.

[00181] Fig. 16A and Fig. 16B illustrate the editing activity of the chRDNA Cas12a guide sequence/nucleoprotein complex with different configurations of polypeptide linkers and nuclear localization sequence (NLS).

[00182] Fig. 17 illustrates the editing activity of the chRDNA Cas12a guide sequence/nucleoprotein complex when targeting multiple genes simultaneously with different polypeptide linker and nuclear localization sequence (NLS) configurations.

Detailed description of the invention

[00183] It should be noted that the terminology used herein is intended only to describe particular embodiments of the invention, and is not intended to limit the scope of the invention. As used in this application and in the appended claims, the articles "a" and "an" and "the" when used with singular nouns may also refer to plural nouns unless the context of the description otherwise requires. Thus, for example, the term "polynucleotide" includes one or more polynucleotides, and the term "vector" includes one or more vectors.

[00184] Unless otherwise defined, all technical and scientific terms used in this application have their commonly understood meanings as understood by those skilled in the art to which the invention pertains. Although other similar or equivalent methods and materials may be used in this description, the materials and methods described in this application are preferred.

[00185] The terms “SITE-Seq®” and “SITE-Seq® analysis” refer to a biochemical method for identifying the sequence of cleavage sites in genomic DNA generated by Cas9-programmed single-stranded guide RNAs (ssRNAs). This assay is described in detail in Cameron P. et al. (2017) Mapping the genomic landscape of CRISPR-Cas9 cleavage . Nature Methods. 14(6), 600–606. https//doi.org/10.1038/nmeth.4284).

[00185] Based on the description of the present invention, one skilled in the art can apply conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant polynucleotides, as described, for example, in the following standard textbooks: Abbas et al. (Cellular and Molecular Immunology, 2017, 9th Edition, Elsevier, ISBN 978-0323479783); Butterfield et al. (Cancer Immunotherapy Principles and Practice, 2017, 1st Edition, Demos Medical, ISBN 978-1620700976); Kenneth Murphy (Janeway's Immunobiology, 2016, 9th Edition, Garland Science, ISBN 978-0815345053); Stevens et al. (Clinical Immunology and Serology: A Laboratory Perspective, 2016, 4th Edition, Davis Company, ISBN 978-0803644663); E. A. Greenfield (Antibodies: A Laboratory Manual, 2014, Second edition, Cold Spring Harbor Laboratory Press, ISBN 978-1-936113-81-1); R.I. Freshney (Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 2016, 7th Edition, Wiley-Blackwell, ISBN 978-1118873656); CA Pinkert (Transgenic Animal Technology, Third Edition: A Laboratory Handbook, 2014, Elsevier, ISBN 978-0124104907); H. Hedrich (The Laboratory Mouse, 2012, Second Edition, Academic Press, ISBN 978-0123820082); Behringer et al. (Manipulating the Mouse Embryo: A Laboratory Manual, 2013, Fourth Edition, Cold Spring Harbor Laboratory Press, ISBN 978-1936113019); McPherson et al. (PCR 2: A Practical Approach, 1995, IRL Press, ISBN 978-0199634248); JM Walker (Methods in Molecular Biology (Series), Humana Press, ISSN 1064-3745); Rio et al. (RNA: A Laboratory Manual, 2010, Cold Spring Harbor Laboratory Press, ISBN 978-0879698911); Methods in Enzymology (Series), Academic Press; Green et al. (Molecular Cloning: A Laboratory Manual, 2012, Fourth Edition, Cold Spring Harbor Laboratory Press, ISBN 978-1605500560); GT Hermanson (Bioconjugate Techniques, 2013, Third Edition, Academic Press, ISBN 978-0123822390).

[00187] Clustered regularly interspaced short palindromic repeats (CRISPR) and related CRISPR-associated proteins (Cas proteins) comprise CRISPR-Cas systems. The classification of CRISPR-Cas systems has undergone many revisions. In the publication by Makarova et al. (Nat. Rev. Microbiol., 2020, 18:67-83), a classification system was proposed that takes into account the signature cas genes specific to individual types and subtypes of CRISPR-Cas systems. This classification also takes into account the sequence similarity between several common Cas proteins, the phylogeny of the most conserved Cas protein, gene organization, and the structure of the CRISPR array. This approach provided a classification scheme that divides CRISPR-Cas systems into two distinct classes: class 1 and class 2.

[00188] In class 2, type V systems, crRNA-target binding involves binding to Cas12 as well as cleavage of the target nucleic acid. The RuvC-like domain of the Cas12a nuclease, for example, cleaves both strands of the target nucleic acid in a staggered manner, resulting in the formation of 5' overhangs, which in turn contrasts with the blunt ends formed by Cas9 cleavage. These 5' overhangs may facilitate DNA insertion using homologous recombination techniques.

[00189] Other type V crRNA-related proteins that are involved in target binding and cleavage include Cas12b (formerly C2c1) and Cas12c (formerly C2c3). The Cas12b and Cas12c proteins are similar in length to the class 2 type II CRISPR Cas9 and class 2 type V CRISPR Cas12a proteins, ranging from approximately 1100 amino acids to approximately 1500 amino acids. The C2c1 and C2c3 proteins also contain RuvC-like nuclease domains and have an architecture similar to Cas12a. The C2c1 proteins are similar to the Cas9 proteins in that they require crRNA and tracrRNA for target binding and cleavage, but have an optimal cleavage temperature of 50°C. C2c1 proteins target the AT-rich PAM, which, like Cas12a, is the 5' target sequence. See, e.g., Shmakov et al. (Molecular Cell, 2015, 60(3):385-397).

[00190] Type V CRISPR subtypes include Cas12 proteins and exhibit a wide diversity of sequences and sizes; however, Cas12 subtypes share a common evolutionary origin from TnpB nucleases encoded by IS605-like transposons. Due to low sequence similarity and likely evolution through multiple independent recombination events of Cas12 proteins, the classification of Cas12 proteins into their respective subtypes has resulted in numerous naming conventions. Table 1 provides the classification and names of type V Cas12 proteins, as well as their approximate size, guide sequence requirements, preferred target polynucleotide, and representative organism from which they are derived.

Table 1
Classification of subtypes of type V Type V Cas nomenclature Other names Effector size Guide RNA Target polynucleotide Representative organism V-A Cas12a MAD7 >1000aa cr-RNA dsDNA Francisella cf. novicida V-B1 Cas12b1 c2c1 >1000aa cr-RNA,
tracr-RNA dsDNA Alicyclobacillus acidoterrestris V-B2 Cas12b2 - Planctomycetes bacterium RBG_13_46_10 V-C Cas12c c2c3 >1000aa cr-RNA,
tracr-RNA dsDNA Oleiphilus spp. V-D Cas12d CasY >1000aa cr-RNA dsDNA Bacterium CG09_39_24 V-E Cas12e CasX ~1000aa cr-RNA,
tracr-RNA dsDNA Deltaproteobacteria bacteria V-F1 Cas12f1 Cas14a, c2c10, V-U3 400-800aa cr-RNA,
tracr-RNA dsDNA Uncultured Archaeon V-F2 Cas12f2 Cas14b cr-RNA Bacillus thuringiensis HD-771 V-F3 Cas12f3 Cas14c cr-RNA Candidatus Micrarchaeota archaeon V-G Cas12g - 700-800aa cr-RNA,
tracr-RNA sRNA Metagenome of a hot spring V-H Cas12h - ~1000aa cr-RNA ssDNA,
dsDNA Metagenome of a high-salinity lake sediment V-I Cas12i - ~1000aa cr-RNA ssDNA,
dsDNA Metagenome of fresh water V-J Cas12j Casϕ (Cas-phi) 700-800aa cr-RNA dsDNA Biggie-phage V-K Cas12K c2c5 >700aa cr-RNA,
tracr-RNA No nuclease activity Cyanotheces spp. PCC 8801 V-U - c2c4, c2c8, c2c9 n.d. n.d. n.d. Gordonia otitidis

[00191] Cas12 homologs can be identified using sequence similarity search methods known to those skilled in the art. Typically, a Cas12 protein is capable of interacting with a cognate Cas12 guide to form a Cas12 guide/nucleoprotein complex capable of binding to a target nucleic acid sequence. In some embodiments of the present invention, the Cas12 protein or homolog thereof is a Cas12a protein or homolog thereof.

[00192] Cas12a proteins include, but are not limited to, Cas12a from Parcubacteria GWC2011_GWC2_44_17 (PbCpf1), Lachnospiraceae MC2017 (Lb3 Cpf1), Butyrivibrio proteoclasticus (BpCpf1), Peregrinibacteria GW2011_GWA_33_10 (PeCpf1), Acidaminococcus spp. BV3L6 (AsCpf1), Porphyromonas macacae (PmCpf1), Lachnospiraceae bacteria ND2006 (LbCpf1), Porphyromonas crevioricanis (PcCpf1), Prevotella disiens (PdCpf1), Moraxella bovoculi 237 (MbCpf1), Smithella sp. SC_K08D17 (SsCpf1), Leptospira inadai (LiCpf1), Lachnospiraceae MA2020 (Lb2Cpf1), Franciscella novicida U112 (FnCpf1), Candidatus methanoplasma termitum (CMtCpf1) and Eubacterium eligens (EeCpf1).

[00193] In type V systems, binding of the target nucleic acid sequence typically involves binding of the Cas12 protein and crRNA, as well as cleavage of the target nucleic acid sequence. In type V systems, the RuvC-like nuclease domain of Cas12 sequentially cleaves both strands of the target nucleic acid sequence, see Swarts et al. (Mol. Cell, 2017, 66:221-233), resulting in the formation of 5' overhangs, in contrast to the blunt ends formed by Cas9 cleavage.

[00194] The cleavage activity of the Cas12 protein of type V systems can be independent of the tracrRNA (e.g., type V-A); and some type V systems require only one crRNA, which has a stem-loop structure that forms an internal duplex. The Cas12 protein binds to the crRNA in a sequence- and structure-specific manner by recognizing the stem-loop and sequences adjacent to the stem-loop, primarily nucleotides of the 5' spacer sequence that hybridize to the target nucleic acid sequence. This stem-loop structure is typically 15 to 22 nucleotides in length. Substitutions that disrupt this stem-loop duplex abolish cleavage activity, whereas other substitutions that do not disrupt the stem-loop duplex do not abolish cleavage activity. Certain V-type systems require hybridization between crRNA and tracrRNA, such as V-F1, V-G, V-C, V-E (CasX), V-K, and V-B-type systems. See, e.g., Yan et al. (Science, 2019, 363(6422):88-91).

[00195] As used herein, the terms "target" and "target polynucleotide" refer to one or more polynucleotides that form a nucleoprotein complex with a Cas protein, wherein the nucleoprotein complex preferentially binds to a target nucleic acid sequence in the polynucleotide (as compared to a polynucleotide that does not contain the target nucleic acid sequence). Such guide sequences may comprise ribonucleotide bases (e.g., RNA), deoxyribonucleotide bases (e.g., DNA), combinations of ribonucleotide bases and deoxyribonucleotide bases (e.g., RNA/DNA), nucleotide analogs, modified nucleotides, and the like, as well as synthetic, naturally occurring, and non-naturally occurring modified backbone residues or linkages. A variety of such guide sequences are known, such as, but not limited to, single-stranded guide RNA (including miniature and truncated single-stranded guide RNAs), crRNA, dual guide RNAs including, but not limited to, crRNA/tracrRNA molecules, etc., the use of which depends on the specific Cas protein. For example, the “CRISPR-Cas12 type V-associated guide sequence” is a guide sequence that specifically binds to the cognate protein Cas12 to form a nucleoprotein complex.

[00195] As used herein, the term "CRISPR polynucleotide" means a polynucleotide sequence comprising a portion of a guide molecule. In some embodiments, a CRISPR polynucleotide comprises a targeting region and/or an activating region.

[00195] With respect to a targeting molecule, the terms "spacer,""spacersequence,""spacerelement," or "targeting region" as used herein refer to a polynucleotide sequence that can specifically hybridize to a target nucleic acid sequence. The targeting region interacts with the target nucleic acid sequence through a hydrogen bond between complementary base pairs (i.e., base pairs). The targeting region binds to a selected target nucleic acid sequence. In some embodiments, the target sequence is a sequence in the genome of a cell, either in vitro or ex vivo (e.g., when producing CAR-T cells) or in vivo (e.g., when the compositions are administered directly to an individual). A targeting molecule can comprise or consist of any sequence selected to target any target sequence. Examples of a target sequence are sequences that are unique to the target genome. Accordingly, a targeting region is a sequence that binds to a target nucleic acid. The targeting region determines the location of site-specific binding and nucleolytic cleavage of the Cas12 protein. Functional length variability for a targeting region is known to those skilled in the art.

[00198] As used herein, the term "activating region" refers to a portion of a polynucleotide that is capable of associating or binding with a Cas12 polypeptide, such as a Cas12a polypeptide.

[00199] As used herein, the terms "minor", "minor site", "minor nucleotide", "non-purine/non-pyrimidine site" and "AP site" are synonymous and refer to a site in a nucleotide sequence that lacks a purine or pyrimidine base. In some embodiments, minor sites comprise a deoxyribose site. In other embodiments, minor sites comprise a ribose site. In further embodiments, minor sites comprise a modified backbone, such as a phosphorothioate backbone or a morpholine backbone. A minor site cannot form hydrogen base pairs with a complementary nucleobase of a DNA or RNA nucleotide because it does not contain a nucleobase.

[00200] As used herein, the terms "base analog," "non-canonical base," and "chemically modified base" refer to a compound that has a structural similarity to a canonical purine or pyrimidine base found in DNA or RNA. A base analog may comprise a modified sugar and/or a modified nitrogenous base compared to a purine or pyrimidine base found naturally in DNA or RNA. In some embodiments, the base analog is inosine or deoxyinosine, such as 2'-deoxyinosine. In other embodiments, the base analog is a 2'-deoxyribonucleoside, 2'-ribonucleoside, 2'-deoxyribonucleotide, or 2'-ribonucleotide, wherein the nitrogenous base comprises a modified base (such as, for example, xanthine, uridine, oxanine (oxanosine), 7-methylguanosine, dihydrouridine, 5-methylcytidine, C3 spacer, 5-methyl-dC, 5-hydroxybutynyl-2'-deoxyuridine, 5-nitroindole, 5-methylisodeoxycytosine, isodeoxyguanosine, deoxyuridine, isodeoxycytidine, other 0-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deazahypoxanthine, other 7-deazapurines, and 2-methylpurines). In some embodiments, the base analog can be selected from the group consisting of 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidite-inosine, 2'-O-methoxyethylinosine, and 2'-OMe-inosine. The term "base analog" also includes, for example, 2'-deoxyribonucleosides, 2'-ribonucleosides, 2'-deoxyribonucleotides, or 2'-ribonucleotides, wherein the nitrogenous base is a substituted hypoxanthine. For example, the substituted hypoxanthine can be replaced by a halogen, such as fluorine or chlorine. In some embodiments, the base analog may be fluorinosine or chlorinosine, such as 2-chloroinosine, 6-chloroinosine, 8-chloroinosine, 2-fluorinosine, 6-fluorinosine, or 8-fluorinosine. In other embodiments, the base analog is deoxyuridine. In other embodiments, the base analog is a nucleic acid mimic (such as, for example, artificial nucleic acids and xeno nucleic acids (XNA)).

[00200] As used herein, the term "hybrid CRISPR guide RNA/DNA" (chRDNA) refers to a polynucleotide guide molecule comprising a targeting region, wherein the polynucleotide comprises RNA together with DNA introduced into the polynucleotide. In the embodiments of the invention provided herein, the crRNA component of the Cas12a guide is chRDNA.

[00202] As used herein, the term "chRDNA Cas12 guide/nucleoprotein complex" refers to a chRDNA guide molecule associated with a Cas12 protein to form a nucleoprotein complex, wherein the nucleoprotein complex is capable of site-directed binding to a target nucleic acid sequence that is complementary to a target nucleic acid-binding sequence present in the chRDNA guide molecule. As used herein, the term "chRDNA Cas12a guide/nucleoprotein complex" refers to a chRDNA guide molecule associated with a Cas12a protein to form a nucleoprotein complex, wherein the nucleoprotein complex is capable of site-directed binding to a target nucleic acid sequence that is complementary to a target nucleic acid-binding sequence present in the chRDNA guide molecule.

[00203] As used herein, the term "stem element" or "stem structure" refers to two strands of nucleic acids that form a double-stranded region (a "stem element"). A "stem-loop element" or "stem-loop structure" refers to a stem structure wherein the 3'-terminal sequences of one strand are covalently linked to the 5'-terminal sequences of a second strand by a nucleotide sequence, typically consisting of single-stranded nucleotides (a "stem-loop element nucleotide sequence"). In some embodiments, the loop element comprises a loop element nucleotide sequence comprising from about 3 to about 20 nucleotides, and preferably from about 4 to about 10 nucleotides. In some embodiments of the invention, the nucleotide sequence of the loop element is a single-stranded nucleotide sequence of unpaired nucleic acid bases that do not interact via hydrogen bonding to create a stem element in the nucleotide sequence of the loop element. The term "hairpin element" as used herein also refers to stem-loop structures. Such structures are well known to those skilled in the art. The base pairing may be precise, however, as is known to those skilled in the art, a stem element does not require precise base pairing. Thus, the stem element may include one or more mispaired bases or unpaired bases. The stem-loop element may further comprise a pseudoknot structure.

[00204] As used herein, the terms "linker element nucleotide sequence," "linker nucleotide sequence," and "linker polynucleotide" are synonymous and refer to a sequence of one or more nucleotides covalently attached to a first nucleic acid sequence (5' linker nucleotide sequence - first nucleic acid sequence-3'). In some embodiments, a linker nucleotide sequence connects two separate nucleic acid sequences to form a single polynucleotide (e.g., first nucleic acid 5' sequence - linker nucleotide sequence - second nucleic acid sequence-3'). Other examples of linker sequences include, but are not limited to, a 5' first nucleic acid sequence - a linker nucleotide sequence-3', and a 5' linker nucleotide sequence - a first sequence of a first nucleic acid - a linker nucleotide sequence-3'. In some embodiments, the nucleotide sequence of the linker element may be a single-stranded nucleotide sequence of unpaired nucleic acid bases that do not interact with each other by hydrogen bonding to create a secondary structure (e.g., a stem-loop structure) within the nucleotide sequence of the linker element. In some embodiments, two single-stranded nucleotide sequences of the linker element may interact with each other by hydrogen bonding between the two nucleotide sequences of the linker element. In some embodiments of the invention, the nucleotide sequence of the linker element may have a length of from about 1 to about 50 nucleotides, and preferably from about 1 to about 15 nucleotides.

[00205] As used herein, the term "related" generally refers to a Cas12 protein (e.g., Cas12a) and one or more type V guide sequences associated with CRISPR-Cas12 (e.g., chRDNA Cas12 guide sequences) that are capable of forming a nucleoprotein complex capable of site-directed binding to a target nucleic acid sequence that is complementary to a target nucleic acid binding sequence present in the one or more guide sequences.

[00205] The terms "wild type," "naturally occurring," and "unmodified" are used herein to refer to the typical (or most common) form, appearance, phenotype, or strain that exists in nature; for example, the typical form of cells, organisms, polynucleotides, proteins, macromolecular complexes, genes, RNA, DNA, or genomes as they occur in and can be isolated from a natural source. The wild type form, appearance, phenotype, or strain serves as the original parent prior to its desired modification. Thus, mutant, variant, engineered, recombinant, and modified forms are not wild type forms.

[00207] The term "isolated," when referring to a polypeptide, means that the molecule is distinct and has been removed from the whole organism in which the molecule naturally occurs or is present in the substantial absence of other biological macromolecules of the same type. The term "isolated," when referring to a polynucleotide, means that the nucleic acid molecule is wholly or partially devoid of sequences normally associated with it in nature; or it refers to a sequence as it occurs in nature but has heterologous sequences associated with it, or to a molecule that is separated from a chromosome.

[00208] The term "purified" as used herein preferably means that the same molecule is present at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight.

[00209] The terms "engineered," "genetically engineered," "genetically modified," "recombinant," "modified," "non-naturally occurring," and "non-native" refer to modification of the genome of an organism or cell by a human. These terms encompass methods of genomic modification that include genome editing as defined herein, as well as gene expression modification methods or gene inactivation methods, enzymatic engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Genetic engineering methods are known to those skilled in the art.

[00210] As used herein, the terms "covalent bond", "covalently attached", "covalently linked", "covalently connected", "covalent bonding" and "molecular bond" are synonymous and refer to a chemical bond that involves the sharing of electron pairs between atoms. Examples of covalent bonds include, but are not limited to, phosphodiester bonds and phosphorothioate bonds.

[00211] As used herein, the terms "non-covalent bond", "non-covalently attached", "non-covalently bound", "non-covalently connected", "non-covalent interaction" and "non-covalent binding" are synonymous and refer to any relatively weak chemical bond that does not share a pair of electrons. Multiple non-covalent bonds often stabilize the conformation of macromolecules and mediate specific interactions between molecules. Examples of non-covalent bonds include, but are not limited to, hydrogen bonds, ionic interactions (e.g., Na ⁺ Cl ^- ), van der Waals interactions and hydrophobic bonds.

[00212] As used herein, the terms "hydrogen bond,""hydrogen-basepairing," and "hydrogen-bonded" are synonymous and refer to canonical hydrogen bonding and non-canonical hydrogen bonding, including, but not limited to, "Watson-Crick hydrogen-bonded base pairs" (W-C hydrogen-bonded base pairs or W-C hydrogen bond); "Hoogsteen hydrogen-bonded base pairs" (Hoogsteen hydrogen bond); and "wop-paired hydrogen-bonded base pairs" (wop-pairing hydrogen bond). W-C hydrogen bonding, including reverse W-C hydrogen bonding, refers to the pairing of purine-pyrimidine bases such as adenine:thymine, guanine:cytosine, and uracil:adenine. Hoogsteen hydrogen bonding, including reverse Hoogsteen hydrogen bonding, refers to a variant of base pairing in nucleic acids in which two nitrogenous bases, one on each strand, are held together by hydrogen bonds in the major groove. This non-WC hydrogen bonding can allow a third strand to wrap around the duplex and form three-stranded helices. Wobble hydrogen bonding, including reverse wobble hydrogen bonding, refers to the pairing between two nucleotides in RNA molecules that does not obey the Watson-Crick base pairing rules. There are four major wobble base pairs: guanine:uracil, inosine (hypoxanthine):uracil, inosine:adenine, and inosine:cytosine. Wobble base pairing interactions are also known to occur between inosine and thymine and inosine and guanine. Inosine bases and deoxyinosine bases can be called "universal base pairing" because they are able to form hydrogen bonds with the canonical bases of DNA and RNA. See, e.g., Watkins et al . ( Nucleic Acid Research , 2005, 33(19):6258–67). The rules for canonical hydrogen bonding and noncanonical hydrogen bonding are known to those skilled in the art. See, e.g., R. F. Gesteland (The RNA World, Third Edition (Cold Spring Harbor Monograph Series), 2005, Cold Spring Harbor Laboratory Press, ISBN 978-0879697396); R. F. Gesteland (The RNA World, Second Edition (Cold Spring Harbor Monograph Series), 1999, Cold Spring Harbor Laboratory Press, ISBN 978-0879695613); RF Gesteland (The RNA World, First Edition (Cold Spring Harbor Monograph Series), 1993, Cold Spring Harbor Laboratory Press, 978-0879694562) ( see, for example, Appendix 1: Structures of Base Pairs Involving at Least Two Hydrogen Bonds, I. Tinoco); W. Saenger (Principles of Nucleic Acid Structure, 1988, Springer International Publishing AG, ISBN 978-0-387-90761-1); S. Neidle (Principles of Nucleic Acid Structure, 2007, First Edition, Academic Press, ISBN 978-01236950791).

[00213] As used herein, the terms “link,” “linked,” and “linking” are synonymous and refer to a covalent bond or non-covalent bond between two macromolecules (e.g., polynucleotides, proteins, etc.).

[00214] As used herein, the terms "nucleic acid sequence", "nucleotide sequence" and "oligonucleotide" are synonymous and refer to a polymeric form of nucleotides. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides that has one 5' end and one 3' end and may contain one or more nucleic acid sequences. The nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and may be of any length. Polynucleotides may perform any function and may have a variety of secondary and tertiary structures. These terms include known analogs of natural nucleotides and nucleotides that are modified in the base, sugar and/or phosphate moieties. Analogs of a particular nucleotide have the same base pairing specificity (e.g., an analog of an A to T base pair). A polynucleotide may contain one modified nucleotide or more modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, chemically modified sugars, and nucleotide analogs. The nucleotide structure may be modified before or after polymer assembly. After polymerization, polynucleotides may be further modified by, for example, conjugation to a labeling component or a target binding component. The nucleotide sequence may include non-nucleotide components. These terms also encompass nucleic acids containing modified backbone residues or linkages that are synthetic, naturally occurring, and/or non-naturally occurring and have similar binding properties as a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide-linked nucleic acids (PNA), locked nucleic acids (LNA™) (Exiqon, Woburn, MA), nucleosides, glycolic nucleic acid, bridged nucleic acids, and morpholine structures.

[00215] Peptide-linked nucleic acids (PNA) are synthetic homologues of nucleic acids in which the polynucleotide-phosphate-sugar backbone is replaced by a flexible pseudopeptide polymer. The nitrogenous bases are linked to the polymer. PNA have the ability to hybridize with high affinity and specificity to complementary RNA and DNA sequences.

[00215] In phosphorothioate nucleic acids, a phosphorothioate (PS) linkage replaces a non-bridging oxygen with a sulfur atom in the polynucleotide-phosphate backbone. This modification renders the internucleotide linkage resistant to nuclease degradation. In some embodiments, phosphorothioate linkages are introduced between the last 3-5 nucleotides in the 5'-terminal or 3'-terminal sequences of a polynucleotide sequence to inhibit exonuclease degradation. The placement of phosphorothioate linkages throughout the oligonucleotide also helps reduce the extent of endonuclease degradation.

[00217] Threoses nucleic acid (TNA) is an artificial genetic polymer. The backbone structure of TNA comprises repeating threose sugars linked by phosphodiester bonds. TNA polymers are resistant to nuclease degradation. TNA can self-assemble through hydrogen bonding between base pairs into duplex structures.

[00218] Linkage inversions can be introduced into polynucleotides using "reverse phosphoramidites" (see, e.g., www.ucalgary.ca/dnalab/synthesis/modifications/linkages). A 3'-3' linkage at the end of a polynucleotide stabilizes the polynucleotide against exonuclease degradation by forming an oligonucleotide having two 5'-OH termini but no 3'-OH terminus. Typically, such polynucleotides have phosphoramidite groups at the 5'-OH position and a dimethoxytrityl (DMT) protecting group at the 3'-OH position. Typically, the DMT protecting group is at the 5'-OH and the phosphoramidite is at the 3'-OH.

[00219] Polynucleotide sequences are shown herein in the conventional 5' to 3' orientation unless otherwise indicated.

[00220] As used herein, the term "sequence identity" generally refers to the percentage of nucleotide base or amino acid identity when comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide, respectively, using algorithms having various weighting parameters. The sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignment in a variety of ways and using computer programs (e.g., BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, etc.) available from websites including, but not limited to, GENBANK (www.ncbi.nlm.nih.gov/genbank/) and EMBL-EBI (www.ebi.ac.uk.). The sequence identity between two polynucleotides or two polypeptide sequences is generally calculated using standard default parameters used in the various methods or computer programs. A high degree of sequence identity between two polynucleotides or two polypeptides is typically about 90% to 100% over the length of the reference polypeptide, such as about 90% or higher, preferably about 95% or higher, more preferably about 98% or higher. A moderate degree of sequence identity between two polynucleotides or two polypeptides is typically about 80% to about 85%, such as about 80% or higher, preferably about 85% over the length of the reference polypeptide. A low degree of sequence identity between two polynucleotides or two polypeptides is typically about 50% to 75%, such as about 50%, preferably about 60%, and more preferably about 75% over the length of the reference polypeptide. For example, a Cas12 protein (e.g., Cas12 containing amino acid substitutions) may have low sequence identity, moderate sequence identity, or high sequence identity over its entire length compared to a reference Cas12 protein (e.g., wild-type Cas12). As another example, a guide molecule may have low sequence identity, moderate sequence identity, or high sequence identity over its entire length compared to a wild-type reference guide molecule that complexes with a reference Cas12 protein (e.g., a polynucleotide that complexes with Cas12).

[00221] As used herein, the term "hybridization,""hybridizes," or "hybridizing" refers to the process of joining two complementary single-stranded nucleic acid molecules (e.g., DNA or RNA) to form a single double-stranded molecule (e.g., DNA/DNA, DNA/RNA, RNA/RNA) through hydrogen bonding base pairing. Hybridization stringency is typically determined by the hybridization temperature and the salt concentration of the hybridization buffer; for example, high temperature and low salt provide highly stringent hybridization conditions. Examples of salt concentration ranges and temperature ranges for various hybridization conditions are: high stringency, about 0.01 to about 0.05 M salt, a hybridization temperature of 5°C to 10°C below Tm; moderate stringency, about 0.16 to about 0.33 M salt, a hybridization temperature of 20°C to 29°C below Tm; and low stringency, about 0.33 M to about 0.82 M salt, a hybridization temperature of 40°C to 48°C below Tm. Tm of duplex nucleic acid sequences are calculated by standard methods well known to those skilled in the art. See, e.g., Maniatis et al . (Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press: New York); Casey et al . ( Nucleic Acids Research , 1977, 4:1539-1552); Bodkin et al . ( Journal of Virological Methods , 1985, 10(1):45-52); and Wallace et al . ( Nucleic Acids Research , 1981, 9(4):879-894). Prediction tools of algorithms for estimating Tm are also widely available. High stringency conditions for hybridization generally refer to conditions under which a polynucleotide complementary to a target sequence preferentially hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Typically, hybridization conditions are moderately stringent, and preferably highly stringent.

[00222] As used herein, the term "complementarity" refers to the ability of a nucleic acid sequence to form hydrogen bonds with another nucleic acid sequence (e.g., via canonical Watson-Crick base pairing). Percent complementarity refers to the percentage of residues in a nucleic acid sequence that can form hydrogen bonds with a second nucleic acid sequence. If two nucleic acid sequences have 100% complementarity, then the two sequences are completely complementary, meaning that all contiguous hydrogen bond residues of the first polynucleotide are complementary to the same number of contiguous residues in the second polynucleotide.

[00223] As used herein, the term "corresponding deoxyribonucleotide base," when used in relation to a ribonucleotide base, refers to a deoxyribonucleotide base (including, for example, a modified or altered version of a canonical deoxyribonucleotide base) that binds via complementary base pairing (Watson-Crick) to the same base as the ribonucleotide base. Thus, for example, for ribonucleotide bases A, C, and G, the corresponding deoxyribonucleotide bases may be A, C, and G, respectively. For ribonucleotide base U, the corresponding deoxyribonucleotide base may be, for example, T.

[00224] As used herein, the term "binding" refers to a non-covalent interaction between macromolecules (e.g., between a protein and a polynucleotide, between a polynucleotide and a polynucleotide, or between a protein and a protein, etc.). Such non-covalent interaction is also referred to as an "association" or "interaction" (e.g., if a first macromolecule interacts with a second macromolecule, then the first macromolecule binds to the second macromolecule in a non-covalent manner). Some portions of the interaction by the binding mechanism may be sequence-specific (the terms "sequence-specific binding," "sequence-specific interaction," "site-specific binding," and "site-specific interaction" are used synonymously herein). Sequence-specific binding generally refers to one or more targeting molecules capable of forming a complex with a protein (e.g., Cas12) to initiate binding of the protein to a nucleic acid sequence (e.g., a DNA sequence) containing a target nucleic acid sequence (e.g., a target DNA sequence), and preferably to a second nucleic acid sequence (e.g., a second DNA sequence) without a target nucleic acid binding sequence (e.g., without a target DNA binding sequence). All components of the binding interaction need not be sequence specific, such as contacts of the protein with phosphate residues in the DNA backbone. Binding interactions can be characterized by a dissociation constant (Kd). "Binding affinity" refers to the strength of the binding interaction. Increased binding affinity correlates with a lower Kd.

[00225] As described herein, a Cas12 protein is said to "target" a polynucleotide if the Cas12 guide/nucleoprotein complex binds or cleaves a polynucleotide within a target nucleic acid sequence within the polynucleotide.

[00225] As used herein, the term "protospacer adjacent motif" or "PAM" refers to double-stranded nucleic acid sequences containing a recognition sequence that binds to the Cas12 protein, wherein amino acids of the Cas12 protein directly interact with the recognition sequence (e.g., the Cas12a protein interacts with the PAM 5'-TTTN-3' or with the PAM 5'-TTTV-3'). The PAM sequences are on the non-target strand and can be a 5' or 3' sequence complementary to the target (e.g., in CRISPR-Cas12a systems, the PAM 5'-TTTN-3' or PAM 5'-TTTV-3' sequence is on the non-target strand and is a 5' sequence complementary to the target). PAMs are recognized by Cas12 effector proteins (e.g., Cas12a protein) prior to unwinding of the target sequence and hydrogen bonding of base pairs between the target sequence and the target nucleic acid binding sequence.

[00227] As used herein, the terms "target", "target sequence", "target nucleic acid sequence", "target nucleic acid sequence" and "target sequence" are synonymous and refer to a nucleic acid sequence that is fully or partially complementary to a target nucleic acid binding sequence of a Cas12 polynucleotide (e.g., a targeting region). Typically, the target nucleic acid binding sequence is selected to be 100% complementary to the target nucleic acid sequence to which the Cas12 nucleoprotein complex is directed to bind; however, a lower percentage of complementarity may be used to weaken binding to the target nucleic acid sequence.

[00228] If a target nucleic acid binding sequence is 100% complementary to a target sequence, excluding non-essential sites contained in the target nucleic acid binding sequence, then such target sequence is referred to herein as a "target sequence." Binding to a target sequence refers to binding of the Cas12 guide/nucleoprotein complex to a nucleic acid sequence that is 100% complementary to a non-essential portion of a target nucleic acid binding sequence site (spacer). If a target nucleic acid binding sequence (spacer) is less than 100% complementary to the target sequence, excluding non-essential sites contained in the target nucleic acid binding sequence, the target sequence may be referred to as an "off-target sequence." Off-target sequence binding refers to binding of the Cas12 guide/nucleoprotein complex to nucleic acid sequences that are less than 100% complementary to a non-essential portion of the target nucleic acid binding sequence (spacer) site. The target nucleic acid sequence may be a double-stranded or single-stranded DNA molecule. The target sequence may be a double-stranded or single-stranded RNA molecule. The target sequence may be an RNA:DNA hybrid molecule. The target sequence may be present on the opposite strand of the PAM sequence.

[00229] As used herein, the term "double-strand break" (DSB) refers to the separation of both strands of a double-stranded segment of DNA. In some cases, when such a break occurs, one strand can be said to have a "sticky end" where nucleotides are exposed, but not hydrogen bonded to nucleotides on the other strand. In other cases, there may be a "blunt end" where both strands remain completely paired with each other.

[00230] As used herein, the terms "donor polynucleotide,""donoroligonucleotide,""donortemplate,""non-viraldonor," and "non-viral template" are synonymous and may be a double-stranded polynucleotide (e.g., DNA), a single-stranded polynucleotide (e.g., DNA or RNA), or a combination thereof. Donor polynucleotides may contain arms of homology flanking an insertion sequence (e.g., a DSB in DNA). The arms of homology on each side may differ in length. Parameters for creating and designing donor polynucleotides are well known to those skilled in the art. See, e.g., Ran et al . ( Nature Protocols , 2013, 8(11):2281-2308); Smithies et al . ( Nature , 1985, 317:230-234); Thomas et al . ( Cell , 1986, 44:419-428); Wu et al . ( Nature Protocols , 2008, 3:1056-1076); Singer et al . ( Cell , 1982, 31:25-33); Shen et al. ( Genetics , 1986, 112:441-457); Watt et al. ( PNAS 1985, 82:4768-4772); Sugawara et al . ( Journal of Molecular Cell Biology , 1992, 12(2):563-575); Rubnitz et al . ( Journal of Molecular Cell Biology , 1984, 4(11):2253-2258); Ayares et al . ( PNAS , 1986, 83(14):5199-5203); and Liskay et al . ( Genetics , 1987, 115(1):161-167). In some embodiments, the donor polynucleotide comprises a chimeric antigen receptor (CAR).

[00231] As used herein, the term "homology-based repair" (HDR) refers to DNA repair that occurs in cells, such as during the repair of a DSB in DNA. HDR requires nucleotide sequence homology and uses a donor polynucleotide to repair the sequence where the DSB has occurred (e.g., in a target DNA sequence). The donor polynucleotide typically has the desired sequence homology to the sequence flanking the DSB, and therefore, the donor polynucleotide can serve as a suitable template for repair. HDR results in the transfer of genetic information, such as from the donor polynucleotide to the target DNA sequence. HDR can result in a change in the target DNA sequence (e.g., an insertion, deletion, or mutation) if the donor polynucleotide sequence differs from the target DNA sequence and part or all of the donor polynucleotide is incorporated into the target DNA sequence. In some embodiments of the invention, all of a donor polynucleotide, a portion of a donor polynucleotide, or a copy of a donor polynucleotide is integrated into a site of a target DNA sequence. For example, a donor polynucleotide can be used to repair a break in a target DNA sequence, where such repair results in the transfer of genetic information (e.g., polynucleotide sequences) from the donor polynucleotide at or in close proximity to the break in DNA. Accordingly, new genetic information (e.g., polynucleotide sequences) can be inserted or copied into the target DNA sequence.

[00232] As used herein, the term "homology-independent target integration" (HITI) refers to DNA repair that occurs in a cell, such as during the repair of a DSB in DNA. HITI, unlike HDR, does not require nucleotide sequence homology and uses a donor polynucleotide to repair the sequence where the DSB has occurred (e.g., in a target DNA sequence). HITI results in the transfer of genetic information, such as from the donor polynucleotide to the target DNA sequence. HITI can result in a change in the target DNA sequence (e.g., an insertion, deletion, or mutation) if the donor polynucleotide sequence differs from the target DNA sequence and part or all of the donor polynucleotide is incorporated into the target DNA sequence. In some embodiments of the invention, all of the donor polynucleotide, a portion of the donor polynucleotide, or a copy of the donor polynucleotide is integrated into a site of a target DNA sequence. For example, the donor polynucleotide can be used to repair a break in a target DNA sequence, where such repair results in the transfer of genetic information (e.g., polynucleotide sequences) from the donor polynucleotide at or in close proximity to the break in the DNA. Accordingly, new genetic information (e.g., polynucleotide sequences) can be inserted or copied into the target DNA sequence.

[00233] A "genomic region" is a segment of a chromosome in the genome of a host cell that is present on either side of a target nucleic acid sequence site or, alternatively, also includes a portion of the target nucleic acid sequence site. The homology arms of a donor polynucleotide have sufficient homology to undergo homologous recombination with corresponding regions of the genome. In some embodiments, the homology arms of a donor polynucleotide have significant sequence homology to a genomic region immediately flanking the target nucleic acid sequence site; however, it has been found that the homology arms can be engineered to have sufficient homology to regions of the genome located further from the target nucleic acid sequence site.

[00234] As used herein, the term "non-homologous end joining" (NHEJ) refers to the repair of a DSB in DNA by directly ligating one end of the break to the other end of the break without the need for a donor polynucleotide. NHEJ is a DNA repair pathway available to cells to repair DNA without the use of a repair template. NHEJ in the absence of a donor polynucleotide often results in random insertion or deletion of nucleotides at the site of a DSB.

[00235] Microhomology-mediated end joining (MMEJ) is a pathway for repairing DNA DSBs. MMEJ involves deletions flanking the DSB and alignment of microhomologous sequences within the break site prior to joining. MMEJ is genetically determined and requires the activity of, for example, CtIP, poly(ADP-ribose) polymerase 1 (PARP1), DNA polymerase theta (Pol θ), DNA ligase 1 (Lig 1), or DNA ligase 3 (Lig 3). Additional genetic components are known to those skilled in the art. See, for example, Sfeir et al. ( Trends in Biochemical Sciences , 2015, 40:701-714).

[00235] As used herein, the term "DNA repair" encompasses any process by which the cellular machinery repairs damage to a DNA molecule contained within a cell. The damage repaired may include single-strand breaks or double-strand breaks (DSBs). There are at least three mechanisms for repairing DSBs: HDR, NHEJ, and MMEJ. "DNA repair" is also used herein to refer to the repair of DNA resulting from human manipulation, where the target locus is modified, for example, by insertion, deletion, or substitution of nucleotides, which are forms of genome editing.

[00237] As used herein, the term "recombination" refers to the process of exchange of genetic information between two polynucleotides.

[00238] As used herein, the terms "regulatory sequences," "regulatory elements," and "control elements" are synonymous and refer to polynucleotide sequences that are located upstream (5' non-coding sequences), within, or downstream (3' untranslated sequences) of an expressed target polynucleotide. Regulatory sequences affect, for example, the timing of transcription, the amount or level of transcription, the processing or stability of RNA, and/or the translation of a nucleotide sequence with a related structure. Regulatory sequences may include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, transcription initiation sites, repressor binding sequences, stem-loop structures, translation initiation sequences, internal ribosome entry sites (IRES), translation leader sequences, transcription termination sequences (e.g., polyadenylation signals and poly-U sequences), translation termination sequences, primer binding sites, etc.

[00239] Regulatory elements include those that regulate constitutive, inducible, or repressible expression of a nucleotide sequence in many types of host cells, and those that regulate expression of a nucleotide sequence only in some host cells (e.g., tissue-specific regulatory sequences). In some embodiments, the vector comprises one or more pol III promoters, one or more pol II promoters, one or more pol I promoters, or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the Rous sarcoma retrovirus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer; see, e.g., Boshart et al . ( Cell , 1985, 41:521-530)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Those skilled in the art will appreciate that the type of expression vector construct may depend on factors such as the choice of host cell to be transformed, the desired level of expression, etc. The vector can be introduced into host cells so as to result in the production of transcripts, proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acid sequences described herein.

[00240] As used herein, the term "gene" refers to a polynucleotide sequence comprising exons and related regulatory sequences. A gene may further comprise introns and/or untranslated regions (UTRs).

[00241] As used herein, the term "operably linked" refers to polynucleotide sequences or amino acid sequences that are in a functional relationship with one another. For example, regulatory sequences (e.g., a promoter or enhancer) are "operably linked" to a polynucleotide encoding a gene product if the regulatory sequences regulate or help modulate the transcription of the polynucleotide. Operatively linked regulatory elements are typically contiguous with the coding sequence. However, enhancers can function even if they are separated from the promoter by up to several thousand base pairs or more. Accordingly, some regulatory elements can be operably linked to a polynucleotide sequence but are not contiguous with the polynucleotide sequence. Similarly, translational regulatory elements help modulate protein expression from a polynucleotide.

[00242] As used herein, the term "expression" refers to the transcription of a polynucleotide from a DNA template, resulting in, for example, messenger RNA (mRNA) or another RNA transcript (e.g., a non-coding transcript such as structural or scaffold RNAs). The term also refers to the process by which transcribed mRNA is translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be referred to collectively as "gene products." Expression may involve splicing of mRNA in a eukaryotic cell if the polynucleotide is derived from genomic DNA.

[00243] A "coding sequence" or a sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are defined by a start codon at the 5' end and a translation stop codon at the 3' end. A transcription termination sequence may be located at a distance from the 3' end of the coding sequence.

[00244] As used herein, the term "modulate" refers to a change in the amount, degree, or magnitude of a function. For example, a Cas12 guide/nucleoprotein complex as described herein can modulate the activity of a promoter sequence by binding to a target nucleic acid sequence in or near the promoter. Depending on the action that occurs upon binding, the Cas12 guide/nucleoprotein complex can induce, enhance, repress, or inhibit transcription of a gene operably linked to the promoter sequence. Thus, "modulation" of gene expression includes both gene activation and gene repression.

[00245] Modulation can be analyzed by determining any property that is directly or indirectly affected by the expression of a target gene. Such properties include, for example, changes in RNA or protein levels, protein activity, product levels, gene expression, or reporter gene activity levels. Accordingly, the terms "modulation of expression," "inhibition of expression," and "activation of expression" of a gene can refer to the ability of the Cas12 guide/nucleoprotein complex to alter, activate, or inhibit transcription of a gene.

[00245] As used herein, the terms "vector" and "plasmid" refer to a polynucleotide vehicle for introducing genetic material into a cell. Vectors may be linear or circular. Vectors may contain a replication sequence capable of causing the vector to replicate in a suitable host cell (e.g., an origin of replication). Upon transformation of a suitable host, the vector may replicate and function independently of the host genome or integrate into the host genome. The design of a vector depends on, among other things, its intended use and the host cell, and the structure of a vector for its particular use in a host cell can be determined by one of skill in the art. The four main types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Vectors typically contain an origin of replication, a multiple cloning site, and/or a selectable marker. An expression vector typically contains an expression cassette. By "recombinant virus" is meant a virus that has been genetically altered, for example by the addition or insertion of a heterologous nucleic acid construct into the viral genome or part of it.

[00247] As used herein, the term "expression cassette" refers to a polynucleotide construct produced using recombinant or synthetic methods and comprising regulatory sequences operably linked to a selected polynucleotide to facilitate expression of the selected polynucleotide in a host cell. For example, the regulatory sequences may facilitate transcription of the selected polynucleotide in the host cell or transcription and translation of the selected polynucleotide in the host cell. The expression cassette may be, for example, integrated into the genome of the host cell or it may be present in a vector to form an expression vector.

[00248] As used herein, the term "targeting vector" refers to a recombinant DNA construct, typically comprising adapted DNA strands homologous to genomic DNA that flank target gene elements or target nucleic acid sequences (e.g., DSBs). The targeting vector comprises a donor polynucleotide. The target gene elements may be modified in a number of ways, including deletions and/or insertions. A defective target gene may be replaced with a functional target gene, or, alternatively, a functional gene may be disabled. Optionally, the donor polynucleotide of the targeting vector comprises a selection cassette comprising a selectable marker that is introduced into the target gene. Targeting regions adjacent to or within the target gene may be used to affect the regulation of gene expression.

[00249] As used herein, the terms "gene editing" or "genome editing" refer to a type of genetic engineering that allows for a genetic modification, such as an insertion, deletion, or substitution of a nucleotide sequence or even a single base at a specific site in a cellular genome. These terms include, but are not limited to, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, and disruptive genetic modification as defined herein.

[00250] As used herein, the term "between" includes end values within a given range (e.g., a range of from about 1 to about 50 nucleotides in length includes a range of from 1 nucleotide to 50 nucleotides).

[00251] As used herein, the term "amino acid" refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and optical D- or L-isomers.

[00252] As used herein, the terms "peptide," "polypeptide," and "protein" are synonymous and refer to polymers of amino acids. A polypeptide may be of any length. It may be branched or linear, may be interrupted by non-amino acids, and may contain modified amino acids. These terms also refer to an amino acid polymer that has been modified, for example, by acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, PEGylation, biotinylation, cross-linking, and/or conjugation (e.g., with a labeling component or a ligand). Polypeptide sequences are shown herein in the general N-terminus to C-terminus orientation, unless otherwise noted. Polypeptides and polynucleotides can be prepared using conventional techniques in the art of molecular biology. In addition, virtually any polypeptide or polynucleotide can be obtained from commercially available sources.

[00253] As used herein, the terms "fusion protein" and "chimeric protein" refer to a single protein created by joining two or more proteins, protein domains, or protein fragments that are not naturally found together in a single protein.

[00254] The fusion protein may also comprise epitope tags (e.g., histidine tags, FLAG® tags (Sigma Aldrich, St. Louis, MO), Myc tags), reporter protein sequences (e.g., glutathione S-transferase, beta-galactosidase, luciferase, green fluorescent protein, blue fluorescent protein, yellow fluorescent protein), and/or nucleic acid sequence binding domains (e.g., a DNA binding domain or an RNA binding domain). The fusion protein may comprise at least one nuclear localization sequence (NLS), such as the simian virus 40 (SV40) NLS or the nucleoplasmin NLS. The fusion protein may also contain activator domains (e.g., heat shock transcription factors, NFKB activators) or repressor domains (e.g., the KRAB domain). As described by Lupo et al. ( Current Genomics , 2013, 14(4):268–278), the KRAB domain is a potent transcriptional repression module and is located at the amino-terminal sequence of most C2H2 zinc finger proteins. See, e.g., Margolin et al . ( PNAS , 1994, 91:4509–4513); and Witzgall et al. ( PNAS , 1994, 91:4514-4518 (1994) . The KRAB domain typically associates with corepressor proteins and/or transcription factors through protein-protein interactions, resulting in the transcriptional repression of genes to which KRAB zinc finger proteins (KRAB-ZFPs) bind. See, e.g., Friedman et al . ( Genes & Development , 1996, 10:2067-2678). In some embodiments, linker nucleic acid sequences are used to join two or more proteins, protein domains, or protein fragments.

[00255] As used herein, the terms "nuclear localization sequence" (NLS) or "nuclear localization signal" refer to a polypeptide sequence of a protein that preferentially increases the level of subcellular localization of the protein in the nucleus of a cell. NLS sequences are typically positively charged amino acid fragments located at the amino terminus ("N-terminus"), at the carboxy terminus ("C-terminus"), or within the protein (or a combination thereof, i.e., where one or more NLSs are located at the N-terminus and one or more NLSs are located at the C-terminus). NLS sequences may be covalently linked directly to the protein, or they may be attached via a linker polypeptide. The length of linker sequences may be optimized based on structural features of the protein (e.g., based on solvent accessibility at the ends, the presence of other important functional peptide sequences at the ends, etc.) to ensure that the NLS sequence is accessible for binding and transfer of the importin cognate protein. In addition, the optimal linker length can be screened empirically (see, e.g., Example 11). NLS sequences can be entirely synthetic, or they can be derived from endogenous or exogenous protein sequences. Computer programs can be used to predict the NLS sequence in a protein (see, e.g., moseslab.csb.utoronto.ca/NLSStradamus/ or nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). Examples of NLS sequences are given in Table 2.

Table 2
Representative nuclear localization sequences Source Subsequence SEQ ID NO. SV40 Large T Antigen PKKKRKV SEQ ID NO:04 Nucleoplasmin KRPAATKKAGQAKKKK SEQ ID NO:05 53BP1 GKRKLITSEEERSPAKRGRKS SEQ ID NO:493 VACM-1/CUL5 PKLKRQ SEQ ID NO:494 CXCR4 R.P.R.K. SEQ ID NO:495 VP1 RRRRRRG SEQ ID NO:496 ING4 KGKKGRTQKEKKAARARSKGKN SEQ ID NO:497 IER5 RCRCAAGVGGGPAGCPAPGSTPLKKPRR SEQ ID NO:498 ERK5 RKPVTAQERQREREEKRRRRQERAKEREKRRQERER SEQ ID NO:499 UL79 TLLLRETMNNLGVSDHAVLSRKTPQPY SEQ ID NO:500 EWS PGKMDKGEHRQERRDRPY SEQ ID NO:501 Hrp1 RSGGNHRRNGRGGRGGYNRRNNGYHPY SEQ ID NO:502 cMyc (1) PAAKRCKLD SEQ ID NO:503 cMyc (2) RQRRNELKRSP SEQ ID NO:504 Mouse c-able IV SALIKKKKKNAP SEQ ID NO:505 Matα2 KIPIK SEQ ID NO:506 MINIYO KSGSRK SEQ ID NO:507

[00255] As used herein, the term "fragment" refers to a portion of a molecule. A fragment may be a functional group or a portion of a molecule with multiple functional groups (e.g., that share common structural properties). As used herein, the terms "fragment" and "functional group" are generally synonymous, however, more particularly, a "functional group" may refer to a portion of a molecule that shares certain common chemical properties. The term "fragment" is often used as a structural description. In some embodiments, the 5' end, the 3' end, or the 5' end and 3' end (e.g., the non-native 5' end and/or the non-native 3' end in the first stem element) may comprise one or more fragments.

[00257] As used herein, the terms "modified protein," "mutated protein," "protein variant," and "engineered protein" generally refer to a protein that has been modified such that it contains a non-native sequence (i.e., the modified protein has a unique sequence compared to the unmodified protein).

[00258] As used herein, the term "transformation" refers to the introduction of an exogenous polynucleotide into a host cell, regardless of the method of introduction. For example, transformation can be achieved by direct uptake, transfection, infection, etc. The exogenous polynucleotide can be maintained as a non-integrated vector, such as an episome, or, alternatively, it can be integrated into the host genome.

[00259] A "host cell" is a cell that has been transformed or is capable of being transformed with an exogenous DNA sequence. A host cell may be from any organism that has one or more cells. Examples of host cells include, but are not limited to, a prokaryotic cell, a eukaryotic cell, a bacterial cell, an archaebacterial cell, a unicellular eukaryotic cell, a protozoan cell, a plant cell, an algal cell, a fungal cell (e.g., a yeast cell, an edible mushroom cell), an animal cell, an invertebrate cell, a vertebrate cell such as a mammalian cell (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.). Additionally, the host cell may be a stem cell or progenitor cell, or an immune cell, such as any of the immune cells described herein. The host cell may be a human cell. For example, the host cells may be lymphocytes or stem cells, such as hematopoietic stem cells. Lymphocytes include T cells for cell-mediated cytotoxic adaptive immunity, such as cytotoxic CD4 ⁺ and/or CD8 ⁺ T cells; natural killer (NK) cells, which participate in cell-mediated cytotoxic innate immunity; and B cells for antibody-mediated humoral adaptive immunity. Also included within the scope of the term are hematopoietic stem cells, which give rise to lymphoid cells. In addition, CAR-T cells, cells containing T cell receptors (TCRs), including TCR-engineered CAR-T cells, tumor infiltrating lymphocytes (TILs), CAR TILs, CAR-NK cells, and the like, can be modified using the methods described herein. In some embodiments, the human cell is outside the human body. In some embodiments, cells of a living organism (e.g., a human body) are modified ex vivo (i.e., outside the living organism). The term ex vivo often refers to a medical procedure in which an organ, cells, or tissue is removed from a living organism (e.g., a human) for treatment or therapy and then returned to the living organism. The term in vivo often refers to a medical procedure in which an organ, cells, or tissue in a living organism (e.g., a human body) is subjected to treatment or therapy.

[00260] The terms "subject," "individual," or "patient" are used synonymously herein and refer to any member of the phylum Chordata, including, but not limited to, humans and other primates, including non-human primates such as rhesus macaques, chimpanzees, and other species of monkeys and apes; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rabbits, mice, rats, and guinea pigs; birds, including poultry, wild birds, and game such as chickens, turkeys, and other birds of the gallinaceous family, ducks, and geese, and the like. The term does not indicate a particular age or sex. Thus, the term includes adults, juveniles, and neonates, as well as males and females. In some embodiments, the host cell is derived from an individual (e.g., lymphocytes, stem cells, progenitor cells, or tissue-specific cells). In some embodiments, such individuals include non-human individuals.

[00261] The terms "effective amount" or "therapeutically effective amount" of a composition or agent, such as a genetically engineered adoptive cell of the invention, refers to an amount of the composition or agent sufficient to provide a desired response. Preferably, the effective amount prevents, removes, or eliminates one or more harmful side effects. Such responses will depend on the particular disease being treated. For example, in a patient undergoing cancer treatment using adoptive cell therapy, the desired response may include the prevention, prevention, or elimination of one or more of the diseases described below, such as treatment or prevention of graft versus host disease (GvHD), host versus graft disease, cytokine release syndrome (CRS), cytokine storm, and a reduction in oncogenic transformations of the introduced genetically modified cells. The exact amount of the agent required for treatment may vary from individual to individual, depending on the species, age and general health of the individual, the severity of the condition being treated and the particular modified lymphocyte used, the route of administration, etc. A suitable "effective" amount in any individual case can be determined by one skilled in the art using routine experimentation.

[00262] "Treatment" or "therapy" for a particular disease, such as cancer or graft-versus-host disease, includes: preventing the disease, such as preventing the development of the disease or reducing the intensity of the disease in an individual who may be predisposed to developing the disease but who has not yet exhibited or been diagnosed with symptoms of the disease; inhibiting the disease, such as slowing the rate of its progression, stopping its progression, or reversing the disease; and/or alleviating the symptoms of the disease, such as reducing the number of symptoms in an individual.

[00263] Cas12 Guides

[00264] The chRDNA Cas12 targeting molecule of the invention is capable of forming a nucleoprotein complex with a related Cas12 protein, such as the Cas12a protein, wherein said complex is capable of targeting a target sequence complementary to the targeting region (spacer sequence).

[00265] Fig. 1A illustrates an exemplary BV3l6 Cas12a Acidaminococcus spp. guide molecule comprising: an activating region (Fig. 1A, 101) comprising a stem-loop duplex (Fig. 1A, 102); and a spacer sequence (Fig. 1A, 103) comprising a target binding sequence (Fig. 1A, 104). Fig. 1B illustrates an alternative Cas12a guide molecule comprising: an activating region (Fig. 1B, 105) comprising a stem-loop duplex (Fig. 1B, 106); and a spacer sequence (Fig. 1B, 107) comprising a target binding sequence (Fig. 1B, 108) and a 3' extension (Fig. 1B, 109). The 3' extension (Fig. 1B, 109) can be connected to the spacer sequence (Fig. 1B, 107) via a linker sequence. Fig. 1C illustrates an alternative Cas12a targeting molecule comprising: an activating region (Fig. 1C, 110) comprising a stem-loop duplex (Fig. 1C, 111) and a linker nucleotide (Fig. 1C, 114) and a 5' extension (Fig. 1C, 115); and a spacer sequence (Fig. 1C, 112) containing a target binding sequence (Fig. 1C, 113).

[00265] In the Cas12 chRDNA targeting molecules of the invention, the targeting region may comprise DNA, RNA, or a mixture of DNA and RNA. In some embodiments, the targeting region may comprise both DNA and RNA. In some embodiments, the targeting region may also comprise other base analogs, modified nucleotides, non-basic sites, and the like, as well as synthetic, natural, and non-naturally occurring modified backbone residues or backbone linkages, or combinations thereof.

[00267] In the Cas12 chRDNA guide molecules of the invention, the activating region may comprise DNA, RNA, or a mixture of DNA and RNA. In some embodiments, the activating region may comprise both DNA and RNA. In certain embodiments, the activating region may also comprise other base analogs, modified nucleotides, non-basic sites, and the like, as well as synthetic, natural, and non-naturally occurring modified backbone residues or linkages, or combinations thereof. In some embodiments, the activating region is adjacent to the targeting region. In some embodiments, the activating region is located downstream of the targeting region. In some embodiments, the activating region is located upstream of the targeting region.

[00268] In some embodiments, the Cas12 chRDNA guide molecules of the invention comprise a nucleic acid sequence comprising ribonucleotide bases and about 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, 20% or less, 21% or less, 22% or less, 23% or less, 24% or less, 25% or less, 26% or less, 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, 40% or less, 41% or less, 42% or less, 43% or less, 44% or less, 45% or less, 46% or less, 47% or less, 48% or less, 49% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, or 75% or less of deoxyribonucleotide bases or variants or modified derivatives thereof.

[00269] The Cas12 chRDNA targeting molecules of the invention may have, for example, a length of 30-75 bases, including non-basic sites. In some embodiments, the Cas12 chRDNA guide molecule is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 bases in length, including non-basic sites. In some embodiments, the Cas12 chRDNA guide molecule is 40 bases in length, including non-basic sites.

[00270] As used herein, the term "percentage of total length" of a polynucleotide sequence, such as a Cas12 chRDNA guide molecule, activation region, or targeting region, refers to the total length of the polynucleotide sequence, including, for example, non-basic sites and modified and variant bases.

[00271] In some embodiments, the activating region is 10 to 25 bases in length, including non-basic sites. In some embodiments, the activating region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases in length, including non-basic sites. In some embodiments, the activating region is 20 bases in length, including non-basic sites.

[00272] In some embodiments, the targeting region is 10 to 30 bases in length, including non-basic sites. In some embodiments, the targeting region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases in length, including non-basic sites. In some embodiments, the targeting region is 20 bases in length, including non-basic sites.

[00273] In the embodiments provided herein, the activating region and/or targeting region comprises ribonucleotide bases and one or more deoxyribonucleotide bases. The activating and/or targeting region may also, in some embodiments, comprise additional modifications, including base analogs, modified nucleotides, non-basic sites, or combinations thereof. In some embodiments, the activating and/or targeting region may comprise synthetic, natural, or non-naturally occurring modified backbone residues or linkages, or combinations thereof.

[00274] One or more deoxyribonucleotide bases may be present at any one or more positions in the targeting region. Thus, for example, for a 30 base long targeting region that includes non-basic sites, one or more deoxyribonucleotide bases may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. For smaller targeting regions, the positions will be shortened accordingly. For example, for a 20 base long targeting region that includes non-basic sites, one or more deoxyribonucleotide bases may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[00275] One or more additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof) may be present at any one or more positions in the targeting region. Thus, for example, for a 30 base long targeting region that includes non-basic sites, one or more additional modifications may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. For smaller targeting regions, the positions will be shortened accordingly. For example, for a 20 base long targeting region that includes non-basic sites, one or more additional modifications may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[00275] One or more deoxyribonucleotide bases may be present at any one or more positions in the activating region. Thus, for example, for an activating region of 25 bases in length that includes non-basic sites, one or more deoxyribonucleotide bases may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. For smaller activating regions, the positions will be reduced accordingly. For example, for a 20 base long activating region that includes non-basic sites, one or more deoxyribonucleotide bases may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[00277] One or more additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof) may be present at any one or more positions in the activating region. Thus, for example, for an activating region of 25 bases in length that includes non-basic sites, one or more additional modifications may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. For smaller activating regions, the positions will be reduced accordingly. For example, for a 20 base long activating region that includes non-basic sites, one or more additional modifications may be present at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[00278] In some embodiments, the amount of deoxyribonucleotide bases as a percentage of the total size of the Cas12 chRDNA guide, including non-basic sites, is preferably 75% or less. In some embodiments, the number of deoxyribonucleotide bases as a percentage of the total size of the Cas12 chRDNA guide, including non-basic sites, is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[00279] In some embodiments, the amount of additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof) as a percentage of the total size of the Cas12 chRDNA guide including non-basic sites is preferably 75% or less. In some embodiments, the amount of additional modifications as a percentage of the total size of the Cas12 chRDNA guide, including non-essential sites, is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[00280] In some embodiments of the invention, the amount of deoxyribonucleotide bases as a percentage of the total size of the targeting region, including non-basic sites, is preferably 75% or less. In some embodiments, the number of deoxyribonucleotide bases as a percentage of the total size of the targeting region including non-basic sites is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[00281] In some embodiments, the amount of additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof) as a percentage of the total size of the targeting region including non-basic sites is preferably 75% or less. In some embodiments, the amount of additional modifications as a percentage of the total size of the targeting region including non-basic sites is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[00282] In some embodiments, the amount of deoxyribonucleotide bases as a percentage of the total size of the activating region, including non-basic sites, is preferably 75% or less. In some embodiments, the number of deoxyribonucleotide bases as a percentage of the total size of the activating region including non-basic sites is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[00283] In some embodiments, the amount of additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof) as a percentage of the total size of the activating region including non-basic sites is preferably 75% or less. In some embodiments, the amount of additional modifications as a percentage of the total size of the activating region including non-basic sites is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[00284] In some embodiments, the amount of deoxyribonucleotide bases in the activating region and/or targeting region is adjusted to provide a statistically significant difference compared to, for example, a corresponding activating region and/or targeting region without deoxyribonucleotide bases. In some embodiments, the statistically significant difference is a difference in editing of a target or non-target molecule.

[00285] In some embodiments, the activating region and the targeting region comprise one or more deoxyribonucleotide bases. In some embodiments, the activating region comprises one or more deoxyribonucleotide bases, and the targeting region does not comprise any deoxyribonucleotide bases (e.g., comprises only RNA and/or modified ribonucleotides). In some embodiments, the targeting region comprises one or more deoxyribonucleotide bases, and the activating region does not comprise any deoxyribonucleotide bases (e.g., comprises only RNA and/or modified ribonucleotides).

[00285] In some embodiments, the activating region and the targeting region each comprise one or more additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof). In some embodiments, the activating region comprises one or more additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof), and the targeting region does not comprise any additional modifications (i.e., comprises only RNA or DNA). In some embodiments, the targeting region comprises one or more additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof), and the activating region does not comprise any additional modifications (i.e., comprises only RNA or DNA).

[00287] Figure 2 illustrates the Cas12a protein (Figure 2, 206) associated with a cognate Cas12a chRDNA guide molecule (Figure 2, 204) containing a target binding sequence (Figure 2, 205). The Cas12a chRDNA guide/nucleoprotein complex unwinds a target polynucleotide containing the target sequence, and the target binding sequence of the Cas12 chRDNA guide molecule (Figure 2, 205) is hydrogen bonded (Figure 2, indicated by the vertical line between the polynucleotides) to the target sequence (Figure 2, 207). In Figure 2, the target polynucleotide comprises a target strand (Fig. 2, 201) containing a target sequence (Fig. 2, 207), and the non-target strand (Fig. 2, 202) comprises a PAM sequence (Fig. 2, 203). The PAM sequence (Fig. 2, 203) is typically located upstream (i.e., in the 5' direction) of the target sequence (Fig. 2, 207) on the non-target strand (Fig. 2, 202). Hydrogen bonding between the target-binding sequence of the Cas12a chRDNA guide molecule (Fig. 2, 205) and the target sequence (Fig. 2, 207) results in stepwise cleavage (Fig. 2, 208) of the target strand (Fig. 2, 201) and the non-target strand (Fig. 2, 202).

[00288] Figures 3A-3I illustrate various canonical and non-canonical nucleotides for use in the Cas12 chRDNA guide molecules of the invention. Table 3 provides a number of indices used in Figures 3A-FIG. 3I.

Table 3
Numerical indicators of canonical and non-canonical nucleotides for their use in Cas12 crRNA and chRDNA guide molecules Fig. Indicator Description Fig. 3A 301 ribose sugar 302 nitrogenous base 303 phosphate backbone 304 2'-hydroxyl group Fig. 3B 305 deoxyribose sugar 306 nitrogenous base 307 phosphate backbone 308 absence of 2'-hydroxyl group Fig. 3C 309 ribose sugar 310 phosphate backbone 311 2'-hydroxyl group Fig. 3D 312 deoxyribose sugar 313 phosphate backbone 314 absence of 2'-hydroxyl group Fig. 3E 315 ribose sugar 316 non-canonical nitrogenous base or mimetic base 317 phosphate backbone 318 2'-hydroxyl group Fig. 3F 319 deoxyribose sugar 320 non-canonical nitrogenous base or mimetic base 321 phosphate backbone 322 absence of 2'-hydroxyl group Fig. 3G 323 ribose sugar 324 nitrogenous base 325 chemically modified skeleton 326 2'-hydroxyl group Fig. 3H 327 deoxyribose sugar 328 nitrogenous base 329 chemically modified skeleton 330 absence of 2'-hydroxyl group Fig. 3I 331 non-canonical or chemically modified sugar 332 nitrogenous base 333 phosphate backbone

[00289] Figure 4 illustrates a Cas12a protein (Figure 4, 406) associated with a cognate Cas12a chRDNA guide molecule (Figure 4, 404) comprising a target binding sequence (Figure 4, 405), wherein the target binding sequence (Figure 4, 405) comprises nucleotides other than RNA nucleotides (Figure 4, 409), such as the canonical and non-canonical nucleotides shown in Figures 3B-3I. The Cas12a chRDNA guide/nucleoprotein complex unwinds the target polynucleotide containing the target sequence, and the target-binding sequence of the Cas12 chRDNA guide molecule (Fig. 4, 405) is hydrogen bonded (Fig. 4, indicated by the vertical line between the polynucleotides) to the target sequence (Fig. 4, 407). In Fig. 4, the target polynucleotide contains the target strand (Fig. 4, 401) containing the target sequence (Fig. 4, 407), and the non-target strand (Fig. 4, 402) contains the PAM sequence (Fig. 4, 403). The PAM sequence (Fig. 4, 403) is typically located upstream (i.e., 5') of the target sequence (Fig. 4, 407) on the non-target strand (Fig. 4, 402). Hydrogen bonding between the target-binding sequence of the Cas12a chRDNA guide molecule (Fig. 4, 405) and the target sequence (Fig. 4, 407) results in stepwise cleavage (Fig. 4, 408) of the target strand (Fig. 4, 401) and the non-target strand (Fig. 4, 402).

[00290] Fig. 5 illustrates an example of a cr-RNA guide molecule of Acidaminococcus spp. Cas12a (strain BV3L6) comprising: an activating region (Fig. 5, 501) containing a stem-loop duplex (Fig. 5, 502); and a spacer (Fig. 5, 503) containing a target binding sequence (Fig. 5, 504). Each nucleotide position in the activating region (Fig. 5, 501) and in the spacer (Fig. 5, 503) is labeled starting from the 5' end of the guide molecule, where the activating region and the target binding region contain RNA.

[00291] Fig. 6 illustrates an example of a cr-RNA guide molecule of Acidaminococcus spp. Cas12a (strain BV3L6) comprising: an activating region (Fig. 6, 601) comprising a stem-loop duplex (Fig. 6, 602); and a spacer (Fig. 6, 603) comprising a target binding sequence (Fig. 6, 604). Each nucleotide position in the activating region (Fig. 6, 601) and in the spacer (Fig. 6, 603) is labeled, starting from the 5' end of the guide molecule, where the activating region comprises a mixture of RNA (shaded white) and DNA (shaded gray), and the target binding sequence comprises a mixture of RNA (shaded white) and DNA (shaded gray).

[00292] Fig. 7 illustrates an example of an Acidaminococcus spp. (strain BV3L6) chRDNA Cas12a guide molecule comprising: an activating region (Fig. 7, 701) containing a stem-loop duplex (Fig. 7, 702); and a spacer (Fig. 7, 703) containing a target binding sequence (Fig. 7, 704). Each nucleotide position in the activating region (Fig. 7, 701) and in the spacer (Fig. 7, 703) is labeled, starting from the 5' end of the guide molecule, where the activating region contains a mixture of RNA (shaded white) and DNA (shaded gray). The Cas12a chRDNA guide molecule further comprises other non-canonical nucleotides such as a chemically modified sugar nucleotide (Fig. 7, 705), a non-basic ribonucleotide (Fig. 7, 706), a deoxyribonucleotide with a chemically modified backbone (Fig. 7, 707), a ribonucleotide with a chemically modified backbone (Fig. 7, 708), and a non-basic deoxyribonucleotide (Fig. 7, 709).

[00293] Figure 8 illustrates the formation of a Cas12a chRDNA guide/nucleoprotein complex, wherein the Cas12 protein (Figure 8, 801) binds to a Cas12a chRDNA guide molecule (Figure 8, 802) to form a Cas12a chRDNA guide/nucleoprotein complex (Figure 8, 803). The Cas12a chRDNA guide/nucleoprotein complex (Figure 8, 803) binds to a target polynucleotide (Figure 8, 804), wherein the target polynucleotide comprises a target sequence that is complementary to a target-binding sequence of the Cas12 chRDNA guide molecule, and hydrogen bonds are formed between the target-binding sequence of the Cas12 chRDNA guide molecule and the target sequence (Figure 8, 805).

[00294] Fig. 9 illustrates the formation of an insertion or deletion (indel) in a target polynucleotide by a Cas12a guide chRDNA/nucleoprotein complex, wherein the Cas12 protein (Fig. 9, 901), complexed with a Cas12 guide chRDNA molecule (Fig. 9, 902), binds to a target polynucleotide (Fig. 9, 903) containing a PAM (Fig. 9, 904), and the target polynucleotide is cleaved (Fig. 9, 905) by the Cas12a guide chRDNA/nucleoprotein complex. Upon targeting, the Cas12a chRDNA guide/nucleoprotein complex dissociates from the target polynucleotide (Fig. 9, 906), where the target polynucleotide comprises an upstream strand (i.e., in the 5' direction) (Fig. 9, 907) and a downstream strand (i.e., in the 3' direction) (Fig. 9, 908) relative to the PAM (Fig. 9, 904). The cellular DNA repair machinery restores the target polynucleotide by insertion or deletion (Fig. 9, 910) of the sequence around the cleavage site in the target polynucleotide. The upstream strand (Fig. 9, 911) and downstream strand (Fig. 9, 912) are joined and the edited target polynucleotide (Fig. 9, 914) contains insertions-deletions (Fig. 9, 913) at the cleavage site, wherein the edited target polynucleotide has a different sequence compared to the unedited target polynucleotide. In some embodiments, the insertion or deletion (indel) in the target polynucleotide formed by the Cas12a chRDNA guide/nucleoprotein complex is observed intracellularly.

[00295] Fig. 10 illustrates the incorporation of a donor polynucleotide sequence into a target polynucleotide, wherein the Cas12 protein (Fig. 10, 1001), formed as a complex with the Cas12 guide molecule chRDNA (Fig. 10, 1002), binds to the target polynucleotide (Fig. 10, 1003) containing a PAM (Fig. 10, 1004), and the target polynucleotide is cleaved (Fig. 10, 1005) by the Cas12a guide chRDNA/nucleoprotein complex. Following targeting, the Cas12a chRDNA guide/nucleoprotein complex dissociates from the target polynucleotide (Fig. 10, 1006), where the target polynucleotide comprises an upstream strand (i.e., in the 5' direction) (Fig. 10, 1007) and a downstream strand (i.e., in the 3' direction) (Fig. 10, 1008) relative to the PAM (Fig. 10, 1004), and where a donor polynucleotide is formed (Fig. 10, 1009). The cellular DNA repair machinery restores the target polynucleotide (Fig. 10, 1010) via the donor polynucleotide (Fig. 10, 1011). The resulting edited target polynucleotide (Fig. 10, 1010) contains a donor sequence (Fig. 10, 1011) at the target site. In some embodiments, the incorporation of the donor polynucleotide sequence into the target polynucleotide occurs intracellularly.

[00295] Figure 11 illustrates a single-stranded nicking of a target polynucleotide, wherein the Cas12 protein (Figure 11, 1101) complexed with the Cas12 guide molecule chRDNA (Figure 11, 1102) containing DNA bases in the target binding sequence (Figure 11, 1106) binds to a target polynucleotide (Figure 11, 1103) containing a PAM (Figure 11, 1104), and the target polynucleotide is nicked (Figure 11, 1105) in only one strand of the target polynucleotide by the Cas12a guide chRDNA/nucleoprotein complex.

[00297] Figure 12 illustrates the use of two Cas12a guide chRDNA/nucleoprotein single-strand break complexes to form a staggered double-strand break in a target polynucleotide, wherein a first Cas12a guide chRDNA/nucleoprotein complex binds to an upstream (i.e., 5') target sequence (Figure 12, 1201) to form a first break in the target polynucleotide (Figure 12, 1202) and a second Cas12a guide chRDNA/nucleoprotein complex binds to a downstream (i.e., 3') target sequence of the target polynucleotide (Figure 12, 1203) to form a second break in the target polynucleotide (Figure 12, 1204). After tandem cleavage, the target polynucleotide after cleavage contains an upstream (i.e., in the 5' direction) strand (Fig. 12, 1205) and a downstream (i.e., in the 3' direction) strand (Fig. 12, 1206) with 5' overhangs. A donor polynucleotide is then formed, and the cellular DNA repair machinery repairs the target polynucleotide (Fig. 12, 1207) via the donor polynucleotide (Fig. 12, 1208). The resulting edited target polynucleotide (Fig. 12, 1209) contains the donor sequence (Fig. 12, 1210) at the tandem single-strand break site. In some embodiments of the invention, the use of two Cas12a guide chRDNA/single-strand break nucleoprotein complexes to generate a staggered DSB in a target polynucleotide occurs intracellularly.

[00298] Figure 13 illustrates the positions in the target binding sequence of the Acidaminococcus spp. (strain BV3L6) Cas12a CHRDNA guide molecule containing DNA bases. The y-axis represents the normalized percentage of editing of multiple DNA targets (see Example 5) at a single position in the target binding sequence (error bars indicate standard deviation). The x-axis indicates each position (5' to 3') of the target binding sequence. The target binding sequence is shown above the graph (Figure 13, 1301) with the preferred positions of DNA base usage (i.e., average normalized editing greater than 70%) shown in gray. The location of the Cas12a chRDNA activating regions is also shown (Figure 13, 1302).

[00299] Figure 14 illustrates positions in the activating region of the Acidaminococcus spp. (strain BV3L6) Cas12a chRDNA guide molecule containing DNA bases (see Example 8). The y-axis represents the normalized percentage of editing of the guide molecule with DNA at one position in the activating region. The x-axis indicates positions (5' to 3') in the activating region. The activating region is shown on the left of the graph (Figure 14, 1401) with the preferred positions of DNA base usage (i.e., greater than 70% of the average normalized editing) indicated in gray. The location of the binding sequence to the Cas12a chRDNA guide target is also indicated (Figure 14, 1402).

[00300] Figures 15A and 15B illustrate flow cytometric analysis of CAR-T cells engineered with Cas12a chRDNA/nucleoprotein guide complexes. Figure 15A shows the percentage of cells expressing anti-BCMA CAR antibodies (Figure 15A, 1501), TRAC protein (Figure 15A, 1502), and B2M protein (Figure 15A, 1503). The x-axis shows cells that were not treated (Fig. 15A, 1504), cells that were transfected with the Cas12a guide chRDNA/nucleoprotein complex targeting the TRAC and B2M genes (Fig. 15A, 1505), and cells that were transfected with both Cas12a guide chRDNA/nucleoprotein complexes targeting both TRAC and B2M genes and transduced with two viruses containing donor DNAs encoding the anti-BCMA CAR antibody gene and the B2M-HLA-E fusion gene, respectively (Fig. 15A, 1506). The y-axis shows the percentage of cells positive for various cell surface markers as determined by flow cytometry. In Fig. 15B shows the results of an in vitro cytotoxicity assay performed using CAR T cells containing anti-BCMA antibodies, B2M-HLA-E (shown as gray circles) and control TRAC-KO T cells (shown as black circles) containing a BCMA-positive target cell line. The y-axis shows the percentage of target cell cytolysis, and the x-axis shows the E:T ratio used. Each raw data point represents the average of 3 co-culture wells at each E:T ratio.

[00301] Figures 16A and 16B illustrate the cell editing activity of a Cas12a chRDNA guide/nucleoprotein complex containing multiple linkers and a nuclear localization sequence (NLS). The y-axis in Figures 16A and 16B indicates the percentage of editing calculated by next-generation sequencing. In Figure 16A, the x-axis shows the linker-NLS configuration, where all input data are presented as replicate data. In Figure 16B, the x-axis shows the concentration in picomoles of Cas12a guide sequence and chRDNA (20:60 or 80:240 pmol) of all four linker-NLS constructs shown in Figure 16A, as well as the “non-optimized” linker-NLS construct (Figures 16B, 1613). All raw data represent data for a unique target guide molecule sequence and are averages of triplicate values for each raw data. Figure 17 illustrates the cell editing activity of the Cas12a guide chRDNA/nucleoprotein complex formed together with GS-SV40 (Figure 7, 1708; SEQ ID NO: 479) and (G4S)2-NPL (Figure 7, 1712; SEQ ID NO: 489) when multiple Cas12a guide molecules were co-delivered in a single transfection reaction. The y-axis in Figure 17 indicates the percentage of editing calculated by next-generation sequencing. Figure 17, the X-axis shows the target gene as the TRAC gene (Fig. 17, 1701; SEQ ID NO: 36), the B2M gene (Fig. 17, 1702; SEQ ID NO: 62), the CISH gene (Fig. 17, 1703; SEQ ID NO: 158), or the CBLB gene (Fig. 17, 1704; SEQ ID NO: 171). Cas12a chRDNA guide/nucleoprotein complexes were used as one targeting complex per transfection reaction (Fig. 17, 1705 and Fig. 17, 1709), two targeting complexes per transfection reaction (Fig. 17, 1706 and Fig. 17, 1710), or four targeting complexes per transfection reaction (Fig. 17, 1707 and Fig. 17, 1711). Each bar in the graph represents the average of two to three replicates.

[00302] Methods for constructing specific Cas12 chRDNA guide molecules that can be modified with deoxyribonucleotide bases and, optionally, additional modifications (such as base analogs, modified nucleotides, non-basic sites, modified backbone residues or linkages, or combinations thereof) are well known in the art. See, for example, Briner et al. ( Molecular Cell , 2014, 56:333-339). To do this, the genomic sequence of the gene to be targeted is first identified. The precise region of the gene selected for targeting depends on the particular application. For example, to activate or repress a target gene, such as by activating CRISPR or inhibiting CRISPR, Cas12a chRDNA guide/nucleoprotein complexes can be targeted to a promoter that regulates the expression of the gene of interest. For gene knockout, Cas12 chRDNA guide molecules can be engineered to target 5' constitutively expressed exons to reduce the likelihood of the target region being removed from the mRNA by alternative splicing. Exons near the N-terminus can be considered as targets because frameshift mutations increase the likelihood of producing a non-functional protein product. Alternatively, related Cas12 chRDNA guide molecules can be engineered to target exons that encode known essential protein domains. In this regard, non-frameshift mutations such as insertions or deletions are more likely to alter protein function if they are present in protein domains that are essential for protein function. For gene editing using HDR, the target sequence must be in the vicinity of the desired edit site. In this case, the location required for editing is determined and a target sequence in the vicinity is selected.

[00303] In some embodiments, the Cas12 chRDNA guide molecule may be engineered such that the Cas12a guide chRDNA/nucleoprotein complex can bind outside of the Cas12 protein cleavage site. In this case, the target nucleic acid may not interact with the Cas12a guide chRDNA/nucleoprotein complex, and the target nucleic acid can be excised (e.g., freed from the Cas12a guide chRDNA/nucleoprotein complex). In some embodiments, the Cas12 chRDNA guide molecule may be engineered such that the Cas12a guide chRDNA/nucleoprotein complex can bind within the Cas12 protein cleavage site. In this case, the target nucleic acid can interact with the Cas12a guide chRDNA/nucleoprotein complex, and the target nucleic acid can be bound (e.g., associated with the Cas12a guide chRDNA/nucleoprotein complex).

[00304] The Cas12 CHRDNA guide molecules can be designed such that the Cas12a guide chRDNA/nucleoprotein complex can hybridize to multiple sites in a nucleic acid sample. A plurality of Cas12a guide chRDNA/nucleoprotein complexes can be contacted with a nucleic acid sample. The plurality of Cas12a guide chRDNA/nucleoprotein complexes can comprise Cas12-chRDNA guide molecules designed to hybridize to the same sequence. The plurality of Cas12a guide chRDNA/nucleoprotein complexes can comprise Cas12 chRDNA guide molecules designed to hybridize to different target sequences.

[00305] The target sequences may be located at different positions within the target nucleic acid. These positions may contain the same or similar target nucleic acid sequences. These positions may contain different target nucleic acid sequences. Such positions may be defined according to their distance from each other. Such positions may be located at a distance of less than 10 kilobase pairs (kb) from each other, less than 8 kb from each other, less than 6 kb from each other, less than 4 kb from each other, less than 2 kb from each other, less than 1 kb from each other, from each other, less than 900 nucleotides from each other, less than 800 nucleotides from each other, less than 700 nucleotides from each other, less than 600 nucleotides from each other, less than 500 nucleotides from each other, less than 400 nucleotides from each other, less than 300 nucleotides from each other, less than 200 nucleotides from each other, or less than 100 nucleotides from each other.

[00305] The Cas12a chRDNA guide/nucleoprotein complexes can cleave a target nucleic acid, which can result in the removal of a target nucleic acid that can be less than 10 kilobase pairs (kb), less than 8 kb, less than 6 kb, less than 4 kb, less than 2 kb, less than 1 kb, less than 900 nucleotides, less than 800 nucleotides, less than 700 nucleotides, less than 600 nucleotides, less than 500 nucleotides, less than 400 nucleotides, less than 300 nucleotides, less than 200 nucleotides, or less than 100 nucleotides in length.

[00307] The Cas12a guide chRDNA/nucleoprotein complexes can be associated with a fragmented target nucleic acid that can be less than 10 kilobase pairs (kb), less than 8 kb, less than 6 kb, less than 4 kb, less than 2 kb, less than 1 kb, less than 900 nucleotides, less than 800 nucleotides, less than 700 nucleotides, less than 600 nucleotides, less than 500 nucleotides, less than 400 nucleotides, less than 300 nucleotides, less than 200 nucleotides, or less than 100 nucleotides in length.

[00308] The Cas12 chRDNA targeting molecules of the invention may be synthesized in vitro by known methods, such as chemical synthesis in solution or on a solid support, or in some cases, they may be produced recombinantly. A single production or synthesis technology or a combination of production and synthesis technologies may be used, in which deoxyribonucleotide bases and/or modifications may be introduced at one or more positions along the entire length of the sequence.

[00309] In some embodiments, a Cas12 CHRDNA guide molecule, its targeting region, or its activating region are designed to comprise deoxyribonucleotide base(s) (and/or modified deoxyribonucleotide base(s)) at specific positions compared to a reference Cas12 CHRDNA guide molecule, reference targeting region, or reference activating region, each of which is comprised of ribonucleotide bases, respectively.

[00310] In some embodiments, the Cas12a chRDNA reference guide molecule comprises the following RNA sequence: UAAUUUCUACUUCUUGUAGAUGAGUCUCUCAGCUGGUACAC. The Cas12a CHRDNA guide molecules of the invention designed based on this reference RNA sequence include Cas12 CHRDNA guide molecules having one or more deoxyribonucleotide bases at one or more of positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, 19, 21, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39 and 40. In some embodiments, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 of these listed positions are deoxyribonucleotide bases. In some embodiments, one, all, or more of the deoxyribonucleotide bases in the targeting region form canonical base pairs with the target sequence. In some embodiments, at least one or more of the deoxyribonucleotide bases in the targeting region do not form a canonical base pair with the target sequence.

[00311] In some embodiments, the Cas12a chRDNA reference guide molecule comprises the following RNA sequence: UAAUUUCUACUCUUGUAGAUAGUGGGGGUGAAUUCAGUGU. The Cas12a CHRDNA guide molecules of the invention designed based on this reference RNA sequence include Cas12 CHRDNA guide molecules having one or more deoxyribonucleotide bases at one or more of positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, 19, 21, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39 and 40. In some embodiments, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 of these listed positions are deoxyribonucleotide bases. In some embodiments, one, all, or more of the deoxyribonucleotide bases in the targeting region form canonical base pairs with the target sequence. In some embodiments, at least one or more of the deoxyribonucleotide bases in the targeting region do not form a canonical base pair with the target sequence.

[00312] In some embodiments, the reference activating region comprises the RNA sequence: UAAUUUCUACUCUUGUAGAU. Activating regions comprising a deoxyribonucleotide base of the invention and designed based on this reference RNA sequence include activating regions having one or more deoxyribonucleotide bases at one or more of positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18, and 19. In some embodiments, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 of these listed positions are deoxyribonucleotide bases.

[00313] In some embodiments, the reference targeting region comprises the RNA sequence: GAGUCUCUCAGCUGGUACAC. Targeting regions comprising a deoxyribonucleotide base of the invention and designed based on this reference RNA sequence include targeting regions having one or more deoxyribonucleotide bases at one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 of these listed positions are deoxyribonucleotide bases.

[00314] In some embodiments, the reference targeting region comprises the RNA sequence: AGUGGGGGUGAAUUCAGUGU. Targeting regions comprising a deoxyribonucleotide base of the invention and designed based on this reference RNA sequence include targeting regions having one or more deoxyribonucleotide bases at one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 of these listed positions are deoxyribonucleotide bases.

[00315] In some embodiments, the activating region has the sequence TrAArUrUrUCrUrACrUCrUTGrUrArGArU (where the "r" precedes the ribonucleotide base; and the absence of an "r" before the base indicates a deoxyribonucleotide base).

[00315] In some embodiments, the targeting region has the sequence GrArGrUrCrUrCrUrCrAGrCrUrGrGrUrArCrAC (where the "r" precedes a ribonucleotide base; and the absence of an "r" before the base indicates a deoxyribonucleotide base).

[00317] In some embodiments, the targeting region has the sequence ArGrUrGrGrGrGrGrUrGArArUrUrCrArGrUrGT (where the "r" precedes the ribonucleotide base; and the absence of an "r" before the base indicates a deoxyribonucleotide base).

[00318] In some embodiments, the Cas12a chRDNA guide sequence has the sequence TrAArUrUrUCrUrACrUCrUTGrUrArGArUGrArGrUrCrUrCrUrCrAGrCrUrGrGrUrArCrAC (the "r" precedes the ribonucleotide base; and the absence of an "r" before the base indicates a deoxyribonucleotide base).

[00319] In some embodiments, the Cas12a chRDNA guide sequence has the sequence TrAArUrUrUCrUrACrUCrUTGrUrArGArUArGrUrGrGrGrGrGrGrUrGArArUrUrCrArGrUrGT (where the "r" precedes a ribonucleotide base; and the absence of an "r" before the base indicates a deoxyribonucleotide base).

[00320] Cas12 proteins

[00321] Cas12 proteins of the invention include, but are not limited to, wild-type Cas12 proteins derived from type V CRISPR-Cas systems, modified Cas12 proteins, variant Cas12 proteins, orthologs of Cas12, and combinations thereof. In some embodiments, the Cas12 protein is a wild-type Cas12a protein, a modified Cas12a protein, a variant Cas12a protein, an ortholog of Cas12a, or a combination thereof.

[00322] The Cas12 protein may be modified. The modification may include amino acid modifications. The modifications may also change the primary amino acid sequence and/or the secondary, tertiary, and/or quaternary amino acid structure. In some embodiments, one or more amino acid sequences of the Cas12 protein may be changed without significantly affecting the structure or function of the Cas12 protein. The type of mutation may not be critical if the change occurs in some regions (e.g., a non-critical region) of the protein. Depending on the location of the substitution, the mutation may not significantly affect the biological properties of the resulting variant. For example, the properties and functions of certain Cas12 variants may be the same type of properties as wild-type Cas12.

[00323] In some cases, whether a mutation can significantly affect the structure and/or function of the Cas12 protein can be determined by sequence and/or structure alignment. Sequence alignment can identify regions of the polypeptide that are similar and/or dissimilar (e.g., conserved, non-conserved, hydrophobic, hydrophilic, etc.). In some cases, a region in the sequence of interest that is similar to other sequences is suitable for modification. In other cases, a region in the sequence of interest that is different from other sequences is suitable for modification. For example, sequence alignment can be performed by database searching, pairwise alignment, multiple sequence alignment, genomic analysis, motif searching, comparative analysis, and/or using programs such as BLAST, CS-BLAST, HHPRED, psi-BLAST, LALIGN, PyMOL, and SEQALN. Structure alignment can be performed using programs such as Dali, PHYRE, Chimera, COOT, O, and PyMOL. Alignment can be performed by database searching, pairwise alignment, multiple sequence alignment, genomic analysis, motif search, or comparative analysis, or any combination thereof.

[00324] Cas12 proteins typically consist of six domains corresponding to the REC1, REC2, PAM-interacting (PI), nuclease (Nuc), Wedge (WED), and RuvC domains. See, for example, Yamano et al. ( Cell , 2016, 165(4):949-962). The WED domain and the RuvC domain can have a tripartite sequence architecture interrupted by sequences from other domains. For example, the Acidaminococcus spp. Cas12a WED domain sequence is interrupted by REC1, REC2, and PI domain sequences. Additionally, some subtypes of Cas12 proteins contain a bridging helix domain that is adjacent to or between the RuvC domain sequences.

[00325] Regions of the Cas12 protein can be modified to modulate the activity of the Cas12 protein. For example, regions of the Acidaminococcus spp. (strain BV3L6) Cas12a protein corresponding to residues of the PI domain (598-718) and the WED domain (526-597 and 719-883) can be modified to alter PAM specificity. See, for example, Tóth et al. ( Nucleic Acid Research , 2020, 48(7):3722-3733). The region of the Acidaminococcus spp. (strain BV3L6) Cas12a protein corresponding to residues of the REC1 (24-319) and REC2 (320-526) domains can be modified to alter target interaction and cleavage kinetics. Regions of the REC1 (226-304) and REC2 (368-435) domains interact directly with the distal end of the PAM target-binding sequence and with the target sequence and can be engineered to alter the efficiency of target sequence cleavage. Regions of the Nuc (1066-1261) and RuvC (940-956, 957-1065, and 1261-1307) domains can be modified to alter the efficiency of cleavage of the target strand, the non-target strand, or the target strand or non-target strand of the target sequence. Engineering of these regions can involve the introduction of mutations, substitution with corresponding regions from other Cas12 orthologs, deletions, insertions, etc.

[00325] Modified Cas12 proteins can be used in combination with chRDNA Cas12 guide molecules to alter the activity or specificity of the Cas12 protein. In some cases, the Cas12 protein can be modified to provide enhanced activity or specificity when complexed with a chRDNA Cas12 guide molecule, wherein the Cas12 modifications occur in the REC1, REC2, RuvC, WED, and/or Nuc domain(s). In some cases, the Cas12 protein can be modified to provide enhanced activity or specificity when complexed with a chRDNA Cas12 guide molecule, wherein the Cas12a modifications occur in regions 226-304, 368-435, 940-956, 978-1158, 1159-1180, and 1181-1298 (numbering is based on the Acidominococus spp. Cas12a sequence).

[00327] Such mutations may be caused by site-directed mutagenesis. Mutations may include substitutions, additions, deletions, or any combination thereof. In some cases, the mutation is a substitution of an amino acid for alanine. In other cases, the mutation is a substitution of one amino acid for another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine). The mutation may be a substitution of an amino acid for an unnatural amino acid (e.g., selenomethionine). The mutation may be a substitution of an amino acid for amino acid mimetics (e.g., phosphomimetics). The mutation may be a conservative mutation. For example, a mutation may be a substitution of an amino acid with amino acids that resemble the mutated amino acids in size, shape, charge, polarity, conformation, and/or rotamers (e.g., cysteine/serine substitutions, lysine/asparagine substitutions, histidine/phenylalanine substitutions).

[00328] In some embodiments, the Cas12 protein is an nCas12 protein. The nCas12 protein is a variant of the Cas12 protein that is nuclease deficient, and is also referred to as "single-strand nick Cas12" or "Cas12 nickase". Such molecules lack some of the endonuclease activity and, therefore, can only "cut" one strand of a target nucleic acid. See, e.g., Jinek et al. ( Science , 2012, 337:816-821). This can be achieved, for example, by introducing mutation(s) into the RuvC nuclease domain. Non-limiting examples of such modifications can include D917A, E1006A, and D1225A in the RuvC nuclease domain of the F. novicida Cas12a protein. Obviously, mutation of other catalytic residues to reduce the activity of the RuvC nuclease domain can also be carried out by those skilled in the art. The resulting nCas12 protein is unable to cleave double-stranded DNA, but retains the ability to form a complex with a guide molecule, bind to a target DNA sequence, and cleave only one strand of the target DNA. Targeting specificity is determined by binding of the Cas12 protein to the PAM sequence and complementary base pairing of the guide molecule to the genomic locus. In some embodiments, the nCas12 protein is nCas12a protein.

[00329] In some embodiments, the Cas12 protein is a dCas12 protein. The dCas12 protein is a variant of the Cas12 protein that is nuclease-inactivated and is also referred to as "catalytically inactive Cas12 protein,""enzymatically inactive Cas12,""catalytically dead Cas12," or "dead Cas12." Such molecules lack endonuclease activity and can therefore be used for guide RNA-based gene regulation. See, e.g., Jinek et al. ( Science , 2012, 337:816-821). Mutations of the catalytic residues to eliminate RuvC domain activity can be made by those skilled in the art. The resulting dCas12 protein is unable to cleave double-stranded DNA, but retains the ability to form complexes with the guide molecule and bind to a target DNA sequence. Targeting specificity is determined by binding of the Cas12 protein to the PAM sequence and complementary base pairing of the targeting molecule to the genomic locus. In some embodiments, the dCas12 protein is dCas12a protein.

[00330] Some Cas12 protein subtypes lack nuclease activity, either due to inactivation of the RuvC-like nuclease domain or a partial or complete absence of the RuvC-like nuclease domain. One such subtype, the VK type and its associated Cas12k protein, are instead associated with the Tn7-like mobile genetic elements tnsB, tnsC, tniQ . See, e.g., Strecker et al. ( Science , 2019, 364(6448):48-53) . Cas12k retains the ability to form complexes with a guide molecule and bind to a target DNA sequence, and its associated Tn7-like proteins facilitate RNA-guided transposition of DNA sequences. In some embodiments of the invention, the Cas12a guide chRDNA/nucleoprotein complex is a Cas12k guide chRDNA/nucleoprotein complex.

[00331] Other amino acid modifications may include amino acids with glycosylated forms, aggregated conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., PEGylated molecules). Covalent variants may be produced by linking functional groups to groups that are in the amino acid chain or at the N- or C-terminal residue. In some cases, mutated site-directed polypeptides may also include allelic variants and species variants.

[00332] In some embodiments, the Cas12 protein may be a fusion or chimeric protein comprising a first domain of the Cas12 protein and a second domain of another protein, such as the Csy4 protein. Modification of the fusion with the Cas12 protein may impart additional activity to the modified Cas12 protein. Such activities may include a nuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer forming activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, a glycosylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a reverse transcriptase activity, a deubiquitination activity, an adenylation activity, a deadenylation activity, a SUMOylation activity, a deSUMOsumoylation activity, a ribosylation activity, a deribosylation activity, and/or a myristoylation or demyristoylation activity that modify a polypeptide linked to a nucleic acid sequence target acids (eg, with histone).

[00333] In some embodiments, a Cas12 protein may comprise one or more NLS sequences (e.g., added to and/or inserted into the Cas12 protein sequence). The NLS sequence may be located, for example, at the N-terminus, at the C-terminus, or within the Cas12 protein (such as the Cas12a protein), including combinations thereof (e.g., one or more NLS at the N-terminus and one or more NLS at the C-terminus).

[00334] In some embodiments, a Cas12 protein, including a Cas12a protein, may comprise a plurality of NLS sequences, such as at least 2, at least 3, at least 4, or at least 5 NLS sequences. The plurality of NLS sequences may be present at one end of the Cas12a protein (e.g., NLS sequences are present only at the N-terminus or only at the C-terminus), or may be present at both ends (e.g., one or more NLS sequences at the N-terminus and one or more NLS sequences at the C-terminus). The NLS sequences may be entirely synthetic, modified, or may be derived from endogenous or exogenous protein sequences. In some embodiments of the invention, the Cas12 protein, including the Cas12a protein, may comprise an NLS sequence or a modified sequence or a sequence derived from an NLS sequence selected from SV40 large T antigen, nucleoplasmin, 53BP1, VACM-1/CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrp1, cMyc(1), cMyc(2), mouse c-ABLE IV, Matα2, and MINIYO.

[00335] In some embodiments, a Cas12 protein, including a Cas12a protein, may comprise an NLS sequence or a modified sequence or a sequence derived from an NLS sequence selected from any of SEQ ID NOs: 04, 05, and 493-507. The modified NLS sequence or derivative thereof may comprise, for example, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid substitution; 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid deletion; and/or 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid addition compared to a reference NLS sequence (e.g., an NLS sequence selected from any of SEQ ID NOs: 04, 05, and 493-507).

[00335] In some embodiments, the NLS sequence may have, for example, a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an NLS sequence selected from any of SEQ ID NOs: 04, 05, and 493-507.

[00337] The NLS sequences can be covalently linked (e.g., to the Cas12 protein, to another NLS sequence(s), or to a fusion protein sequence linked to the Cas12 protein) either directly or via a linker polypeptide. The length of the linker sequence can be optimized based on the structural features of the particular Cas12 protein (e.g., solvent accessibility at the ends, presence of other important functional peptide sequences at the ends, etc.) to ensure that the NLS sequence is accessible for binding and transfer of the related importin protein. Additionally, as described in Example 11, an empirical screening for the desired linker length can be performed.

[00338] In some embodiments, the NLS sequences are covalently linked (e.g., to the Cas12 protein, to another NLS sequence(s), or to a fusion protein sequence linked to the Cas12 protein) via a linker sequence comprising one or more amino acids. In some embodiments, the linker sequence comprises at least one glycine, serine, and/or threonine residue. In some embodiments, the linker sequence comprises at least one glycine residue and at least one serine residue. In some embodiments, the linker sequence comprises a plurality of glycine residues and at least one serine residue. In some embodiments, the linker sequence consists of or comprises the sequence GS. In some embodiments, the linker sequence consists of or comprises the sequence GGGGS. In some embodiments, the linker sequence consists of or comprises the sequence GGGGSGGGGS.

[00339] In some embodiments, the Cas12a protein comprises at least one linker sequence and at least one NLS sequence at the C-terminus. In some embodiments, the at least one NLS sequence is selected from SV40 large T antigen and nucleoplasmin or from sequences that have been modified or that are derived from these sequences.

[00340] In some embodiments of the invention, the Cas12a protein comprises a linker sequence GGGGSGGGGS and a nucleoplasmin NLS sequence at the C-terminus, wherein the nucleoplasmin NLS sequence is located at the C-terminal side of the linker sequence GGGGSGGGGS.

[00341] In some embodiments, the Cas12a protein comprises at least one GS linker sequence, an SV40 large T antigen NLS sequence, and a nucleoplasmin NLS sequence at the C-terminus, wherein the nucleoplasmin NLS sequence is located at the C-terminal side of the SV40 large T antigen NLS sequence. In some embodiments, the first GS linker sequence is present at the N-terminal side of the SV40 large T antigen NLS sequence, and the second GS linker sequence is present between the SV40 large T antigen NLS sequence and the nucleoplasmin NLS sequence.

[00342] In some embodiments, the Cas12a protein comprises one GS linker sequence, a GGGGSGGGGS linker sequence, an SV40 large T antigen NLS sequence, and a nucleoplasmin NLS sequence at the C-terminus, wherein the nucleoplasmin NLS sequence is located at the C-terminal side of the SV40 large T antigen NLS sequence. In some embodiments, the GGGGSGGGGS linker sequence is present at the N-terminal side of the SV40 large T antigen NLS sequence, and the GS linker sequence is present between the SV40 large T antigen NLS sequence and the nucleoplasmin NLS sequence.

[00343] In some embodiments of the invention, the Cas12a protein comprises a linker sequence GGGGSGGGGS and an SV40 large T antigen NLS sequence at the C-terminus, wherein the SV40 large T antigen NLS sequence is located at the C-terminal side of the linker sequence GGGGSGGGGS.

[00344] In some embodiments, the Cas12a protein comprises a sequence comprising a linker and an NLS at the C-terminus. In some embodiments, the sequence comprising a linker and an NLS comprises or consists of an amino acid sequence selected from SEQ ID NOs: 479-490. In some embodiments, the sequence comprising a linker and an NLS comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 479-490.

[00345] Related Cas12a chRDNA guide/nucleoprotein complexes can also be prepared using methods well known to those skilled in the art. The Cas12 protein components can be produced recombinantly, and then the Cas12 chRDNA guide molecules and Cas12 proteins can be combined into complexes using methods known to those skilled in the art. See, for example, Example 2 for a non-limiting example of a method for assembling nucleoprotein complexes containing a guide molecule/Cas12 protein.

[00345] In addition, cell lines can be developed that constitutively express Cas12 proteins, which can be transfected with Cas12 chRDNA targeting components, and these complexes can be purified from the cells using standard purification methods, such as, but not limited to, affinity, ion exchange, and size exclusion chromatography. See, for example, Jinek et al. ( Science , 2012, 337:816-821).

[00347] According to known methods, Cas12 proteins can be produced using expression cassettes encoding the Cas12 protein. Expression cassettes typically contain regulatory sequences that are functional in the host cells into which they are introduced. The regulatory sequences are involved in one or more of the following processes: transcriptional regulation, post-transcriptional regulation, and translational regulation. Expression cassettes can be present in expression vectors and can be introduced into a wide range of host cells, including bacterial cells, yeast cells, plant cells, and mammalian cells.

[00348] Cas12 proteins can be produced in vectors, including expression vectors, containing polynucleotides encoding Cas12 proteins. Vectors suitable for producing Cas12 proteins include plasmids, viruses (including phage), and integrable nucleic acid fragments (i.e., fragments that integrate into the host genome through homologous recombination). The vector replicates and functions independently of the host genome or may, in some cases, integrate into the genome itself. Suitable replicating vectors will contain a replicon and regulatory sequences derived from a species compatible with the intended host cell expression. In some embodiments, a polynucleotide encoding a Cas12 protein is operably linked to an inducible promoter, a repressible promoter, or a constitutive promoter. Expression vectors may also include polynucleotides encoding protein tags (e.g., polyhistidine tags, hemagglutinin tags, fluorescent protein tags, bioluminescent tags, nuclear localization tags). The coding sequences for such protein tags may be fused to the coding sequence, or they may be included in an expression cassette, such as a targeting vector.

[00349] General methods for constructing expression vectors are known to those skilled in the art. Expression vectors for most host cells are commercially available. Several commercial software programs exist that have been developed to facilitate the selection of suitable vectors and their construction, such as insect cell vectors for transforming insect cells and expressing genes in insect cells, bacterial plasmids for transforming bacteria and expressing genes in bacterial cells, yeast plasmids for transforming cells and expressing genes in yeast and other fungi, mammalian vectors for transforming mammalian cells and expressing genes in mammalian cells or in mammals themselves, viral vectors (including retroviral, lentiviral, and adenoviral vectors) for transforming cells as well as for expressing genes; and methods for easily cloning such polynucleotides. SnapGene™ (GSL Biotech LLC, Chicago, II1; snapgene.com/resources/plasmid_files/your_time_is_valuable/), for example, provides an extensive list of vectors, individual vector sequences, and vector maps, as well as commercial sources for many vectors. A large number of mammalian vectors suitable for use are commercially available (e.g., from Life Technologies, Grand Island, NY; NeoBiolab, Cambridge, MA; Promega, Madison, WI; ATUM, Menlo Park, CA; Addgene, Cambridge, MA).

[00350] Vectors derived from mammalian viruses can also be used to express Cas12 protein components by the methods of the invention in mammalian cells. These include vectors derived from viruses such as adenovirus, adeno-associated virus, parvovirus, herpes virus, polyomavirus, cytomegalovirus, lentivirus, retrovirus, vaccinia virus, and simian virus 40 (SV40). See, for example, Kaufman et al . ( Mol. Biotech ., 2000, 16:151-160); and Cooray et al. ( Methods Enzymol ., 2012, 507:29-57). Regulatory sequences operably linked to the Cas12 protein encoding sequences may include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loops, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like. Commonly used promoters are the mammalian constitutive promoters CMV, MND, EF1a, SV40, PGK1 (mouse or human), Ubc, CAG, CAMKIIa, and beta-Act, and other promoters known to those skilled in the art. See, for example, Khan et al. ( Advanced Pharmaceutical Bulletin , 2013, 3: 257-263). In addition, mammalian RNA polymerase III promoters, including H1 and U6, may be used.

[00351] Numerous mammalian cell lines have been used to express gene products, including HEK 293 (human embryonic kidney cells) and CHO (Chinese hamster ovary cells). These cell lines can be transfected using standard methods (e.g., using calcium phosphate or polyethyleneimine (PEI), or by electroporation). Other exemplary mammalian cell lines include, but are not limited to, HeLa, U2OS, 549, HT1080, CAD, P19, NIH 3T3, L929, N2a, human embryonic kidney 293 cells, MCF-7, Y79, SO-Rb50, Hep G2, DUKX-X11, J558L, and baby hamster kidney (BHK) cells. Such cells are examples of cells that can be used to produce Cas12 proteins.

[00352] Vectors can be introduced into and propagated by a prokaryote. Prokaryotic vectors are well known to those skilled in the art. Typically, a prokaryotic vector contains an origin of replication appropriate for the target host cell (e.g., oriC derived from E. Coli ; pUC derived from pBR322; pSC101 derived from Salmonella); origin 15A (derived from p15A); and bacterial artificial chromosomes). Vectors can include a selectable marker (e.g., genes encoding resistance to ampicillin, chloramphenicol, gentamicin, and kanamycin). Zeocin™ (Life Technologies, Grand Island, NY) can be used to select bacteria, fungi (including yeast), plants, and mammalian cell lines. Accordingly, vectors can be constructed that carry only a single Zeocin™ drug resistance gene for selection purposes in a variety of organisms. Suitable promoters for protein expression in prokaryotes are known in the art, such as T5, T7, rhamnose (inducible), arabinose (inducible), and phoA (inducible). In addition, T7 promoters are widely used in vectors that also encode T7 RNA polymerase. Prokaryotic vectors can also include ribosome binding sites of varying strength and secretion signals (e.g., mal, sec, tat, OmpC, and pelB). In addition, vectors can contain RNA polymerase promoters for expression of NATNA. Prokaryotic RNA polymerase transcription termination sequences are also well known (e.g., transcription termination sequences from S. pyogenes ).

[00353] Expression of proteins in prokaryotes is often accomplished in E. coli using vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion proteins. However, protein expression using other prokaryotic systems is within the scope of the present invention.

[00354] In some embodiments, the vector is a yeast expression vector. Examples of Saccharomyces cerivisiae expression vectors include, but are not limited to, pYepSec1, pMFa, pJRY88, pYES2, and picZ. Methods for expressing genes in yeast cells are known to those skilled in the art. See, for example, Christine Guthrie and Gerald R. Fink ("Guide to Yeast Genetics and Molecular and Cell Biology, Part A" in Methods in Enzymology, 2004, Volume 194, Elsevier Academic Press, San Diego, CA). Generally, expression of protein-encoding genes in yeast requires a promoter operably linked to the coding region of interest and a transcription terminator. Various yeast promoters can be used to construct expression cassettes for gene expression in yeast.

[00355] Genome editing of cells using Cas12a guide chRDNA/nucleoprotein complexes

[00355] Delivery of the Cas12 CHRDNA targeting molecules, Cas12 proteins, and Cas12 chRDNA targeting/nucleoprotein complexes of the invention into cells, in vitro, ex vivo , or in vivo , can be achieved using a variety of methods known to those skilled in the art. Non-limiting methods for introducing these components into a cell include viral vector delivery, sonoporation, cell squeezing, electroporation, nucleofection, lipofection, shotgun particle spray technology, microjet bombardment, or treatment with chemical reagents (e.g., cell-penetrating peptides).

[00357] In some embodiments, electroporation can be used to deliver the Cas12 chRDNA guide molecules of the invention into cells. Electroporation can also be used to deliver the Cas12 guide chRDNA/nucleoprotein complexes of the invention. In these methods, the chRDNA guide molecules or the Cas12 guide chRDNA/nucleoprotein complexes are mixed in an electroporation buffer with the target cells to form a suspension. The suspension is then exposed to an electrical pulse with an optimized voltage that creates transient pores in the phospholipid bilayer of the cell membrane, allowing charged molecules (such as nucleic acids and proteins) to pass through the pores and enter the cell. Reagents and equipment for performing electroporation are commercially available.

[00358] Example 3 illustrates nucleofection of activated T cells with Cas12 targeting chRDNA/nucleoprotein complexes. Example 5 illustrates nucleofection of activated T cells with Cas12 targeting chRDNA/nucleoprotein complexes.

[00359] Cas12 guide chRDNA/nucleoprotein complexes can be used to cleave or bind to a target nucleic acid. A Cas12 guide chRDNA molecule can be introduced into cells with the Cas12 protein, which will result in the formation of a Cas12 guide chRDNA/nucleoprotein complex. The Cas12 guide chRDNA/nucleoprotein complex can hybridize to a target nucleic acid, wherein the target nucleic acid contains a PAM. In one embodiment, the present invention encompasses a method of linking a target nucleic acid sequence in a polynucleotide (e.g., double-stranded DNA (dsDNA)), wherein the method comprises providing one or more Cas12 guide chRDNA/nucleoprotein complexes for introduction into a cell and delivering the Cas12-nucleoprotein complex(es) into the cell, thereby facilitating contact of the Cas12 guide chRDNA/nucleoprotein complex(es) with the target polynucleotide sequence. In one embodiment, the first Cas12 guide chRDNA/nucleoprotein complex comprises a Cas12 guide CHRDNA molecule having a first targeting region element complementary to a first target nucleic acid sequence in the polynucleotide; and the second Cas12 guide chRDNA/nucleoprotein complex comprises a Cas12 guide chRDNA molecule having a second targeting region complementary to a second nucleic acid target sequence in the polynucleotide. Contacting the Cas12 guide chRDNA/nucleoprotein complexes with the polynucleotide results in binding of the Cas12 guide chRDNA/nucleoprotein complexes to the target nucleic acid sequences in the polynucleotide. In one embodiment of the invention, the first Cas12 guide chRDNA/nucleoprotein complex binds to the first nucleic acid target sequence; and the second Cas12 guide chRDNA/nucleoprotein complex binds to the second nucleic acid target sequence in the polynucleotide.

[00360] Such methods of binding a target nucleic acid sequence may be performed in vitro (e.g., in a biochemical reaction or in cultured cells; in some embodiments, the cultured cells are cultured human cells that are maintained in culture and are not introduced into a human); in vivo (e.g., in cells of a living organism, provided that in some embodiments, the organism is a non-human organism); or ex vivo (e.g., in cells taken from an individual, provided that in some embodiments, the individual is not a human).

[00361] Delivery of the Cas12 chRDNA targeting molecules, Cas12 proteins, and Cas12 chRDNA targeting/nucleoprotein complexes of the invention can be achieved by packaging the components into a biological compartment. The biological compartment containing these components can be administered in vivo (e.g., in cells of a living organism, provided that in some embodiments of the invention, such organism is not a human organism). Biological compartments can include, but are not limited to, viruses (lentivirus, adenovirus), nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vesicles, polyethylene glycol particles, hydrogels, and micelles.

[00362] For example, the biological compartment may comprise a liposome. The liposome may be a self-assembling structure comprising one or more lipid bilayers, each of which may comprise two monolayers comprising oppositely oriented amphipathic lipid molecules. The amphipathic lipids may comprise a polar (hydrophilic) head group covalently linked to one or two or more non-polar (hydrophobic) acyl or alkyl chains. Energetically mismatched contacts between the hydrophobic acyl chains and the surrounding aqueous environment induce self-assembly of the amphipathic lipid molecules, as a result of which the polar head groups may be oriented toward the surface of the bilayer, and the acyl chains may be oriented toward the interior of the bilayer, which allows for the acyl chains to be effectively protected from their contacts with the aqueous environment.

[00363] Examples of preferred amphipathic compounds used in liposomes can be phosphoglycerides and sphingolipids, representative examples of which are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyloleolylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, egg sphingomyelin, or any combinations thereof.

[00364] The biological compartment may contain a nanoparticle. The nanoparticle may have a diameter of about 40 nanometers to about 1.5 micrometers, about 50 nanometers to about 1.2 micrometers, about 60 nanometers to about 1 micrometer, about 70 nanometers to about 800 nanometers, about 80 nanometers to about 600 nanometers, about 90 nanometers to about 400 nanometers, or about 100 nanometers to about 200 nanometers. In some cases, as the size of the nanoparticles increases, the release rate may be slower or prolonged, and as the size of the nanoparticles decreases, the release rate may be increased.

[00365] In some embodiments, the Cas12 guide chRDNA/nucleoprotein complexes are packaged in a biological compartment. In some cases, the nucleic acid encoding Cas12 and the chemically synthesized guide chRDNA are packaged in a biological compartment. In some cases, the mRNA encoding Cas12 and the chemically synthesized guide chRDNA are packaged in a biological compartment.

[00365] Those skilled in the art are familiar with a variety of methods for assessing and/or quantifying interactions between nucleic acid sequences and polypeptides, including, but not limited to, the following methods: immunoprecipitation assays (ChIP), DNA electrophoretic mobility shift assays (EMSA), DNA release assays, and microplate capture and detection assays. Commercially available kits, materials, and reagents are suitable for use in many of these methods and, for example, can be purchased from the following suppliers: Thermo Scientific (Wilmington, DE), Signosis (Santa Clara, CA), Bio-Rad (Hercules, CA), and Promega (Madison, WI). A general approach to detecting interactions between a polypeptide and a nucleic acid sequence is EMSA (see, e.g., Hellman L. M., et al., Nature Protocols 2(8):1849–1861 (2007)).

[00367] In another embodiment, the invention provides a method of cleaving a nucleic acid target sequence in a polynucleotide (e.g., single-stranded cleavage in dsDNA or double-stranded cleavage in dsDNA), the method comprising providing one or more Cas12 guide chRDNA/nucleoprotein complexes for introduction into a cell and delivering the Cas12 guide chRDNA/nucleoprotein complex(es) into the cell to facilitate contacting of the Cas12 guide chRDNA/nucleoprotein complex(es) with the polynucleotide. In one embodiment, the first Cas12 guide chRDNA/nucleoprotein complex comprises a first Cas12 guide CHRDNA molecule having a first targeting region complementary to a first nucleic acid target sequence in the polynucleotide; and the second Cas12 guide chRDNA/nucleoprotein complex comprises a second Cas12 guide chRDNA molecule having a second targeting region complementary to a second target nucleic acid sequence in the polynucleotide, and these complexes are introduced into the cell. The contacting results in cleavage of the target nucleic acid sequence in the polynucleotide (e.g., dsDNA) by the Cas12 guide chRDNA/nucleoprotein complex(es). In one embodiment, the first Cas12 guide chRDNA/nucleoprotein complex binds to the first target nucleic acid sequence in the dsDNA and cleaves the first strand of the dsDNA; and the second Cas12 guide chRDNA/nucleoprotein complex binds to the second target nucleic acid sequence in the dsDNA and cleaves the second strand of the dsDNA. In some embodiments, the target nucleic acid sequence is DNA or genomic DNA. Such methods of binding a target nucleic acid sequence are carried out in vitro , in a cell (e.g., in cultured cells), ex vivo (e.g., in stem cells taken from an individual), and in vivo .

[00368] The target nucleic acid sequence(s) may be appropriately selected based on, for example, a desired location in a polynucleotide sequence or in a genome; and/or a desired gene sequence in a polynucleotide sequence or in a genome to be deleted or disrupted.

[00369] In a further embodiment of cleaving a target nucleic acid sequence in a polynucleotide, a donor polynucleotide can also be introduced into a cell to facilitate incorporation of at least a portion of the donor polynucleotide into the genomic DNA of the cell. Typically, the donor polynucleotide is introduced in close proximity to a site-directed break in the target nucleic acid to improve insertion (e.g., homologous recombination) of the donor polynucleotide into the site of the double-strand break. In some cases, the donor polynucleotide is introduced in close proximity to the site of the double-strand break in the target nucleic acid by linking it to a Cas12 protein that generates a double-strand break (e.g., Cas12a).

[00370] The donor sequence(s) of the target nucleic acids may be selected appropriately based on, for example, the desired modification. For example, the donor polynucleotide may encode all or part of the protein of interest. In some embodiments, the donor polynucleotide may encode a CAR.

[00371] The present invention also encompasses delivery of a donor polynucleotide into a cell via a virus, wherein the donor polynucleotide encodes a CAR.

[00372] In some embodiments, the donor polynucleotide may be single-stranded. In some embodiments, the donor polynucleotide may be double-stranded. In some embodiments, the donor DNA may be a minicircle. In some embodiments, the donor polynucleotide may be a plasmid. In some embodiments, the plasmid may be supercoiled. In some embodiments, the donor polynucleotide may be methylated. In some embodiments, the donor polynucleotide may be unmethylated. The donor polynucleotide may comprise a modification. Modifications may include modifications described herein, including, but not limited to, biotinylation, chemical conjugation, and the introduction of synthetic nucleotides.

[00373] Therapeutic compositions, uses and methods

[00374] The chRDNA Cas12 targeting molecules and the chRDNA Cas12 targeting/nucleoprotein complexes of the invention can be used to produce modified cells (such as CAR-expressing cells). Such modified cells can be used, for example, in the field of cell therapy (e.g., for the treatment or prevention of a disease by administering cells), and especially for adoptive cell therapy. Such administered cells can be, for example, genetically modified adoptive cells. Genetic modifications can be introduced into the adoptive cells via the chRDNA Cas12 targeting molecules and the chRDNA Cas12 targeting/nucleoprotein complexes described herein using one or more delivery methods. The present invention encompasses, for example, the modification and administration of cells that are autologous or allogeneic to the recipient to which they are to be administered. As used herein, the term "allogeneic" refers to another, genetically non-identical individual of the same species. Thus, for example, an allogeneic cell refers to a cell obtained from a different, genetically non-identical individual of the same species (relative to the recipient into which the cell is injected). In contrast, the term "autologous" refers to the same individual. Thus, for example, an autologous cell injected into an individual refers to a cell (modified or unmodified, or its modified or unmodified progeny) that is derived from the same individual.

[00375] "Adoptive cell" means a cell that can be genetically modified for use in cell therapy. Adoptive cells include, but are not limited to, stem cells, induced pluripotent stem cells, embryonic stem cells, cord blood stem cells, lymphocytes, natural killer cells, fibroblasts, endothelial cells, epithelial cells, pancreatic progenitor cells, and the like.

[00375] "Stem cell" refers to a cell that has the ability to self-renew, i.e., the ability to undergo multiple cycles of cell division while maintaining an undifferentiated state. Stem cells may be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells include embryonic cells, fetal cells, amniotic cells, adult cells, or induced pluripotent stem cells.

[00377] An "induced pluripotent stem cell" (iPSC) refers to a type of pluripotent stem cell that has been artificially derived from a non-pluripotent cell, typically a somatic cell. In some embodiments, the somatic cell is a human somatic cell. Examples of somatic cells include, but are not limited to, skin fibroblasts, bone marrow mesenchymal cells, cardiac muscle cells, keratinocytes, liver cells, gastric cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Additional examples of somatic cells include immune system cells, including but not limited to B cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes/macrophages, myeloid-derived suppressor cells, NK cells, T cells, thymocytes, and hematopoietic stem cells. Pluripotent stem cells can differentiate into a variety of cell types, including somatic cells, NK cells, NK-like cells, T cells, T-cell-like cells, NK T cells, NK T cell-like cells, dendritic cells, dendritic cell-like cells, macrophages, and macrophage-like cells. Pluripotent stem cells can be edited before or after differentiation using the Cas12 chRDNA/nucleoprotein guide complex. iPSCs can be further modified before or after differentiation by introducing into the genome an exogenous gene or sequence, such as a CAR encoding sequence.

[00378] "Hematopoietic stem cell" refers to an undifferentiated cell that has the capacity to differentiate into a hematopoietic cell, such as a lymphocyte.

[00379] "Lymphocyte" refers to a leukocyte (white blood cell) that is part of the vertebrate immune system. The term "lymphocyte" also encompasses the hematopoietic stem cell, which gives rise to lymphoid cells. Lymphocytes include T cells for cell-mediated, cytotoxic adaptive immunity, such as cytotoxic CD4 ⁺ and/or CD8 ⁺ T cells; alpha/beta T cells and gamma/delta T cells; regulatory T cells such as Treg cells; natural killer (NK) cells, which are involved in cell-mediated, cytotoxic innate immunity; and B cells for antibody-regulated humoral adaptive immunity; NK/T cells; cytokine-inducible killer (CIK) cells; and antigen-presenting cells (APCs), such as dendritic cells. The lymphocyte may be a mammalian cell, such as a human cell.

[00380] As used herein, the term "lymphocyte" also includes T cell receptor (TCR) engineered T cells, which cells have been genetically engineered to express one or more specific, naturally occurring or engineered T cell receptors that can recognize protein or (glyco)lipid antigens of target cells. Small fragments of these antigens, such as peptides or fatty acids, are delivered to the surface of the target cell and presented to the T cell receptors as part of the major histocompatibility complex (MHC). Binding of the T cell receptor to the antigen-loaded MHC activates the lymphocyte.

[00391] Tumor-infiltrating lymphocytes (TILs) are also included within the scope of the term "lymphocyte" as used herein. TILs are immune cells that infiltrate the environment in and around a tumor (the "tumor microenvironment"). TILs are typically isolated from tumor cells and the tumor microenvironment and selected in vitro for high reactivity against tumor antigens. TILs are cultured in vitro under conditions that overcome the tolerant effects observed in vivo and then administered to an individual for treatment.

[00382] The term "lymphocyte" also includes genetically modified T cells and NK cells, such as cells modified to produce chimeric antigen receptors (CARs) on the surface of T or NK cells (CAR-T cells and CAR-NK cells).

[00383] Lymphocytes can be isolated from an individual, such as a human, for example, from blood or from solid tumors, such as in the case of TILs, or from lymphoid organs, such as the thymus, bone marrow, lymph nodes, and mucosal-associated lymphoid tissues. Methods for isolating lymphocytes are well known to those skilled in the art. For example, lymphocytes can be isolated from peripheral blood mononuclear cells (PBMCs), which are separated from whole blood using, for example, Ficoll, a hydrophilic polysaccharide that separates blood layers, and density gradient centrifugation. Typically, anticoagulated or defibrinized blood samples are applied to the Ficoll solution and centrifuged to form different layers of cells. The bottom layer includes red blood cells (erythrocytes), which are collected or aggregated by the Ficoll medium and completely settle to the bottom. The next layer contains mainly granulocytes, which also migrate downward through the Ficoll-Paque solution. The next layer includes lymphocytes, which are usually found at the interface between plasma and Ficoll solution, along with monocytes and platelets. To isolate the lymphocytes, this layer is removed, washed with saline to remove platelets, Ficoll, and plasma, and then centrifuged again.

[00384] Other methods for isolating lymphocytes include biopanning, which allows cell populations to be isolated from a solution by binding cells of interest to antibody-coated plastic surfaces. Unwanted cells are then removed by treatment with specific antibodies and complement. In addition, fluorescence-activated cell sorting (FACS) analysis can be used to detect and count lymphocytes. FACS analysis uses a flow cytometer to separate labeled cells based on differences in light scattering and fluorescence.

[00385] In the case of TILs, lymphocytes are isolated from the tumor and cultured, for example, in high doses of IL-2 and selected using cytokine release co-culture assays, either using antibodies against the autologous tumor or against HLA-matched tumor cell lines. Cultures showing evidence of increased specific reactivity compared to an allogeneic MHC-mismatched control can be selected for rapid expansion and then administered to an individual for cancer treatment. See, for example, Rosenberg et al. ( Clin. Cancer Res ., 2011, 17:4550-4557); Dudly et al. ( Science , 2002, 298:850-854); Dudly et al. ( J. Clin. Oncol ., 2008, 26:5233-5239); and Dudly et al. ( J. Immunother ., 2003, 26:332-342).

[00385] Once isolated, lymphocytes can be characterized in terms of specificity, frequency, and function. Frequently used assays include the ELISPOT assay, which allows determination of the frequency of the T cell response.

[00387] Once isolated, the lymphocytes can be activated using methods well known to those skilled in the art to stimulate proliferation and differentiation into specialized effector lymphocytes. Surface markers for activated T cells include, for example, CD3, CD4, CD8, PD1, IL2R, etc. Activated cytotoxic lymphocytes can kill target cells after binding cognate receptors on the surface of the target cells. Surface markers for NK cells include, for example, CD16, CD56, etc.

[00388] After isolation and optional activation, the lymphocytes can be modified using the Cas12 chRDNA/nucleoprotein guide complex of the invention to perform adoptive T cell immunotherapy. Adoptive immunotherapy typically uses a patient's immune cells (autologous cells) to treat cancer. However, the methods for developing adoptive immunotherapy of the invention also allow the use of third-party donor cells (allogeneic cells), allowing for the development of "off-the-shelf" treatments.

[00389] Thus, in some embodiments of the invention, lymphocytes for use in adoptive immunotherapy are isolated from an individual, modified ex vivo , and then reintroduced into the same individual. This method is known as "autologous lymphocyte therapy."

[00390] Alternatively, lymphocytes can be isolated, modified ex vivo, and administered to another individual. This method is known as "allogeneic lymphocyte therapy."

[00391] In some embodiments, Cas12 guide chRDNA/nucleoprotein complexes are used to prepare therapeutic compositions comprising allogeneic cells. In a preferred embodiment, the allogeneic cells are T cells. In a more preferred embodiment, the T cells express a CAR. In an even more preferred embodiment, the CAR targets an antigen associated with cancer.

[00392] In some embodiments, T cells can be modified to provide safer and more effective allogeneic therapy. For example, the T cell receptor α constant (TRAC) is a protein-coding gene that forms part of the αβ TCR. Thus, selected mutations in TRAC, as well as silencing of TRAC expression, can help eliminate graft-versus-host disease during allogeneic cell therapy. See, e.g., Poirot et al. (Cancer Res., 2015, 75:3853-3864). It has been shown that delivery of a CD19-specific CAR to the TRAC locus using the CRISPR-Cas9 system can result in tumor rejection. See, e.g., Eyquem et al. ( Nature , 2017, 543:113). Similarly, the T cell receptor β constant (TRBC) can also be targeted to prevent αβ-TCR expression. See, e.g., Ren et al. ( Clin. Cancer Res ., 2017, 23:2255–2266).

[00393] Programmed cell death protein 1, also known as PD1, PDCD1, and CD279, is a cell surface receptor that plays an important role in suppressing the immune system and promoting self-tolerance by suppressing inflammatory T cell activity. PDCD1 binds to its cognate ligand, "programmed death ligand 1," also known as PD-L1, CD274, and B7 homolog 1 (B7-H1). PD1 protects against autoimmunity through a dual mechanism of promoting programmed cell death (apoptosis) in antigen-specific T cells in lymph nodes while reducing apoptosis in anti-inflammatory suppressor T cells (regulatory T cells). Through these mechanisms, PD1 binding to PD-L1 inhibits the immune system, thereby preventing autoimmune disorders, but at the same time, it prevents the immune system from destroying cancer cells. Accordingly, mutation or suppression of PD1 production may be useful in T-cell therapy.

[00392] PD1 is an example of an "immune checkpoint" molecule. Immune checkpoint molecules serve to suppress or inhibit the immune response. Immune checkpoint molecules include, but are not limited to, PD1, cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152), LAG3 (also known as CD223), Tim3 (also known as HAVCR2), BTLA (also known as CD272), BY55 (also known as CD160), TIGIT (also known as IVSTM3), LAIR1 (also known as CD305), SIGLEC10, 2B4 (also known as CD244), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7, SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3. In some embodiments, one or more immune checkpoint molecules are inactivated using the Cas12 guide chRDNA/nucleoprotein complexes of the invention. In some embodiments, the inactivation of one or more immune checkpoint molecules is combined with the inactivation of one or more TCR components, as described above.

[00395] Beta-2-microglobulin (B2M) is a component of MHC class I molecules present in nucleated cells. Beta-2-microglobulin is secreted by cells, including tumor cells, into the blood and is required for the assembly and expression of the HLA I complex. However, expression of HLA on the surface of allogeneic T cells causes rapid rejection by T cells of the host immune system. Thus, disruption of beta-2-microglobulin expression is also desirable to improve the efficacy of allogeneic T cell therapy. In addition, lack of expression of MHC class I molecules on allogeneic T cells causes clearance by the host immune system. Thus, presentation of only a subset of HLA molecules, preferably HLA-E, on the cell surface is desirable.

[00395] Additional genes can be similarly targeted using the Cas12 chRDNA targeting molecules described herein to improve the efficacy of adoptive immune cell therapy. Non-limiting examples of preferred genes and chromosomal locations (hg38 genome assembly) are provided in Table 4.

Table 4
Targets for adoptive cell therapy Encoded protein/Alternate name Locus coordinates TRAC chr14:22,509,239-22,552,153 TRBV proteins TRBV chr7 locus Beta-2-microglobulin (B2M) chr15:44,711,477-44,718,877 PDCD1/PD1/CD279 chr2:241,849,881-241,858,908 PD-L1 chr9:5,450,525-5,470,547 CTLA-4/CD152 chr2:203,867,786-203,873,960 LAG-3/CD223 chr12:6,772,520-6,778,453 TIGIT/IVSTM3 chr3:114,291,059-114,308,290 TIM3/HAVCR2 chr5:157,085,832-157,109,714 HLA-E HLA-E chr6 locus HLA-A HLA-A locus lchr6 ROR1 chr1:63,774,017-64,181,498 CD47 chr3:108,043,091-108,094,200 HLA-B HLA-B chr6 locus HLA-C HLA-C chr6 locus HLA-DRA HLA-DRA chr6 locus ADAM17 chr2:9,488,486-9,555,788 BTLA/CD272 chr3:112,463,968-112,499,561 P2X7 chr12:121,132,819-121,188,032 CD160 chr1:145,719,520-145,739,153 ADORA2A Chr22:24,417,879-24,442,357 SIGLEC10 chr19:51,411,054-51,417,698 2B4/CD244 chr1:160,830,160-160,862,855 LAIR1/CD305 chr19_KI270938v1_alt:337,398-347,586 CD52 chr1:26,317,957-26,320,523 CD96 chr3:111,542,118-111,652,241 VSIR or C10orf54/VISTA chr10:71,747,559-71,773,498 KIR2DL1 chr19_KI270933v1_alt:71,449-85,961 KIR2DL2 chr19_KI270932v1_alt:56.346-70.673 KIR2DL3 chr19:54,738,515-54,753,052 CEACAM1 chr19:42,507,306-42,528,509 CBLB chr3:105,655,461-105,869,043 CISH chr3:50,606,522-50,611,831 IL-1R8/TIR8/SIGIRR chr11:405,716-417,397 AHR chr7:17,298,622-17,346,150 Adenosine receptor 2A/ADORA2A chr22:24,423,597-24,442,356 GMCSF/CSF2 chr5:132,073,789-132,076,170 VISTA/VSIR chr10:71,747,556-71,773,520 CII2A chr16:10,877,198-10,936,388 NKG2A chr12:10,446,041-10,454,616

[00397] In some embodiments, the gene encoding TRAC is targeted internally using the Cas12 chRDNA guide described herein. In some embodiments, the gene encoding PD1 is targeted internally using the Cas12 chRDNA guide described herein. In some embodiments, the gene encoding B2M is targeted internally using the Cas12 chRDNA guide described herein. In some embodiments, the gene encoding TRAC and the gene encoding B2M are targeted internally using the Cas12 chRDNA guides described herein.

[00398] Cells modified using the Cas12 guide chRDNA/nucleoprotein complexes of the invention can be used, for example, in adoptive cell therapy to treat cancer. In some embodiments, the modified cell is a genetically modified lymphocyte. Such genetically modified lymphocytes, such as CAR-T cells, can be used to treat various types of cancer in an individual, including, but not limited to, prostate cancer; ovarian cancer; cervical cancer; colorectal cancer; bowel cancer; testicular cancer; skin cancer; lung cancer; thyroid cancer; bone cancer; breast cancer; bladder cancer; uterine cancer; vaginal cancer; pancreatic cancer; liver cancer; kidney cancer; brain cancer; spinal cord cancer; oral cancer; parotid tumors; blood cancer; lymphomas such as B-cell lymphomas; leukemias, and the like. Preferably, an effective amount of modified cells is used for such treatment.

[00399] Table 5 lists exemplary B-cell leukemias and lymphomas that are amenable to treatment using adoptive cells (such as CAR-T cells) generated using the Cas12 targeting chRDNA/nucleoprotein complexes of the invention. It should be noted that lymphocytes modified with the Cas12 targeting chRDNA/nucleoprotein complexes disclosed herein are used to treat diseases, a non-limiting list of which is provided below in Table 5.

Table 5
B-cell leukemias/lymphomas and their abbreviations B-cell leukemia/lymphoma Abbreviations Acute lymphoblastic leukemia B-ALL Acute myeloid leukemia AML B-cell precursor lymphoblastic leukemia LBL Reticuloendotheliosis HCL B-cell progenitor prolymphocytic leukemia B-PLL Chronic lymphocytic leukemia/small cell lymphocytic leukemia CLL/SLL Diffuse large B-cell lymphoma DLBCL ALK-positive large B-cell lymphoma ALK+LBCL Large B-cell lymphoma, T-cell/histiocyte-rich T/HRBCL Follicular lymphoma FL Cerebral cortex cell lymphoma MCL Burkitt's lymphoma (EBV) B.L. Multiple myeloma M.M. Lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia MG Lymphoma of the lymphoid tissue of the mucous membrane MALT Nodal marginal zone B-cell lymphoma NMZL Splenic marginal zone B-cell lymphoma SMZL Primary central nervous system lymphoma PCNSL Primary cutaneous follicular center lymphoma PCFCL Primary large B-cell lymphoma of the mediastinum PMLCL Intravascular large B-cell lymphoma ILBCL Lymphomatoid granulomatosis (EBV) LYG Plasmoblastic lymphoma (ARL) PBL Primary effusion lymphoma PEL

[00400] In other embodiments, other cell proliferative disorders can be treated using adoptive cells (such as CAR-T cells) generated using the Cas12-guided chRDNA/nucleoprotein complexes of the invention, including precancerous conditions; blood diseases; and immune disorders such as autoimmune disorders, including, but not limited to, Addison's disease, celiac disease, type 1 diabetes mellitus, Graves' disease, Hashimoto's disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, scleroderma, and systemic lupus erythematosus.

[00401] The adoptive cell therapy procedures described in this application may be combined at the same or different times with one or more additional therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormonal therapy, laser therapy, and radiation therapy.

[00402] Administration of the modified cells of the invention to individuals may be by any convenient route, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally.

[00403] In one embodiment, the modified cell compositions of the invention are preferably administered by intravenous injection. Administration may include administration of 10 ⁴ -10 ⁹ cells per kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight. The cells may be administered in one or more doses. In some embodiments, an effective amount of the modified cells is administered in a single dose. In other embodiments, an effective amount of the cells is administered in more than one dose over a period of time. Determining optimal ranges of effective amounts of a given cell type for treating a particular disease or condition is within the skill of the art.

[00404] Chimeric antigen receptor (CAR) cells

[00405] In some embodiments, the adoptive cell is a CAR-expressing cell. The CAR is a receptor engineered to recognize and bind to a specific antigen or epitope. The receptor is chimeric in that it has both antigen-binding and T-cell activating functions. The CAR is typically a fusion protein comprising an extracellular ligand-binding domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular signaling domain. The extracellular ligand-binding domain may comprise a single-chain variable fragment (scFv) comprising a fusion of two or more variable regions joined by one or more linkers. The CAR may further comprise a hinge region. A CAR is sometimes referred to as a "chimeric receptor," a "T body," or a "chimeric immune receptor (CIR)."

[00405] In some embodiments of the invention, the CAR may be a TRUCK, a universal CAR, a self-guided CAR, a TanCAR, a reinforced CAR, a self-destructive CAR, a conditional CAR, a labeled CAR, a TenCAR, a dual CAR, or an sCAR.

[00407] TRUCK (T cells retargeted for universal cytokine killing) co-express a chimeric antigen receptor (CAR) and an antitumor cytokine. Cytokine expression can be constitutive or induced by T cell activation. CAR specificity targeting and localized production of proinflammatory cytokines recruits endogenous immune cells to tumor sites and may enhance the antitumor response.

[00408] Universal allogeneic CAR-T cells are engineered to no longer express endogenous T cell receptor (TCR) and/or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[00409] Self-homing CARs co-express the CAR and a chemokine receptor that binds to a tumor ligand, resulting in enhanced tumor homing.

[00410] CAR T cells engineered to be resistant to immunosuppression (reinforced CARs) can be genetically modified so that they no longer express various immune checkpoint molecules (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)) as well as the immune checkpoint switch receptor, or they can be administered with a monoclonal antibody that blocks immune checkpoint signaling.

[00411] A self-destructive CAR can be engineered using RNA delivered via electroporation to encode the CAR. Alternatively, inducible T cell apoptosis can be achieved based on ganciclovir binding to thymidine kinase in genetically modified lymphocytes or based on the more recently described human caspase 9 activation system by a small molecule dimerizer.

[00412] A conditional CAR-T cell is inherently insensitive or "turned off" until the addition of a small molecule completes the reaction cycle, which will ensure full transduction of both signal 1 and signal 2, thereby activating the CAR-T cell. Alternatively, T cells can be engineered to express an adaptor-specific receptor that has affinity for subsequently administered second antibodies directed to the target antigen.

[00413] The labeled CAR-T cells express a CAR and a tumor epitope to which an agent, namely a monoclonal antibody, binds. If persistent side effects occur, administration of the monoclonal antibody results in clearance of the CAR-T cells and alleviation of symptoms without any additional extratumoral effects.

[00414] A tandem CAR (TanCAR) T cell expresses a single CAR comprising two linked scFvs that have different affinities and are linked to one or more intracellular costimulatory domains and the signaling domain of CD3ζ. Activation of TanCAR T cells requires the presence of only one antigen on target cells, however, the presence of both antigens facilitates synergistic activation. In some embodiments, the TanCAR scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), a pair of two heavy chain variable regions (VH), or a pair of two light chain variable regions (VL). In another embodiment, the two TanCAR scFvs can be in a stacked configuration. In another embodiment, the two TanCAR scFvs can be arranged in a series or looped configuration. In specific embodiments of the invention, at least one of the scFvs of the tandem CAR is an anti-CD20 scFv and the second scFv is selected to target a specific antigen on cancer cells, such as an anti-BCMA scFv, an anti-CD19 scFv, an anti-CD30 scFv, an anti-CD22 scFv, an anti-CD70 scFv, an anti-ROR1 scFv, or an anti-kappa light chain scFv.

[00415] A dual CAR-T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the CD3ζ domain and the other CAR includes only the costimulatory domain(s). Activation of dual CAR-T cells requires co-expression of both targets on the tumor.

[00415] A safe CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain, whereby sCAR T cells co-expressing the standard CAR are activated only upon binding to target cells that have a standard CAR target but do not have a sCAR target.

[00417] The extracellular (antigen recognition) domain of the CAR is preferably a single chain antibody, and more preferably an scFv. In one embodiment of the invention, the antigen binding domain comprises an scFv. However, any suitable fragment that binds to a given target with high affinity can be used as the antigen recognition region. The extracellular domain of the CAR capable of binding to an antigen can be, for example, any oligopeptide or polypeptide that can bind to a particular antigen.

[00418] Depending on the desired antigen used as a target, the CAR of the invention can be designed to include an appropriate antigen-binding moiety that is specific for the desired target antigen. For example, if BCMA is the desired antigen to serve as a target, an antibody or antibody fragment (e.g., an scFv) targeting BCMA can be used as the antigen-binding moiety for inclusion in the CAR of the invention.

[00419] Preferred cellular targets and scFvs/CAR binding proteins that target these targets are presented below in Table 6.

Table 6
List of representative cellular targets and anti-CAR scFvs Cellular target scFv/part vs CAR B7-H3 against B7-H3 B7-H6 vs B7-H6 B-cell maturation antigen (BCMA) against BCMA CD123 against CD123 CD138 against CD138 CD171/L1CAM vs CD171 CD19 against CD19 CD20 vs CD20 CD22 against CD22 CD30 vs CD30 CD33 against CD33 CD37 against CD37 CD38 against CD38 CD4 against CD4 CD47 against CD47 CD70 vs CD70 CD73 against CD73 CD79b against CD79b CD371 vs CD371 CEA against CEA Claudine 18.1 vs CLDN 18.1 Claudine 18.2 vs CLDN 18.2 CS-1 vs CS1 CSPG4 vs CSPG4 EFGRvIII against EFGRvIII ENPP3 against ENPP3 EpCAM against EpCAM EphA2 against EphA2 Epidermal growth factor receptor against epidermal growth factor receptor ErbB against ErbB ErbB2 (HER2) against ErbB2 (HER2) FAP against FAP FRα against FRα GD2 vs GD2 GD3 vs GD3 Glypican 3 anti-glypican 3 Her2 vs Her2 IL-11Rα against IL-11Rα IL-13Rα2 against IL-13Rα2 IL13 alpha receptor IL13 Kappa Light Chain Against light chain kappa LewisY/LeY Against LeY Mesothelin against mesothelin MUC1 against MUC1 MUC16 against MUC16 NGFR against NGFR NKG2D ligands against NKG2D ligands PD1 against PD1 PD-L1 against PD-L1 P.S.C.A. against PSCA PSMA against PSMA ROR-1 vs ROR-1 SLAMF7 vs SLAMF7 TACI against TACI TAG72 vs TAG72 ULBP and MICA/B proteins NKG2D VEGF2 against VEGF2 VEGFR2 against VEGFR2 WT1 vs WT1

[00420] In certain embodiments of the invention, the cellular target to which the CAR binds is more preferably selected from BCMA, CD19, CD20, CD22, CD47, CD79b, CD371, ROR-1, EphA2, MUC16, glypican 3, PSCA, and claudin 18.2.

[00421] In an even more preferred embodiment of the invention, the cellular target to which the CAR binds is BCMA.

[00422] In an even more preferred embodiment of the invention, the cellular target to which the CAR binds is CD371.

[00423] The intracellular domain of the CAR may be an oligopeptide or polypeptide that is known to function as a domain that transmits a signal that causes activation or inhibition of a biological process in a cell. The intracellular domain may comprise an activation domain comprising all or part of an intracellular signaling domain of a T cell receptor (TCR) and/or a coreceptor, so long as it transmits a signal to an effector function. Cytoplasmic signaling sequences that regulate the primary activation of the TCR complex, which acts in a stimulatory manner, may comprise signaling motifs that are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAMs containing cytoplasmic signal sequences include those derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (FcyRIIa), DAP10, DAP12, CD79a, CD79b, FcyRIγ, FcyRIIIγ, FceRIβ (FCERIB), and FceRIγ (FCERIG).

[00424] In preferred embodiments of the invention, the activation domain of the intracellular signaling domain is derived from CD3ζ.

[00425] The intracellular signaling domain of a CAR of the invention may be engineered to comprise an activation domain, such as a CD3ζ signaling domain, either alone or in combination with any other desired cytoplasmic domain(s) suitable for the CAR of the invention. For example, the intracellular signaling domain of a CAR may comprise an activation domain, such as a portion of the CD3ζ chain, in addition to a costimulatory domain. A costimulatory domain refers to the portion of a CAR that comprises the intracellular domain of a costimulatory molecule.

[00425] A costimulatory molecule is a molecule other than an antigen receptor or its ligands that is required for an effective lymphocyte response to an antigen. Examples of such costimulatory molecules, all or part of which may be used in the costimulatory domain of a CAR of the invention, include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS-1, GITR, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[00427] In preferred embodiments of the invention, the CAR comprises a costimulatory domain derived from at least 4-1BB.

[00428] The transmembrane domain may be obtained from either a natural or synthetic source. If the source is natural, the domain may be obtained from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e., comprise at least a portion of) the transmembrane region(s) of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3ζ, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR and PAG/Cbp.

[00429] Alternatively, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. A short oligo- or polypeptide linker, for example, 2 to 10 amino acids in length, may form a link between the transmembrane domain and the endoplasmic domain of the CAR.

[00430] In a preferred embodiment of the invention, the transmembrane domain is derived from CD8.

[00431] In some embodiments of the invention, a CAR has more than one transmembrane domain, which may be a repeat of the same transmembrane domain or may be different from the other transmembrane domains.

[00432] The hinge region may comprise a polypeptide hinge region of varying length, such as a region of one or more amino acids, a portion of CD8, or a portion of IgG4, and combinations thereof.

[00433] In a preferred embodiment of the invention, the hinge region is derived from CD8.

[00434] CARs can also be incorporated into TILs, NK cells, macrophages, dendritic cells, induced pluripotent stem cells (iPSCs), or TCRs, resulting in CAR-TILs, CAR-NK cells, CAR-M, CAR-DCs, and TCR-engineered CAR-T cells, respectively. A description of CAR-T cells, methods for producing CAR-T cells, and their uses can be found, for example, in Brudnoet al. (Nature Rev. Clin. Oncol., 2018, 15:31-46); Maudeet al.(N. Engl. J. Med., 2014, 371:1507-1517); and Sadelain et al. (Cancer Disc., 2013, 3:388-398).

[00435] In some embodiments of the invention, a CAR expression cassette is transduced into an adoptive cell and the cassette is integrated into a Cas12-mediated rupture site. Example 9 of the present application illustrates transduction of primary cells with an adeno-associated virus (AAV) vector containing a CAR cassette.

[00435] In some embodiments, the CAR expression cassette comprises a promoter for driving expression of the CAR. Commonly used promoters include the mammalian constitutive promoters CMV, MND, EF1a, SV40, PGK1 (mouse or human), Ubc, CAG, CAMKIIa, and beta-Act, and other promoters known to those of skill in the art. See, for example, Khan et al . ( Advanced Pharmaceutical Bulletin , 2013, 3:257-263). Alternatively, the CAR expression cassette can comprise a ribosome excision sequence (also called a self-cleaving peptide) and can be introduced in frame of an endogenously expressed gene. Commonly used ribosome excision sequences include T2A, P2A, E2A, and F2A. A description of ribosome excision sequences and their uses can be found, for example, in Chng et al. ( MAbs , 2015, 7(2):403-412). Similarly, non-CAR expression clusters may contain similar promoters or ribosome cutting sequences.

[00437] In some embodiments, the Cas12a guide chRDNA/nucleoprotein complexes are used to treat genetic diseases caused by pathogenic autosomal "dominant negative" mutations that are present on one allele in a patient. In some cases, the underlying genetic mutation may be a single nucleotide polymorphism (SNP) on one of the alleles. The Cas12a guide chRDNA/nucleoprotein complexes can be engineered to target the SNP allele but not the wild-type allele, resulting in the disruption of only the SNP allele. See, for example, Example 7, which describes a non-limiting example of a method for engineering Cas12a guide chRDNA/nucleoprotein complexes to target a wild-type sequence that are capable of reducing editing of off-target sequences containing SNPs.

[00438] In some embodiments, Cas12a guide chRDNA/nucleoprotein complexes can be used to selectively edit (e.g., knock out or revert to wild-type with homology-based repair) a SNP-containing allele without modifying the wild-type allele. In some embodiments, such editing can result in gene disruption. In other embodiments, such editing can restore the allele back to a "wild-type" state, such as through homology-based repair. For example, a number of genetic diseases that result in progressive vision loss are caused by pathogenic autosomal "dominant negative" mutations. Examples of SNP correction strategies for dominant negative disease include, but are not limited to, targeting SNP mutations in the rhodopsin gene that cause retinitis pigmentosa, see, e.g., Li et al. ( CRISPR J ., 2018, 1(1):55–64); targeting SNP mutations in the transforming growth factor beta-inducible (TGFBI) gene that cause corneal dystrophy, see, for example, Christie et al . ( Scientific Reports , 2017, 7(1):16–174).

[00439] The Cas12a chRDNA/nucleoprotein targeting complexes of the invention can be delivered, for example, to ocular tissues that are affected by autosomal, pathogenic "dominant negative" genetic mutations. In some embodiments, the Cas12a chRDNA/nucleoprotein targeting complex is designed to selectively disrupt a disease allele, but without targeting the wild-type allele, in order to treat the underlying pathology. Such diseases can include, but are not limited to, macular dystrophies, rod-cone dystrophies, cone-rod dystrophies, or chorioretinopathies. It is evident that the Cas12a chRDNA/nucleoprotein targeting complexes disclosed herein are designed to treat diseases that include, but are not limited to, genetic diseases that cause progressive vision loss.

[00440] Experimental part

Non-limiting embodiments of the invention are illustrated in the following Examples. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, concentrations, percentage changes, etc.), but some experimental errors and variations should be taken into account. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure. It should be noted that these Examples are provided for illustrative purposes only and are not intended to limit the scope of the various embodiments of the invention. Not all of the following steps set forth in each Example are required, and the order in which the steps are performed in each Example need not be as presented herein. The letter "r," when preceding a nucleotide, indicates RNA; all other nucleotides represent DNA (see, e.g., Tables 15 and 17), unless otherwise indicated. Phosphorothioate linkages are represented by "*" between adjacent bases.

Example 1

Obtaining cytotoxic T cells (CD4 ⁺ and CD8 ⁺ ) from PBMC and primary cell culture

[00441] This Example illustrates the production of CD4 ⁺ and CD8 ⁺ T cells from donor peripheral blood mononuclear cells (PBMCs).

[00442] CD4 ⁺ and CD8 ⁺ T cells were obtained from donor PBMCs essentially as described below. T cells were isolated from peripheral blood mononuclear cells (PBMCs) using RoboSep-S (STEMCELL Technologies, Cambridge, MA) and the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies, Cambridge, MA) and activated for 3 days in the presence of beads containing anti-CD3/CD28 antibodies (Dynabeads™; Gibco 11132D) in ImmunoCult-XF complete medium (ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies, Cambridge, MA), and in the presence of CTS SR immune cells (Gibco A2596102), and antibiotic-antifungal agents (100X, Corning 30-004-Cl)) supplemented with recombinant human (rh) IL-2 (100 units/mL). After 3 days, beads were removed by magnetic separation, and cells were expanded for 1 day in complete ImmunoCult-XF medium supplemented with IL-2 (100 units/ml).

Example 2

Cloning, expression, production and assembly of Cas12a chRDNA/nucleoprotein guide complexes

[00443] This Example describes a method for cloning, expressing, and purifying Cas12a guide chRDNA/nucleoprotein complexes, as well as methods for producing Cas12a guide components.

[00444] A. Cloning of Cas12 protein

[00445] The catalytically active sequence of the Cas12a protein from Acidaminococcus spp. (strain BV3L6) (SEQ ID NO:1) was codon optimized for expression in E. coli cells. A glycine-serine linker and one nuclear localization sequence (NLS) (SEQ ID NO:4) were added at the C-terminus. Oligonucleotide sequences encoding the Cas12a-NLS protein (referred to as the AsCas12a and Cas12a proteins in the following Examples) were provided to commercial manufacturers for synthesis. The DNA sequences were then cloned into suitable bacterial expression vectors using standard cloning techniques.

[00445] B. Expression and purification of Cas12a protein

[00447] The AsCas12a protein was expressed in E. coli using an expression vector and purified by affinity chromatography, ion exchange chromatography, and size exclusion chromatography essentially as described, for example, by Swarts et al. ( Molecular Cell , 2017, 66:221-233).

[00448] C. Production of Cas12a guide components

[00449] Cas12a guides were prepared by linking a targeting region to a specific activation region of Cas12a guides. The targeting region or spacer preferably comprises a 20-nucleotide target binding sequence. The target binding sequence was complementary to a target sequence that was downstream (in the 3' direction) of the 5'-TTTV or 5'-TTTN PAM. Representative sequences of the activation region of the Cas12a guide molecule are SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10 for Acidaminococcus spp., L. bacterium , and F. novicida Cas12a, respectively.

[00450] Cas 12a guide sequences (such as crRNA and chRDNA) were provided to a commercial manufacturer for synthesis.

[00451] Guide RNA components (such as crRNA) can be produced by in vitro transcription (e.g., using the T7 Rapid High Yield RNA Synthesis Kit; New England Biolabs, Ipswich, MA) from double-stranded (ds) DNA templates by incorporating a T7 promoter at the 5' end of the dsDNA template sequences.

[00452] D. Assembly of the Cas12a guide/nucleoprotein complex

[00453] Acidaminococcus spp. Cas12a (AsCas12a) tagged at the C-terminus with a nuclear localization sequence (NLS) was recombinantly expressed in E. coli and purified by chromatographic methods. Nucleoprotein complexes were formed at a concentration of 80 pmol Cas12a protein:240 pmol guide molecule, unless otherwise stated. Prior to assembly with Cas12a protein, each guide component (e.g., crRNA or chRDNA) was adjusted to the desired total concentration (240 pmol) in a final volume of 1 μl, incubated for 2 min at 95°C, removed from the thermowell, and allowed to equilibrate to room temperature. Cas12a protein was diluted to the appropriate concentration in binding buffer (60 mM Tris-acetate, 150 mM potassium acetate, 30 mM magnesium acetate, pH 7.9) to a final volume of 1.5 μl and mixed with 1 μl of targeting components, followed by incubation at 37°C for 10 min.

Example 3

Nucleofection of T cells (CD4 ⁺ and CD8 ⁺ ) from PBMCs under the influence of Cas12a/nucleoprotein guide complexes

[00454] This Example describes nucleofection of activated T cells using a Cas12a/nucleoprotein guide complex.

[00455] The Cas12a guide/nucleoprotein complexes of Example 2 were transfected into primary activated T cells (CD4 ⁺ and CD8 ⁺ ) (prepared as described in Example 1) using the Nucleofector™ 96-well shuttle system (Lonza, Allendale, NJ). The Cas12a guide/nucleoprotein complex was dispensed in a final volume of 2.5 μl into individual wells of a 96-well plate. The suspended T cells were pelleted by centrifugation for 10 min at 200×g, washed with calcium- and magnesium-free phosphate-buffered saline (PBS), and the cell pellet was resuspended in 10 ml calcium- and magnesium-free PBS. Cells were counted using a Countess® II Automated Cell Counter (Life Technologies; Grand Island, NY).

[00455] 2.2⁷cells were transferred to a 15 ml conical tube and the cells were pelleted. The PBS was removed by aspiration and the cells were resuspended in Nucleofector™ solution P4 or P3 (Lonza, Allendale, NJ) up to density 2⁵-1⁶cells/mL per sample. Then, 20 μL of the cell suspension was added to each well containing 2.5 μL of Cas12a guide/nucleoprotein complexes, and the entire volume from each well was transferred to a well of a 96-well Nucleocuvette™ plate (Lonza, Allendale, NJ). The plate was loaded onto a 96-well Nucleofector™ shuttle (Lonza, Allendale, NJ), and the cells were nucleofected using the CA137 Nucleofector™ program (Lonza, Allendale, NJ). Following nucleofection, 77.5 μl of ImmunoCult-XF complete medium supplemented with IL-2 (100 units/ml) was added to each well, and the entire volume of transfected cell suspension was transferred to a 96-well cell culture plate containing 100 μl of pre-warmed ImmunoCult-XF complete medium supplemented with IL-2 (100 units/ml). The plate was transferred to a tissue culture incubator and maintained at 37°C in 5% CO₂within 48 hours prior to subsequent analysis.

Example 4

Assembly of human genes using Cas12a guide/nucleoprotein complexes

[00457] This Example describes the construction and use of Cas12a guide/nucleoprotein complexes to target genes encoding human T cell constant region alpha (TRAC), beta-2 microglobulin (B2M), programmed cell death protein 1 (PDCD1), cytokine-inducible SH2-containing protein (CISH), and Cb1 proto-oncogene B (CBL-B) in human T cells.

[00458] A. Construction of AsCas12a crRNA guide molecules

[00459] All 20 nucleotide sequences located downstream (in the 3' direction) of the 5'-TTTV PAM motif in the coding regions of the genes encoding human TRAC, B2M, PDCD1, CISH, and CBL-B were selected for targeting (SEQ ID Nos: 12-189). Target selection criteria include, but are not limited to, homology to other regions of the genome; percentage G-C content; melting temperature; and the presence of a homopolymer within the spacer.

[00460] The identified 20 nucleotide sequences were added downstream (in the 3' direction) of the AsCas12a activating region sequence (SEQ ID NO:6).

[00461] The sequences were submitted to commercial manufacturers for synthesis. Individual Cas12a guide/nucleoprotein complexes were then prepared as described in Example 2 and transfected into primary T cells as described in Example 3.

[00462] B. Determining the Efficiency of Genome Editing

[00463] (1) Generation of dsDNA target sequence for deep sequencing gDNA was isolated from nucleofected primary T cells 48 hours after transfection using Cas12a guide/nucleoprotein complexes and 50 μl of QuickExtract™ DNA Extraction Solution (Epicentre, Madison, WI) per well, followed by incubation at 37°C for 10 minutes, 65°C for 30 minutes, and 95°C for 3 minutes to stop the reaction. The isolated gDNA was diluted with 50 μl of sterile water and the samples were stored at -80°C.

[00464] Using the isolated gDNA, the first PCR was performed using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA) at a concentration of 1×, primers designed to amplify the region around the Cas12a target were used at 0.5 μM each, and 3.75 μl of gDNA were used in a final volume of 10 μl. Amplification was performed with an initial cycle at 98°C for 1 minute, 35 cycles of 10 seconds at 98°C and 20 seconds at 60°C, 30 seconds at 72°C; and a final extension at 72°C for 2 minutes. PCR reactions were diluted 1:100 in water.

[00465] To facilitate multiplex sequencing of each sample, a unique set of barcoded PCR index primers was used. Barcoded PCR was performed using a reaction mixture containing 1× Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA), 0.5 μM primers each, and 1 μl of a 1:100 dilution of the first PCR mix in a final volume of 10 μl. Reactions were amplified at 98°C for 1 minute, followed by 12 cycles of 10 seconds at 98°C, 20 seconds at 60°C, and 30 seconds at 72°C, with a final extension reaction at 72°C for 2 minutes.

[00465] (2) SPRIselect Cleaning

[00467] PCR reaction mixtures were pooled and transferred to a single centrifuge tube for amplicon purification using SPRIselect beads (Beckman Coulter, Pasadena, CA) for sequencing.

[00468] A 0.9x volume of SPRIselect beads was added to the amplicon, mixed, and incubated at room temperature for 10 minutes. The microcentrifuge tube was placed on a magnetic tube rack until the solution was cleared. The supernatant was removed and discarded, and the residual beads were washed with 1 volume of 85% ethanol, and the beads were incubated at room temperature for 30 seconds. After incubation, the ethanol was aspirated and the beads were air dried at room temperature for 10 minutes. The microcentrifuge tube was removed from the magnetic rack, and 0.25x volume of Qiagen EB buffer (Qiagen, Venlo, Netherlands) was added to the beads, mixed vigorously, and incubated for 2 minutes at room temperature. The microcentrifuge tube was returned to the magnetic stand, incubated until the solution cleared, and the supernatant containing purified amplicons was transferred to a clean microcentrifuge tube. Purified amplicons were quantified using the Nanodrop™ 2000 System (Thermo Scientific, Wilmington, DE) and library quality was analyzed using the Fragment Analyzer™ System (Advanced Analytical Technologies, Ames, IA) and the DNF-910 dsDNA Reagent Kit (Advanced Analytical Technologies, Ames, IA).

[00469] (3) Deep sequencing methods

[00470] Pooled amplicons were normalized to a concentration of 4 nM, calculated based on Nanodrop™ 2000 System values and average amplicon size. The library was analyzed on a MiSeq sequencer (Illumina, San Diego, CA) with the MiSeq v2 Reagent Kit (Illumina, San Diego, CA) for 300 cycles with two 151-cycle paired-end runs and two 8-cycle index reads.

[00471] (4) Analysis of deep sequencing data

[00472] The identity of the products in the sequencing data analysis was determined based on the barcode index sequences adapted to the amplicons in the barcoded PCR. A computational computer program was used to process the MiSeq data, which allows for the execution of, for example, the tasks described below:

a. Reads were aligned to the human genome (GRCh38/38 constructs) using the Bowtie program (bowtie-bio.sourceforge.net/index.shtml);

b. Aligned reads were compared with the expected wild-type genomic locus sequence, and reads that did not match any part of the wild-type locus were discarded;

c. Reads corresponding to the wild-type sequence were counted;

d. Reads with indels (base insertions or deletions) were classified by indel type and counted; and

e. The total number of indel reads was divided by the sum of wild-type and indel reads to obtain the percentage of mutated reads.

[00473] By identifying indel sequences in regions targeted by the Cas12a guide/nucleoprotein complexes, the final genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined. The results of the in-cell editing experiment are provided below in Table 7.

Table 7
Percentage of indels detected using Cas12a crRNA/nucleoprotein complexes SEQ ID NO: Target ID %
editing StDev SEQ ID NO: Target ID %
editing StDev SEQ ID NO:12 TRAC-tgt1 4.1% 0.7% SEQ ID NO:101 B2M-tgt51 87.6% 1.3% SEQ ID NO:13 TRAC-tgt2 9.7% 1.9% SEQ ID NO:102 B2M-tgt52 88.7% 0.9% SEQ ID NO:14 TRAC-tgt3 89.6% 1.0% SEQ ID NO:103 B2M-tgt53 50.1% 4.2% SEQ ID NO:15 TRAC-tgt4 42.5% 4.4% SEQ ID NO:104 B2M-tgt54 94.7% 0.2% SEQ ID NO:16 TRAC-tgt5 4.6% 0.8% SEQ ID NO:105 B2M-tgt55 88.0% 0.8% SEQ ID NO:17 TRAC-tgt6 87.3% 0.9% SEQ ID NO:106 B2M-tgt56 1.9% 0.2% SEQ ID NO:18 TRAC-tgt7 43.8% 2.9% SEQ ID NO:107 B2M-tgt57 1.8% 0.4% SEQ ID NO:19 TRAC-tgt8 1.0% 0.9% SEQ ID NO:108 B2M-tgt58 75.6% 1.5% SEQ ID NO:20 TRAC-tgt9 32.5% 4.1% SEQ ID NO:109 B2M-tgt59 42.8% 0.9% SEQ ID NO:21 TRAC-tgt10 32.3% 2.2% SEQ ID NO:110 B2M-tgt60 21.5% 0.3% SEQ ID NO:22 TRAC-tgt11 26.3% 2.6% SEQ ID NO:111 B2M-tgt61 7.1% 0.5% SEQ ID NO:23 TRAC-tgt12 94.3% 0.6% SEQ ID NO:112 B2M-tgt62 38.6% 0.8% SEQ ID NO:24 TRAC-tgt13 4.6% 0.5% SEQ ID NO:113 B2M-tgt63 85.6% 3.7% SEQ ID NO:25 TRAC-tgt14 5.4% 1.1% SEQ ID NO:114 B2M-tgt64 91.5% 2.4% SEQ ID NO:26 TRAC-tgt15 42.2% 1.7% SEQ ID NO:115 B2M-tgt65 79.7% 2.3% SEQ ID NO:27 TRAC-tgt16 0.5% 0.1% SEQ ID NO:116 B2M-tgt66 35.9% 1.5% SEQ ID NO:28 TRAC-tgt17 90.6% 0.9% SEQ ID NO:117 B2M-tgt67 75.5% 1.4% SEQ ID NO:29 TRAC-tgt18 7.2% 0.6% SEQ ID NO:118 B2M-tgt68 55.9% 1.5% SEQ ID NO:30 TRAC-tgt19 27.1% 3.9% SEQ ID NO:119 B2M-tgt69 0.6% 0.1% SEQ ID NO:31 TRAC-tgt20 53.1% 3.0% SEQ ID NO:120 B2M-tgt70 9.8% 0.4% SEQ ID NO:32 TRAC-tgt21 57.3% 3.4% SEQ ID NO:121 B2M-tgt71 50.8% 1.2% SEQ ID NO:33 TRAC-tgt22 51.8% 2.4% SEQ ID NO:122 B2M-tgt72 5.2% 0.5% SEQ ID NO:34 TRAC-tgt23 64.2% 4.5% SEQ ID NO:123 B2M-tgt73 29.7% 1.4% SEQ ID NO:35 TRAC-tgt24 5.7% 1.0% SEQ ID NO:124 B2M-tgt74 71.0% 1.1% SEQ ID NO:36 TRAC-tgt25 96.9% 0.7% SEQ ID NO:125 B2M-tgt75 4.0% 0.5% SEQ ID NO:37 TRAC-tgt26 42.3% 2.6% SEQ ID NO:126 B2M-tgt76 14.1% 1.1% SEQ ID NO:38 TRAC-tgt27 19.4% 1.4% SEQ ID NO:127 B2M-tgt77 16.6% 0.5% SEQ ID NO:39 TRAC-tgt28 95.2% 0.3% SEQ ID NO:128 B2M-tgt78 8.3% 1.1% SEQ ID NO:40 TRAC-tgt29 72.8% 0.9% SEQ ID NO:129 B2M-tgt79 3.4% 0.7% SEQ ID NO:41 TRAC-tgt30 94.9% 0.6% SEQ ID NO:130 B2M-tgt80 13.7% 2.0% SEQ ID NO:42 TRAC-tgt31 16.4% 2.6% SEQ ID NO:131 B2M-tgt81 7.2% 1.0% SEQ ID NO:43 TRAC-tgt32 63.5% 7.4% SEQ ID NO:132 B2M-tgt82 1.6% 0.2% SEQ ID NO:44 TRAC-tgt33 81.4% 2.2% SEQ ID NO:133 B2M-tgt83 83.6% 1.0% SEQ ID NO:45 TRAC-tgt34 0.1% 0.0% SEQ ID NO:134 PDCD1-tgt1 75.3% 4.0% SEQ ID NO:46 TRAC-tgt35 0.4% 0.0% SEQ ID NO:135 PDCD1-tgt2 19.2% 1.1% SEQ ID NO:47 TRAC-tgt36 21.7% 1.7% SEQ ID NO:136 PDCD1-tgt3 0.0% 0.0% SEQ ID NO:48 TRAC-tgt37 33.2% 2.6% SEQ ID NO:137 PDCD1-tgt4 35.6% 1.7% SEQ ID NO:49 TRAC-tgt38 95.6% 0.6% SEQ ID NO:138 PDCD1-tgt5 25.6% 2.6% SEQ ID NO:50 TRAC-tgt39 72.8% 2.5% SEQ ID NO:139 PDCD1-tgt6 22.5% 0.7% SEQ ID NO:51 B2M-tgt1 98.0% 0.8% SEQ ID NO:140 PDCD1-tgt7 6.4% 0.3% SEQ ID NO:52 B2M-tgt2 39.0% 2.5% SEQ ID NO:141 PDCD1-tgt8 14.7% 1.0% SEQ ID NO:53 B2M-tgt3 68.3% 2.4% SEQ ID NO:142 PDCD1-tgt9 90.0% 1.5% SEQ ID NO:54 B2M-tgt4 18.4% 1.2% SEQ ID NO:143 PDCD1-tgt10 52.6% 4.3% SEQ ID NO:55 B2M-tgt5 34.0% 3.9% SEQ ID NO:144 PDCD1-tgt11 32.6% 2.6% SEQ ID NO:56 B2M-tgt6 33.2% 3.6% SEQ ID NO:145 PDCD1-tgt12 8.3% 0.6% SEQ ID NO:57 B2M-tgt7 42.0% 1.8% SEQ ID NO:146 PDCD1-tgt13 10.9% 2.1% SEQ ID NO:58 B2M-tgt8 32.4% 1.0% SEQ ID NO:147 PDCD1-tgt14 7.4% 0.4% SEQ ID NO:59 B2M-tgt9 22.6% 1.6% SEQ ID NO:148 PDCD1-tgt15 83.0% 1.0% SEQ ID NO:60 B2M-tgt10 40.6% 4.4% SEQ ID NO:149 PDCD1-tgt16 0.2% 0.0% SEQ ID NO:61 B2M-tgt11 21.5% 2.2% SEQ ID NO:150 PDCD1-tgt17 0.1% 0.1% SEQ ID NO:62 B2M-tgt12 95.8% 0.7% SEQ ID NO:151 PDCD1-tgt18 0.3% 0.1% SEQ ID NO:63 B2M-tgt13 17.3% 1.3% SEQ ID NO:152 PDCD1-tgt19 87.7% 1.0% SEQ ID NO:64 B2M-tgt14 3.9% 0.5% SEQ ID NO:153 PDCD1-tgt20 92.8% 0.8% SEQ ID NO:65 B2M-tgt15 69.4% 3.7% SEQ ID NO:154 PDCD1-tgt21 0.9% 0.0% SEQ ID NO:66 B2M-tgt16 12.2% 1.7% SEQ ID NO:155 PDCD1-tgt22 0.1% 0.1% SEQ ID NO:67 B2M-tgt17 1.5% 0.5% SEQ ID NO:156 CISH-tgt1 56.0% 1.4% SEQ ID NO:68 B2M-tgt18 25.4% 1.3% SEQ ID NO:157 CISH-tgt2 20.0% 0.9% SEQ ID NO:69 B2M-tgt19 53.8% 4.1% SEQ ID NO:158 CISH-tgt3 78.3% 1.6% SEQ ID NO:70 B2M-tgt20 8.0% 0.9% SEQ ID NO:159 CISH-tgt4 40.8% 1.3% SEQ ID NO:71 B2M-tgt21 6.1% 0.7% SEQ ID NO:160 CISH-tgt5 82.2% 1.1% SEQ ID NO:72 B2M-tgt22 10.3% 0.3% SEQ ID NO:161 CISH-tgt6 21.6% 2.7% SEQ ID NO:73 B2M-tgt23 29.6% 2.7% SEQ ID NO:162 CISH-tgt7 79.6% 1.7% SEQ ID NO:74 B2M-tgt24 82.8% 2.6% SEQ ID NO:163 CISH-tgt8 61.9% 1.6% SEQ ID NO:75 B2M-tgt25 24.4% 3.6% SEQ ID NO:164 CISH-tgt9 79.7% 1.2% SEQ ID NO:76 B2M-tgt26 0.2% 0.0% SEQ ID NO:165 CISH-tgt10 70.9% 2.5% SEQ ID NO:77 B2M-tgt27 31.3% 2.6% SEQ ID NO:166 CBLB-tgt1 12.2% 6.4% SEQ ID NO:78 B2M-tgt28 27.3% 4.0% SEQ ID NO:167 CBLB-tgt2 14.6% 1.7% SEQ ID NO:79 B2M-tgt29 2.8% 0.5% SEQ ID NO:168 CBLB-tgt3 48.5% 1.6% SEQ ID NO:80 B2M-tgt30 31.7% 2.2% SEQ ID NO:169 CBLB-tgt4 71.9% 1.5% SEQ ID NO:81 B2M-tgt31 14.8% 1.9% SEQ ID NO:170 CBLB-tgt5 27.1% 2.8% SEQ ID NO:82 B2M-tgt32 54.4% 4.4% SEQ ID NO:171 CBLB-tgt6 85.8% 1.8% SEQ ID NO:83 B2M-tgt33 0.4% 0.2% SEQ ID NO:172 CBLB-tgt7 16.8% 1.8% SEQ ID NO:84 B2M-tgt34 77.1% 1.8% SEQ ID NO:173 CBLB-tgt8 4.1% 0.9% SEQ ID NO:85 B2M-tgt35 15.3% 1.5% SEQ ID NO:174 CBLB-tgt9 1.5% 0.0% SEQ ID NO:86 B2M-tgt36 60.4% 5.9% SEQ ID NO:175 CBLB-tgt10 6.4% 0.9% SEQ ID NO:87 B2M-tgt37 3.4% 0.7% SEQ ID NO:176 CBLB-tgt11 17.2% 2.1% SEQ ID NO:88 B2M-tgt38 3.7% 0.4% SEQ ID NO:177 CBLB-tgt12 31.2% 3.0% SEQ ID NO:89 B2M-tgt39 48.2% 4.0% SEQ ID NO:178 CBLB-tgt13 0.4% 0.0% SEQ ID NO:90 B2M-tgt40 7.0% 0.8% SEQ ID NO:179 CBLB-tgt14 0.2% 0.1% SEQ ID NO:91 B2M-tgt41 1.1% 0.4% SEQ ID NO:180 CBLB-tgt15 7.0% 0.5% SEQ ID NO:92 B2M-tgt42 1.0% 0.1% SEQ ID NO:181 CBLB-tgt16 5.2% 0.4% SEQ ID NO:93 B2M-tgt43 3.0% 0.4% SEQ ID NO:182 CBLB-tgt17 22.2% 2.1% SEQ ID NO:94 B2M-tgt44 35.6% 3.0% SEQ ID NO:183 CBLB-tgt17 0.0% 0.0% SEQ ID NO:95 B2M-tgt45 4.9% 0.8% SEQ ID NO:184 CBLB-tgt17 0.0% 0.0% SEQ ID NO:96 B2M-tgt46 3.5% 0.4% SEQ ID NO:185 CBLB-tgt20 2.4% 0.3% SEQ ID NO:97 B2M-tgt47 6.3% 0.9% SEQ ID NO:186 CBLB-tgt21 6.2% 0.9% SEQ ID NO:98 B2M-tgt48 7.9% 0.6% SEQ ID NO:187 CBLB-tgt22 17.6% 5.2% SEQ ID NO:99 B2M-tgt49 38.7% 2.6% SEQ ID NO:188 CBLB-tgt23 19.8% 2.8% SEQ ID NO:100 B2M-tgt50 6.4% 0.8% SEQ ID NO:189 CBLB-tgt24 0.1% 0.0%

StDev=Standard Deviation, n=3

[00474] The data presented in Table 7 demonstrated that Cas12a crRNA/nucleoprotein complexes are capable of editing multiple genes in primary human T cells. Other genes, such as those described elsewhere herein, can be similarly targeted using AsCas12a or other Cas12a proteins (such as those from L. bacterium or F. novicida ).

Example 5

Construction of Cas12a chRDNA guide molecules using DNA in the target binding sequence

[00475] The following Example describes the design of AsCas12a chRDNA guide molecules to incorporate DNA bases into the target binding sequence.

[00475] A. In silico construction of Cas12a chRDNA targeting molecule

[00477] The 20 nucleotide target binding sequence of the AsCas12a guide molecule was chosen for design, and a single DNA base was introduced into each position of the target binding sequence. The arrangement of DNA bases in the target binding sequence of the AsCas12a chRDNA guide molecules is shown in Table 8 (DNA bases are shown as "d" and RNA bases are shown as "R"; a control crRNA is also shown).

Table 8
AsCas12a chRDNA targeting molecule binding sequences SEQ ID NO: DNA position Subsequence SEQ ID NO:190 Cr-RNA RRRRRRRRRRRRRRRRRRRR SEQ ID NO:191 1 dRRRRRRRRRRRRRRRRRRRR SEQ ID NO:192 2 RdRRRRRRRRRRRRRRRRRR SEQ ID NO:193 3 RRRRRRRRRRRRRRRRRRR SEQ ID NO:194 4 RRRdRRRRRRRRRRRRRRRR SEQ ID NO:195 5 RRRRRRRRRRRRRRRRRRR SEQ ID NO:196 6 RRRRRdRRRRRRRRRRRRRR SEQ ID NO:197 7 RRRRRRRRRRRRRRRRR SEQ ID NO:198 8 RRRRRRRdRRRRRRRRRR SEQ ID NO:199 9 RRRRRRRRRRRRRRRRR SEQ ID NO:200 10 RRRRRRRRRdRRRRRRRRRR SEQ ID NO:201 11 RRRRRRRRRRdRRRRRRRRR SEQ ID NO:202 12 RRRRRRRRRRRdRRRRRRRR SEQ ID NO:203 13 RRRRRRRRRRRRdRRRRRRR SEQ ID NO:204 14 RRRRRRRRRRRRRdRRRRRR SEQ ID NO:205 15 RRRRRRRRRRRRRRdRRRRR SEQ ID NO:206 16 RRRRRRRRRRRRRRRdRRRR SEQ ID NO:207 17 RRRRRRRRRRRRRRRRdRRR SEQ ID NO:208 18 RRRRRRRRRRRRRRRRRdRR SEQ ID NO:209 19 RRRRRRRRRRRRRRRRRRdR SEQ ID NO:210 20 RRRRRRRRRRRRRRRRRRRd

[00478] Three target sequences in the gene encoding human B2M (B2M-tgt12, B2M-tgt1, B2M-intron-tgt12), a target sequence in the gene encoding human TRAC (TRAC-tgt12), and a target sequence in the gene encoding human DNA methyltransferase 1 (DNMT1) (DNMT1-tgt1) were selected for editing. A Cas12a chRDNA guide molecule for each target, containing a target binding sequence including one DNA base at each position (see Table 8), as well as a Cas12a crRNA control sequence, were submitted to a commercial manufacturer for synthesis.

[00479] B. Transfection and Cell Analysis

[00480] Individual Cas12a guide/nucleoprotein complexes for screening were prepared essentially as described in Example 2. The nucleoprotein complexes were transfected into primary T cells as described in Example 3, and the final genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined as described in Example 4. The results of the in-cell editing experiment are presented in Table 9.

Table 9
Levels of editing via Cas12a/nucleoprotein guide complexes B2M-tgt12 -target DNMT1-tgt1-target SEQ ID NO: DNA position % edit StDev SEQ ID NO: DNA position % edit StDev SEQ ID NO:211 cr-RNA 87.0% 1.1% SEQ ID NO:253 cr-RNA 67.1% 2.5% SEQ ID NO:212 1 86.4% 1.4% SEQ ID NO:254 1 68.3% 1.3% SEQ ID NO:213 2 84.6% 2.7% SEQ ID NO:255 2 2.1% 0.2% SEQ ID NO:214 3 84.8% 2.2% SEQ ID NO:256 3 36.4% 0.8% SEQ ID NO:215 4 87.0% 1.2% SEQ ID NO:257 4 12.3% 0.3% SEQ ID NO:216 5 86.0% 1.1% SEQ ID NO:258 5 0.3% 0.1% SEQ ID NO:217 6 86.6% 1.0% SEQ ID NO:259 6 28.0% 0.6% SEQ ID NO:218 7 86.3% 0.4% SEQ ID NO:260 7 8.2% 0.3% SEQ ID NO:219 8 85.0% 1.6% SEQ ID NO:261 8 67.3% 1.2% SEQ ID NO:220 9 85.3% 1.5% SEQ ID NO:262 9 63.8% 1.9% SEQ ID NO:221 10 87.0% 2.2% SEQ ID NO:263 10 62.6% 1.3% SEQ ID NO:222 11 85.5% 2.0% SEQ ID NO:264 11 58.5% 4.1% SEQ ID NO:223 12 86.3% 0.9% SEQ ID NO:265 12 45.6% 0.4% SEQ ID NO:224 13 85.7% 1.3% SEQ ID NO:266 13 18.9% 1.0% SEQ ID NO:225 14 80.8% 1.4% SEQ ID NO:267 14 64.1% 1.1% SEQ ID NO:226 15 85.1% 1.4% SEQ ID NO:268 15 62.3% 1.0% SEQ ID NO:227 16 55.3% 2.1% SEQ ID NO:269 16 42.9% 1.1% SEQ ID NO:228 17 86.2% 1.9% SEQ ID NO:270 17 59.2% 1.5% SEQ ID NO:229 18 86.0% 2.2% SEQ ID NO:271 18 43.4% 1.0% SEQ ID NO:230 19 87.1% 2.3% SEQ ID NO:272 19 63.4% 1.6% SEQ ID NO:231 20 86.2% 1.4% SEQ ID NO:273 20 64.6% 2.2% TRAC-tgt12 B2M-tgt1 SEQ ID NO: DNA position % edit StDev SEQ ID NO: DNA position % edit StDev SEQ ID NO:232 crRNA 90.57% 1.16% SEQ ID NO:274 crRNA 95.2% 0.6% SEQ ID NO:233 1 85.87% 0.52% SEQ ID NO:275 1 94.8% 1.0% SEQ ID NO:234 2 83.90% 0.93% SEQ ID NO:276 2 20.7% 1.4% SEQ ID NO:235 3 28.67% 1.42% SEQ ID NO:277 3 90.9% 1.2% SEQ ID NO:236 4 61.27% 1.97% SEQ ID NO:278 4 49.5% 2.3% SEQ ID NO:237 5 16.43% 1.14% SEQ ID NO:279 5 20.0% 0.7% SEQ ID NO:238 6 2.75% 0.02% SEQ ID NO:280 6 90.4% 0.6% SEQ ID NO:239 7 70.90% 0.71% SEQ ID NO:281 7 61.9% 2.1% SEQ ID NO:240 8 22.27% 0.99% SEQ ID NO:282 8 95.5% 1.0% SEQ ID NO:241 9 78.90% 1.13% SEQ ID NO:283 9 94.5% 1.4% SEQ ID NO:242 10 83.50% 0.67% SEQ ID NO:284 10 93.5% 1.7% SEQ ID NO:243 11 86.97% 1.21% SEQ ID NO:285 11 94.9% 1.0% SEQ ID NO:244 12 76.30% 0.24% SEQ ID NO:286 12 94.4% 0.6% SEQ ID NO:245 13 58.53% 1.67% SEQ ID NO:287 13 84.7% 3.3% SEQ ID NO:246 14 79.27% 1.21% SEQ ID NO:288 14 95.7% 0.9% SEQ ID NO:247 15 87.40% 1.42% SEQ ID NO:289 15 92.9% 5.0% SEQ ID NO:248 16 73.87% 0.87% SEQ ID NO:290 16 94.9% 1.1% SEQ ID NO:249 17 90.97% 1.47% SEQ ID NO:291 17 95.4% 0.9% SEQ ID NO:250 18 67.97% 0.78% SEQ ID NO:292 18 94.2% 1.1% SEQ ID NO:251 19 90.27% 0.90% SEQ ID NO:293 19 95.6% 0.8% SEQ ID NO:252 20 95.10% 0.70% SEQ ID NO:294 20 93.8% 1.0%

B2M-intron-tgt1 SEQ ID NO: DNA position % edit StDev SEQ ID NO:295 Cr-RNA 86.4% 6.6% SEQ ID NO:296 1 87.5% 2.0% SEQ ID NO:297 2 13.6% 1.0% SEQ ID NO:298 3 53.8% 1.0% SEQ ID NO:299 4 54.7% 3.9% SEQ ID NO:300 5 7.7% 0.4% SEQ ID NO:301 6 56.4% 6.8% SEQ ID NO:302 7 28.2% 3.0% SEQ ID NO:303 8 87.3% 0.4% SEQ ID NO:304 9 85.0% 1.7% SEQ ID NO:305 10 86.8% 1.7% SEQ ID NO:306 11 87.7% 0.6% SEQ ID NO:307 12 80.1% 1.2% SEQ ID NO:308 13 53.1% 2.6% SEQ ID NO:309 14 85.2% 2.7% SEQ ID NO:310 15 84.1% 3.0% SEQ ID NO:311 16 66.5% 2.3% SEQ ID NO:312 17 87.8% 1.1% SEQ ID NO:313 18 77.1% 2.0% SEQ ID NO:314 19 86.2% 1.1% SEQ ID NO:315 20 87.4% 1.6%

StDev=standard deviation; n=3

[00481] The editing results presented in Table 9 demonstrate that Cas12a chRDNA guide molecules containing DNA in the spacer are capable of editing at rates comparable to crRNA across multiple targets (cf. SEQ ID NO: 211 vs. SEQ ID NO: 224; SEQ ID NO: 232 vs. SEQ ID NO: 234; or SEQ ID NO: 274 vs. SEQ ID NO: 293). The editing rates of the chRDNA guide constructs presented in Table 9 were normalized to the editing rates of crRNA for each target. The average normalized editing rates are presented in Fig. 13, where the position in the target binding sequence is plotted against the average normalized editing. Preferred DNA base positions (i.e., greater than 70% of the average normalized edit) are indicated by gray shading and include positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, and 20. The data presented in this example, as well as the data presented in Table 9 and Fig. 13, can be used to determine which positions in the target binding sequence of the Cas12a guide molecule can be engineered into DNA.

Example 6

Cas12a chRDNA guide molecules with multiple DNA bases in the target binding sequence

[00482] This example describes the design and testing of Cas12a chRDNA guide molecules with multiple DNA bases in the target binding sequence.

[00483] A. In silico construction of Cas12a chRDNA guides

[00484] The sequence of three 20 nucleotide targets in the gene encoding human B2M (B2M-tgt12, B2M-tgt1, B2M-intron-tgt12), a target in the gene encoding human TRAC (TRAC-tgt12), and a target in the gene encoding human DNA methyltransferase 1 (DNMT1-tgt1) were selected for editing. For each target, 1 to 7 nucleotides of DNA were introduced into the target binding sequence for each AsCas12a guide molecule. Design criteria for introducing DNA base positions include, but are not limited to, prior single position screening data (see Example 5), prior alignment of DNA-tolerant positions (see Fig. 13), distance between individual DNA bases in the target binding sequence, and known locations of mismatches in the off-target sequence. The CHRDNA Cas12a guide constructs as well as the crRNA control sequence were submitted to a commercial manufacturer for synthesis.

[00485] B. Transfection and Cell Analysis

[00485] Individual Cas12a guide/nucleoprotein complexes for screening were prepared essentially as described in Example 2. The Cas12a guide/nucleoprotein complexes were transfected into primary T cells as described in Example 3, and the final genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined as described in Example 4. The results of the in-cell editing experiment and the DNA base positions in the target binding sequence for each Cas12a chRDNA guide molecule are presented in Table 10.

Table 10 Editing rates of Cas12a guide/nucleoprotein complexes Target SEQ ID NO: DNA position % edit StDev TRAC-tgt12 SEQ ID NO:316 - 89.00% 1.45% SEQ ID NO:317 20, 19 90.00% 0.64% SEQ ID NO:318 20.17 90.80% 1.84% SEQ ID NO:319 19, 15 80.17% 2.23% SEQ ID NO:320 11, 10 87.10% 1.06% SEQ ID NO:321 20, 11, 1 90.27% 0.78% SEQ ID NO:322 17, 11 87.03% 2.36% SEQ ID NO:323 2, 1 23.90% 0.30% SEQ ID NO:324 15, 10, 11 60.93% 0.93% SEQ ID NO:325 20, 17, 10, 1 59.93% 6.09% SEQ ID NO:326 18, 15, 2 10.87% 0.23% SEQ ID NO:327 18, 15, 11, 10, 3 5.20% 0.20% SEQ ID NO:328 10, 17, 15, 10, 1 74.17% 0.93% SEQ ID NO:329 20, 19, 17, 15, 11, 10, 2 1.29% 0.11% B2M-tgt12 SEQ ID NO:330 - 95.20% 0.60% SEQ ID NO:331 20.17 90.83% 0.17% SEQ ID NO:332 15, 11 90.77% 1.27% SEQ ID NO:333 15, 14 92.17% 1.53% SEQ ID NO:334 11, 10 90.83% 1.05% SEQ ID NO:335 20, 11, 1 93.60% 0.92% SEQ ID NO:336 17, 11 92.57% 1.52% SEQ ID NO:337 2, 1 90.70% 0.64% SEQ ID NO:338 20, 17, 15, 2 65.30% 8.02% SEQ ID NO:339 15, 14, 11, 10 72.63% 1.62% SEQ ID NO:340 20, 17, 11, 10, 2, 1, 50.37% 8.52% SEQ ID NO:341 17, 15, 14, 11, 10, 9, 3, 1 21.53% 1.15% SEQ ID NO:342 19, 16, 13, 11, 10, 1 0.51% 0.09% SEQ ID NO:343 16, 9, 3, 2, 1 4.41% 0.25% SEQ ID NO:344 20-11 0.00% 0.05% B2M-tgt1 SEQ ID NO:345 - 97.30% 0.20% SEQ ID NO:346 20, 11, 1 85.00% 0.30% SEQ ID NO:347 19, 14, 8 94.90% 0.60% SEQ ID NO:348 19, 17, 14, 8 52.70% 4.80% SEQ ID NO:349 12, 11, 8 65.70% 1.70% SEQ ID NO:350 18, 16, 10 59.20% 5.30% SEQ ID NO:351 19, 17, 15, 1 74.10% 2.40% SEQ ID NO:352 19-16 4.50% 0.60% B2M-intron-tgt1 SEQ ID NO:353 - 79.80% 3.00% SEQ ID NO:354 20, 11, 1 79.10% 3.90% SEQ ID NO:355 20, 17, 11 78.30% 2.00% SEQ ID NO:356 8-Nov 65.40% 1.70% SEQ ID NO:357 17, 10, 8, 1 74.20% 2.10% SEQ ID NO:358 20, 19, 17 61.40% 4.00% SEQ ID NO:359 20, 19, 9, 8 66.60% 1.90% SEQ ID NO:360 20, 19, 17, 11, 10, 1 42.40% 6.10% DNMT1-tgt1 SEQ ID NO:361 - 69.00% 1.06% SEQ ID NO:362 1, 2 63.43% 3.69% SEQ ID NO:363 14, 2 53.00% 0.26% SEQ ID NO:364 8, 19 65.80% 2.35% SEQ ID NO:365 8, 14 59.93% 1.42% SEQ ID NO:366 1, 8, 20 67.43% 0.85% SEQ ID NO:367 10-Aug 51.73% 1.01% SEQ ID NO:368 8, 9, 15 54.90% 2.96% SEQ ID NO:369 1, 8, 19 54.10% 4.11%

StDev=standard deviation; n=3

[00487] The editing results presented in Table 10 demonstrated that Cas12a chRDNA guide molecules containing multiple DNA bases in the target binding sequence are capable of editing at a rate comparable to crRNA for multiple targets (cf. SEQ ID NO: 316 with SEQ ID NO: 321; SEQ ID NO: 330 with SEQ ID NO: 335, or SEQ ID NO: 345 with SEQ ID NO: 347).

Example 7

Reducing the Rate of Off-Target Molecule Editing via Cas12a chRDNA Guides

This Example describes the identification of off-target Cas12a molecules using SITE-Seq® analysis (Cameron, P., et al., (2017). Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nature Methods, 14 (6), 600-606. https://doi.org/10.1038/nmeth.4284) and the reduced rate of off-target editing by Cas12a chRDNA guide compared to Cas12a crRNA guide.

[00488] A. SITE-Seq® Analysis

[00489] Primary human T cells were cultured as described in Example 1. After expansion of the cells in 50 ml conical tubes, high molecular weight genomic DNA (gDNA) was isolated from the primary human T cells using the Blood and Cell Culture DNA Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.

[00490] A 20-nucleotide target in the gene encoding human ribosomal protein L32 (RPL32) (RPL32-tgt1) and in the gene encoding DNMT1 (DNMT1-tgt1) was selected for evaluation using SITE-Seq® analysis (Cameron, P., et al., (2017). Nature Methods, 14 (6), 600-606). Each Cas12a targeting component was serially diluted to the appropriate nucleoprotein concentration, incubated at 95°C for 2 minutes, and then allowed to slowly warm to room temperature over 5 minutes. Cas12a nucleoprotein complexes for the targets RPL32 (SEQ ID NO: 375) and DNMT1 (SEQ ID NO: 404) were prepared by combining incubated Cas12a guides with Cas12a protein at a ratio of 3:1 in cleavage reaction buffer (60 mM Tris-acetate, 150 mM potassium acetate, 30 mM magnesium acetate, pH 7.9) and incubated at 37°C for 10 min. Separate cleavage of 10 μg genomic DNA (gDNA) occurred at 6 concentrations of Cas12a guide/nucleoprotein complex (8 nM, 16 nM, 32 nM, 48 nM, 64 nM, 96 nM, and 128 nM) in a total volume of 50 μl. Negative control reactions (0 nM) were performed in parallel and did not include any nucleoprotein complex. All cleavage reactions, including the negative control, were assembled in triplicate in a 96-well plate. Cleavage reactions were performed by incubation at 37°C for 4 h.

[00491] Library preparation and sequencing were performed essentially as described by Cameron et al. ( Nature Meth. 2017, 14:600–606), with the exception of the dA tailing step following cleavage of the post-nucleoprotein complex. An additional enzymatic end repair step was required due to cleavage of the Cas12a nucleoprotein complex resulting in the formation of a staggered (5′-overhang) end. Addition volumes remained the same, and the original dA tailing kit components were replaced with End Prep enzyme mix (3 µl) and 10x end repair reaction buffer (5 µl) from the NEBNext® Ultra™ End Repair/dA-Tailing Module from New England BioLabs (NEB# E7442L). Samples were incubated at 20°C for 30 min and then at 65°C for 30 min and the standard procedure was repeated. NGS sequencing was performed using the Illumina NextSeq platform (Illumina, San Diego, CA) and ~3 million reads were obtained for each sample. Any sites obtained by performing SITE-Seq® analysis (Cameron, P., et al., (2017) Nature Methods, 14 (6), 600-606) without off-target motifs located within 1 nucleotide of the cleavage site were considered false positives and were discarded. The number of recovered target sites calculated by performing the SITE-Seq® off-target molecule analysis experiment is shown in Table 11.

Table 11 Number of sites identified using SITE-Seq ® analysis SEQ ID NO: Concentration of nucleoprotein complex 8 nM 16 nM 32 nM 48 nM 64 nM 96 nM 128 nM SEQ ID NO:370 0 6 51 158 221 262 280 SEQ ID NO:404 2 4 10 22 57 88 109

[00492] The data presented in Table 11 demonstrated that SITE-Seq® analysis (Cameron, P., et al., (2017). Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nature Methods, 14 (6), 600-606) allowed the identification of biochemical off-target molecules of the Cas12a guide for subsequent intracellular assessment.

[00493] B. Intracellular Validation of Sites Obtained by SITE-Seq® Analysis for Off-Target Molecules

[00494] To measure the frequency of indels at the SITE-Seq® off-target sites presented in Table 11, deep sequencing analyses of targets were performed on a subset of sites isolated from RPL32 and DNMT1 samples for the lowest concentrations (i.e., 8 nM and 16 nM) of the Cas12a nucleoprotein complex. Two off-target sites from the RPL32 sample (SEQ ID NO: 371 and SEQ ID NO: 372) and one off-target site in DNMT1 (SEQ ID NO: 374) were selected to assess the frequency of intracellular off-target editing by crRNA. Forward and reverse amplicon primers were designed for each off-target site and ordered from a commercial manufacturer.

[00495] Primary human T cells were cultured as described in Example 1. RPL32 (SEQ ID NO: 375) and DNMT1 (SEQ ID NO: 404) Cas12a nucleoprotein complexes were generated essentially as described in Example 2. The nucleoprotein complexes were transferred into primary T cells as described in Example 3, and the resulting genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined as described in Example 4. The untransfected pool of cells was used as a wild-type reference. Mutant reads (% insertions-deletions) were defined as any non-reference variant queries within 20 base pairs (bp) of the cleavage site. In this case, sites with low sequencing coverage (<1000 reads in Cas12a pooled samples or <200 reads in reference samples) or >2% query variants in reference samples were discarded. Sites were considered off-target for cells if they accumulated >0.1% mutant reads in Cas12a pooled samples. Results of targeted deep sequencing of recovered off-target sites performed using SITE-Seq® (Cameron, P., et al., (2017). Nature Methods, 14 (6), 600–606) are presented in Table 12, where mismatched nucleotides are underlined.

Table 12 Intracellular validation of off-target sites in SITE-Seq ® analysis SEQ ID NO. Name Coordinates hg38 Subsequence Average StDev SEQ ID NO:370 RPL32_on chr3:129389653-129389676 TTTGGGGTGATCAGACCCAACAGC 47.1% 1.7% SEQ ID NO:371 RPL_32-off.1 chr2:88944276-88944299 TTTGGGGTGATCAGACCCAACA C C 26.4% 0.3% SEQ ID NO:372 RPL_32-off.2 chr6:33355564-33355587 TTTGGGGTGATCAGACCCAACA C C 23.6% 0.6% SEQ ID NO:373 DNMT1_on chr19:10133770-10133793 TTTCCTGATGGTCCATGTCTGTTA 41.1% 0.6% SEQ ID NO:374 DNMT1_off-1 chr1:213204014-213204037 TTTCCTGATGGTCCATGTCTG AAT 0.2% 0.0%

StDev=standard deviation; n=3

[00495] The data presented in Table 12 demonstrated that SITE-Seq® analysis identified off-target sites that were edited in T cells by Cas12a crRNA guides.

[00497] C. In silico construction of chRDNA guides

[00498] A 20 nucleotide target in the gene encoding human RPL32 (RPL32-tgt1) and in the gene encoding DNMT1 (DNMT1-tgt1) was selected for editing. For each target, 1 to 4 DNA nucleotides were designed into the target binding sequence of each AsCas12a chRDNA guide. Design criteria for introducing DNA base positions include, but are not limited to, prior single position screening data (see Example 5), prior alignment of DNA-tolerated positions (see Fig. 13), distance between individual DNA bases in the target binding sequence, and known locations of mismatches in the off-target sequence. The CHRDNA Cas12a guide constructs as well as the crRNA Cas12a control sequence were submitted to a commercial manufacturer for synthesis.

[00499] D. Transfection and Cell Analysis

[00500] Individual Cas12a guide/nucleoprotein complexes were prepared essentially as described in Example 2. The nucleoprotein complexes were transfected into primary T cells as described in Example 3, and the resulting genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined as described in Example 4. The results of the editing experiments, namely, target on/off switching (see Table 12) in the cells and assessment of the location of DNA bases in the target binding sequence for each Cas12a chRDNA guide molecule, are shown for the RPL32 (Table 13) and DNMT1 (Table 14) targets. In addition, Cas12a chRDNA guide molecules with a minor deoxyribose site were also tested, and the location of the minor site is shown as dN.

Table 13
RPL32 crRNA and chRDNA guide for on- and off-target editing SEQ ID NO. Position
DNA Target Non-target 1 Non-target 2 Average StDev Average StDev Average StDev SEQ ID NO:375 - 81.0% 0.8% 35.8% 0.8% 41.1% 0.1% SEQ ID NO:376 1 58.8% 2.4% 23.9% 1.7% 21.4% 1.5% SEQ ID NO:377 8 37.5% 3.1% 10.5% 1.3% 8.8% 1.5% SEQ ID NO:378 9 52.7% 1.7% 13.0% 0.7% 11.9% 0.6% SEQ ID NO:379 10 55.1% 1.9% 19.9% 1.8% 17.9% 1.8% SEQ ID NO:380 11 66.6% 3.8% 23.7% 3.1% 24.4% 3.4% SEQ ID NO:381 12 64.8% 3.3% 13.5% 1.6% 14.9% 1.9% SEQ ID NO:382 14 67.2% 2.6% 17.0% 1.7% 16.7% 1.0% SEQ ID NO:383 15 70.0% 1.6% 16.0% 1.6% 18.3% 1.5% SEQ ID NO:384 16 45.1% 1.9% 0.8% 0.2% 1.1% 0.2% SEQ ID NO:385 17 56.1% 2.0% 14.2% 0.5% 14.3% 1.2% SEQ ID NO:386 18 38.8% 3.2% 5.8% 0.6% 6.6% 0.7% SEQ ID NO:387 19 54.7% 1.7% 18.0% 0.7% 17.7% 1.4% SEQ ID NO:388 20 56.1% 2.0% 22.3% 1.9% 20.1% 0.4% SEQ ID NO:389 13 64.5% 2.4% 0.3% 0.0% 0.4% 0.0% SEQ ID NO:390 11, 12 80.3% 1.0% 5.4% 0.2% 6.0% 0.4% SEQ ID NO:391 12, 14 79.1% 0.8% 1.6% 0.1% 1.8% 0.3% SEQ ID NO:392 14, 15 72.8% 2.6% 1.4% 0.2% 1.6% 0.3% SEQ ID NO:393 11, 15 81.8% 0.9% 6.0% 0.1% 6.8% 0.3% SEQ ID NO:394 11, 12, 14 77.7% 0.3% 0.9% 0.2% 1.1% 0.2% SEQ ID NO:395 12, 14, 15 64.5% 2.5% 0.2% 0.1% 0.2% 0.1% SEQ ID NO:396 11, 14, 15 74.4% 2.0% 0.9% 0.1% 1.0% 0.1% SEQ ID NO:397 11, 12, 14, 15 8.3% 0.5% 0.0% 0.0% 0.0% 0.0% SEQ ID NO:398 dN(20) 59.2% 2.9% 4.5% 0.9% 4.9% 0.6% SEQ ID NO:399 14, dN(20) 40.3% 1.1% 0.6% 0.1% 0.5% 0.1% SEQ ID NO:400 15, dN(20) 20.2% 2.2% 0.1% 0.0% 0.2% 0.1% SEQ ID NO:401 12, dN(20) 50.2% 3.3% 0.5% 0.1% 0.6% 0.1% SEQ ID NO:402 11, dN(20) 25.6% 1.0% 0.7% 0.2% 0.7% 0.1% SEQ ID NO:403 14, dN(20) 19.5% 1.6% 0.1% 0.0% 0.1% 0.0%

StDev=standard deviation; n=3

Table 14
DNMT1 crRNA and chRDNA guide for on- and off-target editing SEQ ID NO. DNA position Target Non-target 1 Average StDev Average StDev SEQ ID NO:404 - 68.4% 1.3% 0.9% 0.2% SEQ ID NO:405 1, 20 68.2% 0.4% 0.6% 0.0% SEQ ID NO:406 8, 19 63.6% 4.6% 0.5% 0.1% SEQ ID NO:407 9 63.8% 1.9% 0.3% 0.0% SEQ ID NO:408 8, 14 61.1% 1.5% 0.2% 0.1% SEQ ID NO:409 10, 17 56.4% 1.8% 0.1% 0.0% SEQ ID NO:410 1, 8, 20 57.5% 4.4% 0.1% 0.1% SEQ ID NO:411 1, 8, 19 52.3% 1.1% <0.1% 0.1% SEQ ID NO:412 8, 9, 15 52.5% 0.6% <0.1% 0.0%

StDev=standard deviation; n=3

[00501] The editing results in Table 12 and Table 13 demonstrated that Cas12a chRDNA guide molecules are capable of reducing editing of off-target sites (cf. the results in Table 13 for off-target editing of SEQ ID NO: 375 with SEQ ID NO: 391; SEQ ID NO: 375 with SEQ ID NO: 394; cf. the results in Table 14 for off-target editing of SEQ ID NO: 404 with SEQ ID NO: 408; or SEQ ID NO: 404 with SEQ ID NO: 412), even at sites with a single nucleotide mismatch (see, e.g., Table 12, SEQ ID NO: 371 and SEQ ID NO: 372).

Example 8

Cas12a chRDNA guide molecules with DNA bases in the activating region

[00502] This example describes the design and testing of AsCas12a chRDNA guide molecules with DNA bases in the activating region.

[00503] A. In silico construction of chRDNA targeting molecules

[00504] The 20 nucleotide guide activating region of the AsCas12a guide molecule was chosen for design, where a different DNA base was used at each position in the activating region. The arrangement of DNA bases in the activating region of the AsCas12a guide molecule is shown in Table 15.

Table 15
AsCas12a chRDNA targeting molecules with DNA in the activating region SEQ ID NO: Position
DNA Subsequence SEQ ID NO:417 Cr-RNA rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:418 1 TrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:419 2 rUArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:420 3 rUrAArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:421 4 rUrArATrUrUrCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:422 5 rUrArArUTrUrCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:423 6 rUrArArUrUTrCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:424 7 rUrArArUrUrUCrUrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:425 8 rUrArArUrUrUrCTrArCrUrCrUrUrGrUrArGrArU SEQ ID NO:426 9 rUrArArUrUrUrCrUArCrUrCrUrUrGrUrArGrArU SEQ ID NO:427 10 rUrArArUrUrUrCrUrACrUrCrUrUrGrUrArGrArU SEQ ID NO:428 11 rUrArArUrUrUrCrUrArCTrCrUrUrGrUrArGrArU SEQ ID NO:429 12 rUrArArUrUrUrCrUrArCrUCrUrUrGrUrArGrArU SEQ ID NO:430 13 rUrArArUrUrUrCrUrArCrUrCTrUrGrUrArGrArU SEQ ID NO:431 14 rUrArArUrUrUrCrUrArCrUrCrUTrGrUrArGrArU SEQ ID NO:432 15 rUrArArUrUrUrCrUrArCrUrCrUrUGrUrArGrArU SEQ ID NO:433 16 rUrArArUrUrUrCrUrArCrUrCrUrUrGTrArGrArU SEQ ID NO:434 17 rUrArArUrUrUrCrUrArCrUrCrUrUrGrUArGrArU SEQ ID NO:435 18 rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrAGrArU SEQ ID NO:436 19 rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGArU SEQ ID NO:437 20 rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrAT

[00505] A target in the gene encoding human DNMT1 (SEQ ID NO: 361) was selected for editing. The DNMT target binding sequence was linked downstream (i.e., 3') to activator region sequences containing one DNA base at each position (see Table 15). The sequences were submitted to a commercial manufacturer for synthesis.

[00505] B. Transfection and Cell Analysis

[00507] Individual Cas12a guide/nucleoprotein complexes for screening were prepared essentially as described in Example 2. The nucleoprotein complexes were transfected into primary T cells as described in Example 3, and the resulting genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined as described in Example 4. The results of the in-cell editing experiment are provided in Table 16.

Table 16
Editing of DNMT1 using Cas12 chRDNA guides with DNA in the activating region SEQ ID NO: DNA position Average StDev SEQ ID NO:438 Cr-RNA 60.8% 1.0% SEQ ID NO:439 1 53.1% 0.5% SEQ ID NO:440 2 29.6% 0.3% SEQ ID NO:441 3 58.5% 0.1% SEQ ID NO:442 4 1.4% 0.1% SEQ ID NO:443 5 44.8% 0.6% SEQ ID NO:444 6 2.2% 0.1% SEQ ID NO:445 7 60.8% 1.0% SEQ ID NO:446 8 25.4% 4.9% SEQ ID NO:447 9 43.4% 2.2% SEQ ID NO:448 10 64.8% 3.3% SEQ ID NO:449 11 19.5% 0.6% SEQ ID NO:450 12 61.6% 1.4% SEQ ID NO:451 13 46.4% 1.6% SEQ ID NO:452 14 65.0% 1.4% SEQ ID NO:453 15 61.4% 1.1% SEQ ID NO:454 16 56.8% 0.6% SEQ ID NO:455 17 58.5% 2.0% SEQ ID NO:456 18 58.1% 1.0% SEQ ID NO:457 19 63.0% 0.6% SEQ ID NO:458 20 28.4% 0.4%

StDev=standard deviation; n=3

[00508] The editing results presented in Table 16 demonstrate that Cas12a chRDNA molecules containing DNA in the activating region are capable of editing at rates comparable to crRNA across multiple targets (cf. SEQ ID NO: 438 with SEQ ID NO: 445; SEQ ID NO: 438 with SEQ ID NO: 450; or SEQ ID NO: 438 with SEQ ID NO: 457). The editing rates of the chRDNA constructs presented in Table 16 were normalized to the editing rates of crRNA. The average normalized editing rates are presented in Fig. 14, where position in the activating region is plotted against average normalized editing. Preferred DNA base positions (i.e., greater than 70% of the average normalized edit) are indicated by gray shading and include positions 1, 3, 5, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, and 19.

[00509] D. CHRDNA with multiple DNA bases in the activating region

[00510] Based on the results presented in Table 10, individual DNA positions were combined into activating region constructs containing multiple DNA bases. The design and arrangement of DNA bases in the activating region of the AsCas12a guide are shown in Table 17.

Table 17
Cas12a chRDNA targeting molecules with multiple DNA bases in the activating region SEQ ID NO: DNA positions Subsequence SEQ ID NO:459 1, 3, 7, 10, 12, 14, 15, 19 TrAArUrUrUCrUrACrUCrUTGrUrArGArU SEQ ID NO:460 1, 15-19 TrArArUrUrUrCrUrArCrUrCrUrUGTAGArU SEQ ID NO:461 1, 13, 16, 17 TrArArUrUrUrCrUrArCrUrCTrUrGTArGrArU SEQ ID NO:462 7, 10, 12, 14, 15, 17, 19 rUrArArUrUrUCrUrACrUCrUTGrUArGArU SEQ ID NO:463 1, 3, 12, 14, 16, 17, 19 TrAArUrUrUrCrUrArCrUCrUTrGTArGArU SEQ ID NO:464 7, 10, 12, 14, 17, 19 rUrArArUrUrUCrUrACrUCrUTrGrUArGArU SEQ ID NO:465 1, 3, 7, 10, 12, 14, 15, 19 TrAArUrUrUCrUrACrUCrUTrGrUArGArU

[00511] The activating region constructs shown in Table 17 were combined with a 20 nucleotide target sequence in the gene encoding human TRAC, constructed using the DNA nucleotides in the target binding sequence (SEQ ID NO: 321). The TRAC target binding sequence was linked downstream (i.e., 3') to the activating region sequences shown in Table 17, and the chRDNA constructs, as well as the crRNA control sequence, were submitted to a commercial manufacturer for synthesis.

[00512] E. Transfection and Cell Analysis

[00513] Individual Cas12a guide/nucleoprotein complexes for screening were prepared essentially as described in Example 2. Individual Cas12a guide/nucleoprotein complexes were transfected into primary T cells as described in Example 3, and the resulting genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined as described in Example 4. The results of the intracellular editing experiment and the DNA base locations in the activating region and target binding sequence of each chRDNA are presented in Table 18.

Table 18
TRAC editing using Cas12a chRDNA guide molecules with DNA in the activating region and in the target binding sequence SEQ ID NO: DNA positions in the activating region DNA positions in the target binding region Average StDev SEQ ID NO:466 0 1, 10, 20 78.7% 1.2% SEQ ID NO:467 1, 3, 7, 10, 12, 14, 15, 19 1, 10, 20 82.3% 1.1% SEQ ID NO:468 1, 15-19 1, 10, 20 75.9% 1.3% SEQ ID NO:469 1, 13, 16, 17 1, 10, 20 76.9% 2.4% SEQ ID NO:470 7, 10, 12, 14, 15, 17, 19 1, 10, 20 71.3% 1.0% SEQ ID NO:471 1, 3, 12, 14, 16, 17, 19 1, 10, 20 73.3% 1.9% SEQ ID NO:472 7, 10, 12, 14, 17, 19 1, 10, 20 67.0% 2.2% SEQ ID NO:473 1, 3, 7, 10, 12, 14, 15, 19 1, 10, 20 59.3% 4.1%

StDev=standard deviation; n=3

[00514] The editing results in Table 18 demonstrated that Cas12a chRDNA guide molecules with DNA in both the activating region and the target binding sequence are capable of editing at a rate comparable to that of crRNA (cf. SEQ ID NO: 466 with SEQ ID NO: 467; SEQ ID NO: 466 with SEQ ID NO: 468, or SEQ ID NO: 466 with SEQ ID NO: 469).

Example 9

Cloning of the AAV donor cluster, AAV production and AAV transduction of primary cells

[00515] This Example describes constructing and cloning a donor DNA cassette into an AAV vector, producing the AAV, delivering Cas12a targeting chRDNA/nucleoprotein complexes into primary cells, and transducing the primary cells with the AAV to site-specifically integrate the CAR expression cassette into the primary cells.

[00515] An AAV can be engineered to deliver donor DNA polynucleotides into mammalian cells. If AAV delivery occurs in combination with a genomic cleavage event, and the donor DNA polynucleotide in the AAV is flanked by homology arms, the donor DNA polynucleotide can be readily inserted into the genomic cleavage site using HDR, as described, for example, by Eyquem et al. (Nature, 2017, 543:113-117).

[00517] A. In silico construction of AAV donor clusters and production of rAAV

[00518] The design of CAR receptors has been described. See, for example, Kochenderfer et al. ( J. Immunotherapy , 2009, 32:689-702). The CAR construct was designed to contain an N-terminal secretion signal (CD8a signal peptide), a BCMA-specific scFv fragment, followed by the CD8 hinge and transmembrane regions, the 4-1BB effector region, the CD3ζ effector region, and a C-terminal BGH polyadenylation signal sequence. A mammalian promoter sequence was introduced upstream of the CAR polynucleotide. A target site in the endogenous TRAC locus (SEQ ID NO: 23) was selected for site-specific integration of the donor DNA polynucleotides into the host cell genome after site-specific cleavage. Homology arms of 500 bp in length were then identified at the 5' and 3' cleavage site. The 5' and 3' homology arms were attached to the end of the donor DNA polynucleotides, wherein the donor DNA polynucleotides were oriented in the reverse orientation (i.e., 3' to 5') relative to the homology arms. The resulting donor DNA polynucleotide is shown in SEQ ID NO: 413.

[00519] A construct of the histocompatibility antigen B2M and HLA class I, alpha chain E (HLA-E) has been described. See, for example, Gornalusse et al. ( Nature Biotechnology , 2017, 35(8):765-772). The fusion construct was designed to contain an N-terminal B2M secretion signal followed by a peptide sequence derived from HLA-G, a first linker sequence, a B2M sequence, a second linker sequence, an HLA-E sequence, and a C-terminal BGH polyadenylation signal sequence. A mammalian EF1α promoter sequence was inserted upstream of the B2M-HLA-E polynucleotide. For site-specific integration of the donor DNA polynucleotide into the host cell genome after site-specific cleavage, a target site in the endogenous B2M locus (SEQ ID NO: 62) was selected. Homology arms of 500 bp in length were then identified at the 5' and 3' cleavage site. The 5' and 3' homology arms were attached to the end of the donor DNA polynucleotides, where the donor DNA polynucleotides were oriented in the reverse orientation (i.e., from 3' to 5') relative to the homology arms. The resulting donor DNA polynucleotide is shown in SEQ ID NO: 414.

[00520] Oligonucleotide sequences encoding donor DNA polynucleotides were provided to a commercial manufacturer for synthesis into a suitable recombinant AAV (rAAV) plasmid. An rAAV plasmid containing SEQ ID NO: 413 and a separate rAAV plasmid containing SEQ ID NO: 414 were provided to the commercial manufacturer for packaging into two separate AAV6 viruses.

[00521] B. Transduction of primary T cells with rAAV

[00522] Primary activated T cells were prepared from PBMCs as described in Example 1. Cas12a guided chRDNA/nucleoprotein complexes targeting the genes encoding TRAC (SEQ ID NO: 415) and B2M (SEQ ID NO: 416) were prepared as described in Example 2. T cells were transfected with Cas12a guided chRDNA/nucleoprotein complexes targeting TRAC (SEQ ID NO: 415), and 1 minute to 4 hours after nucleofection, the cells were infected with AAV6 virus packaged with the CAR donor sequence (SEQ ID NO: 413) at an MOI of 1×10 ⁶ . In addition, T cells were transfected with Cas12a chRDNA/nucleoprotein guide complexes targeting B2M (SEQ ID NO: 416), and 1 min to 4 h after nucleofection, the cells were infected with AAV6 virus packaged with the B2M-HLA-E donor sequence (SEQ ID NO: 414) at an MOI of 1×10 ⁶ . T cells were cultured in ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/ml) for 24 h after transduction. The following day, transduced T cells were transferred to 50 ml conical tubes and centrifuged at 300×g for approximately 7–10 min to pellet the cells. The supernatant was discarded, the pellet was carefully resuspended, and T cells were pooled in an appropriate volume of ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/ml).

[00523] The listed T cells were resuspended at 1× ¹⁰⁶ cells/mL in ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/mL) and placed into as many T-175 suspension flasks as required (maximum volume per flask is 250 mL).

[00524] C. Expression of CAR-T cells containing anti-BCMA antibodies, B2M-HLA-E

[00525] In vitro characterization of CAR-T cells containing anti-BCMA, B2M-HLA-E antibodies, control cells (TRAC knockout (KO) and B2M knockout), and wild-type T cells was performed 7 days after transduction.

[00525] CAR T cells were assessed for expression by flow cytometry, namely, either anti-BCMA CAR expression using recombinant BCMA protein conjugated to phycoerythrin (PE); TRAC expression using Alexa Fluor® 647-conjugated anti-TCR a/b specific antibody (ThermoFisher Scientific, Waltham, MA), or B2M expression using PE-conjugated anti-B2M specific antibody. The results of the flow cytometry analysis are shown in Fig. 15A, where CAR-positive (Fig. 15A, 1501), TRAC-positive (Fig. 15A, 1502), and B2M-positive results (Fig. 15A, 1503) are shown for cells that were not treated (wild-type T cells; Fig. 15A, 1504), cells that were transfected with both Cas12a chRDNA/nucleoprotein targeting complexes only (TRAC KO/B2M KO; Fig. 15A, 1505), and cells that were transfected with both Cas12a chRDNA/nucleoprotein targeting complexes and transduced with both viruses (assayed for anti-BCMA and B2M-HLA-E antibodies for CAR T cells; Fig. 15A, 1506). The y-axis represents the percentage of cells positive by FACS for various cell surface markers. The results are also presented in Table 19.

Table 19
Flow cytometry analysis of Cas12a/AAV-treated cells Wild-type T cells With TRAC/B2M knockout CAR-T cells with antibodies against BCMA, B2M-HLA-E CAR positive 2.6% 5.2% 87.9% TRAC- positive 96.3% 7.8% 3.9% B2M positive 100.0% 8.7% 50.5%

[00527] The results shown in Fig. 15A and Table 19 demonstrate knockout of endogenous TRAC and B2M expression mediated by chRDNA Cas12a targeting and introduction and expression of anti-BCMA CAR and exogenous B2M-HLA-E proteins mediated by AAV6.

[00528] D. Cytotoxicity of CAR-T cells containing anti-BCMA, B2M-HLA-E antibodies in vitro

[00529] The cytotoxicity of CAR-T cells containing the anti-BCMA antibody, B2M-HLA-E, was assessed in vitro against the cytotoxicity of the multiple myeloma cell line NCI-H929, which presents the BCMA antigen. TRAC knockout T cells were used as a control for CAR-mediated cytolysis. Briefly, target cells (NCI-H929 (T)) were labeled with CellTrace™ violet (CTV; Thermo Fisher C34557) to differentiate these cells from anti-BCMA, B2M-HLA-E (E) antibody-containing effector CAR-T cells and the cells were co-cultured at E:T ratios of 0:1, 1:20, 1:10, 1:5, 1:3, 1:1, 3:1 and 10:1 (3 co-culture wells/E:T ratio). Cytotoxicity was measured by gating the CTV cell population (target cells) and live cells assessed using propidium iodide (PI) after 48 hours of co-culture. Data were analyzed by flow cytometry (Intellicyt iQue Screener Plus). Specific lysis was calculated using the following equation for each well: Specific lysis = 1 - (number of live target cells in the test sample/number of live target cells in the control sample).

[00530] The results of the in vitro cytotoxicity assay are shown in Fig. 15B for CAR-T cells containing anti-BCMA antibodies, B2M-HLA-E (gray circles), and for control TRAC knockout T cells (black circles). The y-axis represents the percentage of target cells killed, and the x-axis represents the E:T ratio used. The data shown in Fig. 15B are also presented in Table 20.

Table 20
CAR-mediated cytotoxicity against BCMA E:T TRAC knockout T cells CAR-T cells with antibodies against BCMA, B2M-HLA-E Average StDev Average StDev 0:1 12% 1% 14% 1% 1:20 12% 1% 76% 4% 1:10 14% 1% 85% 1% 1:5 15% 1% 91% 1% 1:3 19% 1% 93% 1% 1:1 30% 7% 95% 1% 3:1 53% 7% 92% 0% 10:1 71% 8% 82% 3%

StDev=standard deviation; n=3

[00531] The results shown in Fig. 15B and Table 20 demonstrated that CAR-T cells generated using Cas12a chRDNA targeting molecules are capable of antigen-specific killing of target cells.

[00532] The methods provided herein can be applied to the production of other cells using Cas12a chRDNA targeting molecules for site-specific introduction of donor polynucleotides containing a chimeric antigen receptor (CAR). Additional donor polynucleotides expressing non-CAR polypeptides (i.e., B2M-HLA-E fusion constructs) can be similarly introduced in accordance with the guidance provided herein.

Example 10

Generation of CAR-T cells containing anti-BCMA antibodies and expressing a B2M-HLA-E fusion construct under the control of the endogenous B2M promoter

[00533] This example describes a construct for transducing primary cells with an AAV virus to site-specifically integrate a CAR polynucleotide and a B2M-HLA-E polynucleotide expression cassette at Cas12a chRDNA-mediated breakpoint sites into the genome of the primary cells.

[00534] A. In silico construction of AAV donor clusters and rAAV production

[00535] The anti-BCMA CAR was constructed as described in Example 9.

[00535] A donor cluster polynucleotide for the P2A-B2M-HLA-E fusion construct was constructed using a polynucleotide encoding the N-terminal B2M secretion signal followed by: a polynucleotide encoding a peptide sequence derived from HLA-G; a polynucleotide encoding a first linker sequence; a polynucleotide encoding a B2M sequence; a polynucleotide encoding a second linker sequence; a polynucleotide encoding an HLA-E sequence; and a polynucleotide encoding a C-terminal BGH polyadenylation signal sequence. The polynucleotide encoding the P2A ribosome excision sequence was inserted upstream of B2M-HLA-E such that expression of the fusion construct was under the control of the endogenous B2M promoter. For site-specific integration of donor DNA polynucleotides into the host cell genome after site-specific cleavage, a target site in the endogenous B2M locus (SEQ ID NO: 62) was selected. Homology arms of 500 bp in length were then identified at the 5' and 3' ends of the cleavage site. The 5' and 3' homology arms were attached to the 5' and 3' ends of the donor DNA polynucleotides, where the donor DNA polynucleotides were oriented in the forward direction (i.e., from 5' to 3') relative to the homology arms. The resulting donor DNA polynucleotide is presented in SEQ ID NO: 479.

[00537] Oligonucleotide sequences encoding donor DNA polynucleotides were provided to a commercial manufacturer for synthesis into a suitable recombinant AAV (rAAV) plasmid. An rAAV plasmid containing SEQ ID NO:413 and a separate rAAV plasmid containing SEQ ID NO:479 were provided to the commercial manufacturer for packaging into two separate AAV6 viruses.

[00538] B. Primary transduction of T cells by rAAV

[00539] Primary activated T cells were prepared from PBMCs as described in Example 1. Cas12a guided chRDNA/nucleoprotein complexes targeting the genes encoding TRAC (SEQ ID NO: 415) and B2M (SEQ ID NO: 416) were prepared as described in Example 2. T cells were transfected with Cas12a guided chRDNA/nucleoprotein complexes targeting TRAC (SEQ ID NO: 415), and 1 minute to 4 hours after nucleofection, the cells were infected with AAV6 virus packaged with the CAR donor sequence (SEQ ID NO: 413) at an MOI of 1×10 ⁶ . In addition, T cells were transfected with Cas12a chRDNA/nucleoprotein guide complexes targeting B2M (SEQ ID NO: 416), and 1 min to 4 h after nucleofection, the cells were infected with AAV6 virus packaged with the P2A-B2M-HLA-E donor sequence (SEQ ID NO: 479) at an MOI of 1×10 ⁶ . T cells were cultured in ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/ml) for 24 h after transduction. The following day, transduced T cells were transferred to 50 ml conical tubes and centrifuged at 300×g for approximately 7–10 min to pellet the cells. The supernatant was discarded, the pellet was carefully resuspended, and T cells were pooled in an appropriate volume of ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/ml).

[00540] The listed T cells were resuspended at 1× ¹⁰⁶ cells/mL in ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/mL) and placed into as many T-175 suspension flasks as required (maximum volume per flask is 250 mL).

[00541] C. Expression of CAR-T cells containing anti-BCMA antibodies, B2M-HLA-E

[00542] In vitro characterization of CAR-T cells containing anti-BCMA, P2A-B2M-HLA-E antibodies, as well as control cells (TRAC knockout (KO) and B2M knockout), and wild-type T cells was performed 7 days after transduction.

[00543] CAR T cells were assessed for expression by flow cytometry, namely, either anti-BCMA CAR expression using recombinant BCMA protein conjugated to phycoerythrin (PE); TRAC expression using Alexa Fluor® 647-conjugated TCR a/b specific antibody (ThermoFisher Scientific, Waltham, MA), or B2M expression using PE-conjugated anti-B2M specific antibody. The results of the flow cytometry analysis are presented in Table 21, where CAR-positive, TRAC-positive, and B2M-positive results are shown for untreated cells (wild-type T cells), cells that were transfected with both Cas12a chRDNA/nucleoprotein targeting complexes only (TRAC KO/B2M KO), and cells that were transfected with both Cas12a chRDNA/nucleoprotein targeting complexes and transduced with both viruses (with anti-BCMA and B2M-HLA-E antibody analysis for CAR T cells).

Table 21
Flow cytometry analysis of Cas12a/AAV-treated cells Wild-type T cells With TRAC/B2M knockout CAR-T cells with antibodies against BCMA, B2M-HLA-E CAR positive 2.5% 4.5% 91.6% TRAC- positive 94.4% 5.1% 3.5% B2M positive 99.0% 4.5% 21.0%

[00544] The results presented in Table 21 demonstrated knockout of endogenous TRAC and B2M expression mediated by the Cas12a-directed chRDNA and the introduction and exogenous expression of the anti-BCMA CAR donor cluster and the expression of the exogenous B2M-HLA-E donor cluster under the control of the endogenous B2M promoter.

[00545] The methods provided herein can be applied to identify Cas12 chRDNA guide constructs for other targets. The activation region and target binding sequences of other Cas12 chRDNA guides can be screened using methods similar to those described herein.

Example 11

Cas12a guide/nucleoprotein complexes with an alternative linker-NLS configuration

[00545] This example describes the construction and comparison of Cas12a guide/nucleoprotein complexes having different linker and nuclear localization signal (NLS) configurations with the "non-optimized" construct containing glycine-serine linkers and the NLS of simian vacuolation virus 40 (SV40; SEQ ID NO:04) large T antigen and used in the previous Examples of this application.

[00547] A. In silico construction of Cas12a-NLS linker sequences

[00548] The Cas12a protein (SEQ ID NO:01) of Acidaminococcus sp . (strain BV3L6) was selected for construction, and two NLS sequences, SV40 (SEQ ID NO:04) and the nucleoplasmin sequence (NPL; SEQ ID NO:05), were selected for covalent attachment to the Cas12a protein using glycine-serine (GS) amino acid linkers or glycine-glycine-glycine-glycine-serine (G4S) amino acid linker pairs. Constructs incorporating two NLSs with variable linkers were also created for testing. The linker-NLS sequence constructs are presented in Table 22:

Table 22
Linker-NLS sequence constructs SEQ ID NO: Name Amino acid sequence SEQ ID NO:479 GS-SV40* GSPKKKRKV SEQ ID NO:480 GS-SV40-GS-SV40 GSPKKKRKVGSPKKKRKV SEQ ID NO:481 GS-NPL GSKRPAATKKAGQAKKKK SEQ ID NO:482 GS-NPL-GS-NPL GSKRPAATKKAGQAKKKKGSKRPAATKKAGQAKKKK SEQ ID NO:483 GS-SV40-GS-NPL GSPKKKRKVGSKRPAATKKAGQAKKKK SEQ ID NO:484 GS-NPL-GS-SV40 GSKRPAATKKAGQAKKKKGSPKKKRKV SEQ ID NO:485 (G4S)2-SV40-GS-NPL GGGGSGGGGSPKKKRKVGSKRPAATKKAGQAKKKK SEQ ID NO:486 (G4S)2-NPL-GS-SV40 GGGGSGGGGSKRPAATKKAGQAKKKKGSPKKKRKV SEQ ID NO:487 (G4S)2-SV40 GGGGSGGGGSPKKKRKV SEQ ID NO:488 (G4S)2-SV40-GS-SV40 GGGGSGGGGSPKKKRKVGSPKKKRKV SEQ ID NO:489 (G4S)2-NPL GGGGSGGGSKRPAATKKAGQAKKKK SEQ ID NO:490 (G4S)2-NPL-GS-NPL GGGGSGGGGSKRPAATKKAGQAKKKKGSKRPAATKKAGQAKKKK *=NLS construct used in previous examples

[00549] The NLS sequences shown in Table 22 were cloned at the C-terminus of the Cas12a protein (SEQ ID NO:01) of Acidaminococcus sp . (strain BV3L6), and the recombinant protein was expressed as described in Example 2.

[00550] B. Cellular Activity of Cas12a Linker-NLS Constructs

[00551] Purified recombinant Cas12a protein containing the linker-NLS sequences shown in Table 22 was complexed with chRDNA guide targeting the TRAC gene (SEQ ID NO:467) as described in Example 2, and these complexes were transferred into primary T cells as described in Example 3. At 48 hours post-transfection, the final genome editing efficiency of each Cas12a chRDNA guide/nucleoprotein complex was determined as described in Example 4. The results of the cell editing experiment are shown in Fig. 16A and presented in Table 23.

Table 23
Screening for Cas12a Linker-NLS Editing in Cells SEQ ID NO: Name Tags FIG16A Average StDev SEQ ID NO:489 (G4S)2-NPL 1601 87.1% 0.7% SEQ ID NO:485 (G4S)2-SV40-GS-NPL 1602 86.1% 1.4% SEQ ID NO:487 (G4S)2-SV40 1603 85.9% 1.6% SEQ ID NO:483 GS-SV40-GS-NPL 1604 82.9% 2.7% SEQ ID NO:490 (G4S)2-NPL-GS-NPL 1605 83.6% 1.1% SEQ ID NO:488 (G4S)2-SV40-GS-SV40 1606 80.8% 0.8% SEQ ID NO:482 GS-NPL-GS-NPL 1607 77.2% 3.6% SEQ ID NO:486 (G4S)2-NPL-GS-SV40 1608 73.9% 0.7% SEQ ID NO:480 GS-SV40-GS-SV40 1609 71.1% 2.6% SEQ ID NO:484 GS-NPL-GS-SV40 1610 68.1% 1.2% SEQ ID NO:479 GS-SV40* 1611 65.9% 1.4% SEQ ID NO:481 GS-NPL 1612 65.3% 3.5

StDev=standard deviation; n=3; *=NLS construct used in previous examples

[00552] The data presented in Fig. 16A, as well as in Table 23 of this Example, showed that alternative linkers and NLS sequences can provide enhanced editing compared to a construct having a single GS-SV40 NLS configuration.

[00553] C. Cellular Activity of Alternative Cas12a-NLS Linker Sequences on Targets

[00554] The four main Cas12a-NLS linker configurations shown in Table 23 (SEQ ID NO:489, SEQ ID NO:485, SEQ ID NO:487, and SEQ ID NO:483) and the “non-optimized” construct (SEQ ID NO:479) were selected for comparison using a mixed panel of crRNA and chRDNA. The targeting region, including the DNA base positions in the chRDNA constructs, is shown in Table 24.

Table 24
Cas12a targets for comparison with linker-NLS SEQ ID NO. Target Type of guide sequence chRDNA design SEQ ID NO:396 RPL32-tgt1 chRDNA 11, 14, 15 SEQ ID NO:404 DNMT1-tgt3 crRNA - SEQ ID NO:321 TRAC-tgt12 chRDNA 1, 11, 20 SEQ ID NO:491 TRAC-tgt25 crRNA - SEQ ID NO:492 TRAC-tgt28 crRNA - SEQ ID NO:335 B2M-tgt12 chRDNA 1, 11, 20 SEQ ID NO:347 B2M-tgt1 chRDNA 8, 14, 19 SEQ ID NO:355 B2M-intron-tgt12 chRDNA 11, 17, 20

[00555] The targeting regions shown in Table 24 were fused to the 3' end of the activating region (SEQ ID NO:459) and submitted to a commercial manufacturer for synthesis.

[00555] Individual configurations of the Cas12a-NLS linker complexed with each guide molecule were prepared essentially as described in Example 2, except that the complexes were assembled at two concentrations of 20:60 and 80:240 pmol Cas12a as the guide sequence for each combination of Cas12a-NLS linker and guide molecule. The Cas12a guide/nucleoprotein complexes were transfected into primary T cells as described in Example 3, and the final genome editing efficiency of the Cas12a guide chRDNA/nucleoprotein complexes was determined as described in Example 4.

[00557] The results of editing the configuration of the Cas12a-NLS linker in complex with the guide molecule shown in Table 24 are shown in Fig. 16B and Tables 25 and 26.

Table 25
Editing activity of Cas12a linker-NLS constructs for all targets SEQ ID NO: SEQ ID NO:396 SEQ ID NO:404 SEQ ID NO:321 SEQ ID NO:491 Target name RPL32-tgt1 DNMT1-tgt3 TRAC-tgt12 TRAC-tgt25 Cas12a-NLS Fig. 16B-identifier Cas12a-guide [pmol] GS-SV40* (SEQ ID NO:479) Fig. 16B - 1613 20:60 Average 21.8% 61.3% 71.3% 93.4% StDev 0.0% 3.8% 5.5% 2.2% 80:240 Average 69.8% 68.6% 77.4% 96.1% StDev 0.4% 1.6% 3.4% 0.0% GS-SV40-GS-NPL (SEQ ID NO:483) Fig. 16B - 1614 20:60 Average 85.5% 80.2% 90.5% 96.1% StDev 3.7% 16.5% 4.1% 1.0% 80:240 Average 91.2% 91.4% 92.9% 97.6% StDev 0.1% 0.1% 0.7% 0.4% (G4S)2-SV40 (SEQ ID NO:487) Fig. 16B - 1615 20:60 Average 32.0% 77.1% 87.5% 94.4% StDev 45.3% 11.2% 2.9% 1.5% 80:240 Average 89.9% 87.4% 91.8% 95.6% StDev 0.9% 0.3% 0.8% 0.8% (G4S)2-NPL (SEQ ID NO:489) Fig. 16B - 1616 20:60 Average 40.1% 84.6% 87.0% 93.6% StDev 56.7% 0.4% 1.9% 0.4% 80:240 Average 91.8% 90.9% 93.4% 96.4% StDev 1.1% 0.4% 0.1% 0.0% (G4S)2-SV40-GS-NPL (SEQ ID NO:485) Fig. 16B - 1617 20:60 Average 88.8% 80.4% 83.4% 95.5% StDev 1.7% 3.7% 1.5% 0.3% 80:240 Average 87.9% 89.0% 90.4% 97.0% StDev 0.3% 5.6% 5.7% 0.5%

Table 26
Editing activity of Cas12a linker-NLS constructs for all targets SEQ ID NO: SEQ ID NO:492 SEQ ID NO:335 SEQ ID NO:347 SEQ ID NO:355 Target name TRAC-tgt28 B2M-tgt12 B2M-tgt1 B2M-intron-tgt12 Cas12a-NLS Fig. 16B - identifier Cas12a-guide [pmol] GS-SV40* (SEQ ID NO:479) Fig. 16B - 1613 20:60 Average 84.4% 92.9% 88.7% 80.3% StDev 5.2% 1.6% 1.4% 0.6% 80:240 Average 89.9% 95.5% 95.9% 91.3% StDev 0.1% 0.2% 0.0% 0.4% GS-SV40-GS-NPL (SEQ ID NO:483) Fig. 16B - 1614 20:60 Average 93.7% 95.8% 90.2% 92.4% StDev 0.8% 0.6% 0.8% 2.3% 80:240 Average 94.9% 97.1% 97.2% 96.0% StDev 1.7% 0.1% 0.2% 0.1% (G4S)2-SV40 (SEQ ID NO:487) Fig. 16B - 1615 20:60 Average 92.1% 93.7% 91.7% 89.8% StDev 0.5% 1.7% 0.6% 1.8% 80:240 Average 94.6% 95.0% 96.5% 94.2% StDev 1.2% 0.0% 1.3% 0.9% (G4S)2--NPL (SEQ ID NO:489) Fig. 16B - 1616 20:60 Average 91.2% 93.7% 89.7% 89.8% StDev 0.6% 1.3% 0.8% 0.8% 80:240 Average 94.1% 96.1% 96.9% 94.9% StDev 0.5% 0.0% 0.7% 0.9% (G4S)2-SV40-GS-NPL (SEQ ID NO:485) Fig. 16B - 1617 20:60 Average 88.1% 93.9% 80.3% 88.1% StDev 0.4% 0.4% 2.9% 2.6% 80:240 Average 94.6% 96.2% 95.6% 93.9% StDev 0.3% 0.5% 0.7% 0.5% StDev=standard deviation; n=3; *=NLS construct used in previous examples

[00558] The data presented in Fig. 16B and Tables 25 and 26 demonstrated increased activity of various NLS configurations against several targets in human primary T cells (see, for example, the average editing scores in Figs. 16B-1613 compared to the average editing scores in Figs. 16B-1616 or Figs. 16B-1617). Alternative NLS sequences, linkers, and Cas nucleases can be screened in a manner similar to the methods described herein.

Example 12

Reaction of formation of multiplex complexes directing Cas12a chRDNA/nucleoprotein

[00559] This example describes the co-delivery of multiple chRDNA Cas12a in a single transfection reaction (multiplex complex formation reaction) into a cell and a comparison of the rates of multiplex editing by the Cas12a chRDNA guide/nucleoprotein complexes with GS-SV40 (SEQ ID NO:479; "non-optimized NLS") and (G4S)2-NPL (SEQ ID NO:489; "optimized NLS").

[00560] A. In silico construction of Cas12a chRDNA-NLS linker sequences

[00561] The Acidaminococcus sp . (strain BV3L6) Cas12a protein (SEQ ID NO:01) was constructed with either the first C-terminal linker-NLS sequence (SEQ ID NO:479) or the second C-terminal linker-NLS sequence (SEQ ID NO:489), and the recombinant Cas12a protein was expressed as described in Example 2.

[00562] B. Activity of formation of multiplex complex with Cas12a-NLS linker.

[00563] Purified recombinant Cas12a proteins containing either the linker-NLS sequence of SEQ ID NO:479 or the linker-NLS sequence of SEQ ID NO:489 were complexed with either the chRDNA gene, aimed atTRAC(SEQ ID NO:508), or with the chRDNA gene targetingB2M(SEQ ID NO:416), or with the chRDNA gene targetingCISH(SEQ ID NO:509), or with the chRDNA gene targetingCBLB(SEQ ID NO:510) as described in Example 2, except that these complexes were assembled in a ratio of 40:80 pmol Cas12a:targeting molecule. Each Cas12a chRDNA guide/nucleoprotein complex was used as a single targeting complex; Cas12a chRDNA guide/nucleoprotein complexes targetingTRACAnd B2M, were combined into one mixture; Cas12a guide chRDNA/nucleoprotein complexes targetingCISHAnd CBLB, were combined into one mixture; or Cas12a chRDNA/nucleoprotein guide complexes targetingTRAC,B2M,CISHAnd CBLB, were combined into one mixture. Each Cas12a chRDNA/nucleoprotein composition was transferred into primary T cells as described in Example 3. Forty-eight hours after transfection, the final genome editing efficiency of each Cas12a chRDNA/nucleoprotein complex was determined as described in Example 4. The results of the in-cell editing experiment are shown in Fig. 17 and presented in Table 27.

Table 27
Cellular editing activity via Cas12a-NLS linker multiplex constructs Unoptimized NLS Optimized NLS Number of multiplexed targeting molecules Name
targets SEQ ID NO. rep-1 rep-2 rep-1 rep-2 One guide molecule TRAC SEQ ID NO:36 75.07% 3.35% 86.33% 0.91% B2M SEQ ID NO:62 83.60% 86.50% 89.60% 91.00% CISH SEQ ID NO:158 51.40% 59.00% 83.10% 89.60% CBLB SEQ ID NO:171 47.50% 44.90% 74.00% 81.40% Two guide molecules TRAC SEQ ID NO:36 81.70% 80.80% 93.90% 92.20% B2M SEQ ID NO:62 88.00% 91.30% 93.70% 92.20% Two guide molecules CISH SEQ ID NO:158 64.80% 63.30% 94.50% 93.10% CBLB SEQ ID NO:171 66.20% 60.20% 90.90% 88.30% Four guide molecules TRAC SEQ ID NO:36 78.40% 82.00% 91.20% 93.10% B2M SEQ ID NO:62 90.40% 92.20% 92.40% 94.20% CISH SEQ ID NO:158 53.20% 58.70% 90.80% 91.80% CBLB SEQ ID NO:171 51.60% 58.00% 86.40% 88.20%

[00564] The data presented in Fig. 17 and Table 27 demonstrated the enhanced activity of the linker-NLS construct when used in a multiplex complex formation reaction in human primary T cells. See, for example , the average edit of the runs in Fig. 17, 1707 with the non-optimized GS-SV40-NLS linker (SEQ ID NO: 479, Fig. 17, 1708) and the average edit of the runs in Fig. 17, 1711 with the optimized (G4S)2-NPL-NLS linker (SEQ ID NO: 489, Fig. 17, 1712). Alternative NLS sequences, linkers, Cas12 nucleases, and multiplex reaction targets can be screened in a manner similar to the methods described herein.

Example 13

Editing by Cas12a chRDNA/nucleoprotein guide complexes involving chemical modifications

[00565] This example describes the editing activities of chRDNA Cas12a containing phosphorothioate chemical modifications in the activating and targeting region of Cas12a guide RNA in cells.

[00565] A. In silico construction of chRDNA Cas12a with chemical modifications

[00567] A TRAC -12 targeting sequence (SEQ ID NO:316) was selected for design. Two phosphorothioate linkages were introduced at the 5' end of the Cas12a guide molecule (i.e. , the 5' end nucleotides of the activating region), and two phosphorothioate linkages were introduced at the 3' end of the Cas12a guide molecule (i.e., the 3' end nucleotides of the targeting region). A series of Cas12a guide sequences were then designed with DNA bases in the activating region in addition to phosphorothioate modifications protecting the termini. The Cas12a chRDNA sequences with chemical modifications are shown in Table 28. The sequences were submitted to a commercial manufacturer for synthesis (the phosphorothioate linkages and DNA base positions listed correspond to the numbers shown in Fig. 5).

Table 28
Cas12a chRDNA with chemical modification SEQ ID NO: Phosphorothioate DNA Subsequence Activating
region Spacer Activating
region SEQ ID NO:511 - - - rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrUrCrUrCrUrCrArGrCrUrGrGrUrArCrArC SEQ ID NO:512 1,2 38, 39 rU*rA*rArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrUrCrUrCrUrCrArGrCrUrGrGrUrArC*rA*rC SEQ ID NO:513 1,2 38, 39 1 T*rA*rArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrUrCrUrCrUrCrArGrCrUrGrGrUrArC*rA*rC SEQ ID NO:514 1, 2, 7 38, 39 7 rU*rA*rArUrUrUC*rUrArCrUrCrUrUrGrUrArGrArUrGrArGrUrCrUrCrUrCrArGrCrUrGrGrUrArC*rA*rC SEQ ID NO:515 1, 2, 10 38, 39 10 rU*rA*rArUrUrUrCrUrAC*rUrCrUrUrGrUrArGrArUrGrArGrUrCrUrCrUrCrArGrCrUrGrGrUrArC*rA*rC SEQ ID NO:516 1, 2, 12 38, 39 12 rU*rA*rArUrUrUrCrUrArCrUC*rUrUrGrUrArGrArUrGrArGrUrCrUrCrUrCrArGrCrUrGrGrUrArC*rA*rC SEQ ID NO:517 1, 2, 14 38, 39 14 rU*rA*rArUrUrUrCrUrArCrUrCrUT*rGrUrArGrArUrGrArGrUrCrUrCrUrCrArGrCrUrGrGrUrArC*rA*rC Phosphorothioate bonds are indicated by the symbol "*".

[00568] B. Transfection and Cell Analysis

[00569] Individual Cas12a guide/nucleoprotein complexes were prepared essentially as described in Example 2. The nucleoprotein complexes were transferred into primary T cells as described in Example 3, and the final genome editing efficiency of the Cas12a guide/nucleoprotein complexes was determined as described in Example 4. The results of the in-cell editing experiment are shown in Table 29.

Table 29
Chemically Modified Cas12a chRDNA Editing in Cells SEQ ID NO. Position of the phosphorothioate bond Position of DNA bases Average StDev Activating region Targeting area Activating region SEQ ID NO:511 - - - 74.8% 1.6% SEQ ID NO:512 1, 2 38, 39 78.6% 4.7% SEQ ID NO:513 1, 2, 1 38, 39 1 71.4% 1.9% SEQ ID NO:514 1, 2, 7 38, 39 7 75.8% 3.7% SEQ ID NO:515 1, 2, 10 38, 39 10 79.8% 2.4% SEQ ID NO:516 1, 2, 12 38, 39 12 70.2% 0.7% SEQ ID NO:517 1, 2, 14 38, 39 14 72.4% 5.0%

StDev=standard deviation; n=2

[00570] The data presented in Table 29 demonstrated the editing activity of Cas12a guide molecules containing either phosphorothioate linkages only or phosphorothioate linkages and DNA bases. These guide molecules provide robust editing in primary human T cells compared to all guide RNAs (see, e.g., Table 29, i.e., where the average edit of SEQ ID NO:511 is indicated compared to the average edit of SEQ ID NO:512 or SEQ ID NO:515). Alternative combinations and positions of chemical modification can be screened by methods similar to those described herein.

Example 14

Transfection of human induced pluripotent stem cells with Cas12a chRDNA/nucleoprotein complexes

[00571] This Example describes editing of human induced pluripotent stem cells (iPSCs) via Cas12a chRDNA/nucleoprotein complexes.

[00572] A. In silico construction of chRDNA guides

[00573] The AsCas12a guide sequence targeting the CISH gene (SEQ ID NO:509) and containing DNA bases in the activating region of the guide molecule was selected for further design and introduction of additional DNA bases into the targeting region (SEQ ID NO:518 - SEQ ID NO:529). The sequences were submitted to a commercial manufacturer for synthesis.

[00574] B. Transfection and Cell Analysis

[00575] Individual Cas12a guide/nucleoprotein complexes were prepared essentially as described in Example 2. The nucleoprotein complexes were transferred into primary T cells as described in Example 3.

[00575] iPSCs were treated and transfected in a manner similar to the treatment and transfection of primary T cells described in Example 3, but with the following modifications. iPSCs were cultured in mTeSR-plus medium (STEMCELL Technologies, Cambridge, MA) supplemented with Rho-associated protein kinase inhibitor containing a supercoiled group (“ROCKi”, MilliporeSigma, Burlington, MA) at a final concentration of 10 μM for 3 hours at 37°C prior to transfection. The mTeSR-plus/ROCKi medium was removed, iPSCs were washed with 10 ml PBS followed by the addition of 3 ml Accutase (STEMCELL Technologies, Cambridge, MA), and the cells were incubated for 5-10 minutes at 37°C. Then 7 ml of mTeSR pulse and ROCKi were added to the cells, after which the cells were mixed and counted. The cells were then centrifuged, the medium was removed, the cells were washed with 10 ml of PBS, centrifuged again and the PBS was removed. The cells were resuspended in Nucleofector™ solution P3 (Lonza, Allendale, NJ) up to density 2⁵cells/ml mixed with Cas12a guide chRDNA/nucleoprotein complexes and transfected using the CA158 pulse code. The resulting genome editing efficiencies of Cas12a guide/nucleoprotein complexes were determined as described in Example 4 and the data are presented in Table 29.

Table 29
Cas12a chRDNA-mediated iPSC editing SEQ ID NO. DNA positions in the activating region DNA positions in the target recognition sequence Average StDev SEQ ID NO:509 1, 3, 7, 10, 12, 14, 15, 19 - 91.5% 0.4% SEQ ID NO:518 1, 3, 7, 10, 12, 14, 15, 19 1 75.5% 3.9% SEQ ID NO:519 1, 3, 7, 10, 12, 14, 15, 19 8 92.0% 0.0% SEQ ID NO:520 1, 3, 7, 10, 12, 14, 15, 19 9 92.0% 2.1% SEQ ID NO:521 1, 3, 7, 10, 12, 14, 15, 19 10 96.0% 0.7% SEQ ID NO:522 1, 3, 7, 10, 12, 14, 15, 19 11 95.0% 0.0% SEQ ID NO:523 1, 3, 7, 10, 12, 14, 15, 19 12 94.5% 0.4% SEQ ID NO:524 1, 3, 7, 10, 12, 14, 15, 19 14 92.5% 1.1% SEQ ID NO:525 1, 3, 7, 10, 12, 14, 15, 19 15 89.5% 4.6% SEQ ID NO:526 1, 3, 7, 10, 12, 14, 15, 19 17 91.0% 2.8% SEQ ID NO:527 1, 3, 7, 10, 12, 14, 15, 19 18 88.0% - SEQ ID NO:528 1, 3, 7, 10, 12, 14, 15, 19 19 85.5% 3.2% SEQ ID NO:529 1, 3, 7, 10, 12, 14, 15, 19 20 81.5% 0.4%

StDev=standard deviation; n=1-2

[00577] The data presented in Table 29 demonstrated that Cas12a guide chRDNA/nucleoprotein complexes can be used to engineer human iPSCs. Other cell types can be edited by methods similar to those described herein.

[00578] As will be apparent to those skilled in the art, modifications and changes to the above embodiments of the invention may be made without departing from the spirit and scope of the present invention. Such modifications and changes are within the scope of the present invention.

--->

SEQUENCE LISTING

<110> CARIBOU BIOSCIENCES, INC.

<120> DNA-CONTAINING POLYNUCLEOTIDES AND GUIDES FOR CRISPR TYPE V

SYSTEMS, AND METHODS OF MAKING AND USING THE SAME

<130> CBI039.30

<140> PCT/US2021/055394

<141> 2021-10-18

<150> 63/229,870

<151> 2021-08-05

<150> 63/127,648

<151> 2020-12-18

<150> 63/093,459

<151> 2020-10-19

<160> 529

<170> PatentIn version 3.5

<210> 1

<211> 1307

<212> PRT

<213> Acidaminococcus sp.

<220>

<223> Acidaminococcus sp. (strain BV3L6)

<220>

<223> Cas12a

<400> 1

Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln

20 25 30

Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys

35 40 45

Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln

50 55 60

Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile

65 70 75 80

Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile

85 90 95

Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly

100 105 110

Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile

115 120 125

Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys

130 135 140

Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg

145 150 155 160

Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg

165 170 175

Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg

180 185 190

Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe

195 200 205

Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn

210 215 220

Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val

225 230 235 240

Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp

245 250 255

Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu

260 265 270

Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn

275 280 285

Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro

290 295 300

Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu

305 310 315 320

Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr

325 330 335

Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu

340 345 350

Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His

355 360 365

Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr

370 375 380

Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys

385 390 395 400

Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu

405 410 415

Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser

420 425 430

Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala

435 440 445

Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys

450 455 460

Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu

465 470 475 480

Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe

485 490 495

Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser

500 505 510

Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val

515 520 525

Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp

530 535 540

Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn

545 550 555 560

Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys

565 570 575

Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys

580 585 590

Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys

595 600 605

Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr

610 615 620

Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys

625 630 635 640

Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln

645 650 655

Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala

660 665 670

Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr

675 680 685

Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr

690 695 700

Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His

705 710 715 720

Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu

725 730 735

Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys

740 745 750

Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu

755 760 765

Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln

770 775 780

Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His

785 790 795 800

Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr

805 810 815

Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His

820 825 830

Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn

835 840 845

Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe

850 855 860

Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln

865 870 875 880

Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu

885 890 895

Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg

900 905 910

Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu

915 920 925

Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu

930 935 940

Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val

945 950 955 960

Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile

965 970 975

His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu

980 985 990

Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu

995 1000 1005

Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu

1010 1015 1020

Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly

1025 1030 1035

Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala

1040 1045 1050

Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro

1055 1060 1065

Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe

1070 1075 1080

Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu

1085 1090 1095

Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe

1100 1105 1110

Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly

1115 1120 1125

Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn

1130 1135 1140

Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys

1145 1150 1155

Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr

1160 1165 1170

Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu

1175 1180 1185

Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu

1190 1195 1200

Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu

1205 1210 1215

Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly

1220 1225 1230

Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys

1235 1240 1245

Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp

1250 1255 1260

Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu

1265 1270 1275

Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile

1280 1285 1290

Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn

1295 1300 1305

<210> 2

<211> 1228

<212> PRT

<213> Unknown

<220>

<223> Description of Unknown:

Lachnospiraceae bacterium ND2006

<220>

<223> Cas12a

<400> 2

Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp

20 25 30

Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys

35 40 45

Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp

50 55 60

Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu

65 70 75 80

Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn

85 90 95

Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn

100 105 110

Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu

115 120 125

Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe

130 135 140

Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn

145 150 155 160

Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile

165 170 175

Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys

180 185 190

Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys

195 200 205

Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe

210 215 220

Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile

225 230 235 240

Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn

245 250 255

Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys

260 265 270

Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser

275 280 285

Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe

290 295 300

Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys

305 310 315 320

Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile

325 330 335

Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe

340 345 350

Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp

355 360 365

Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp

370 375 380

Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu

385 390 395 400

Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu

405 410 415

Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser

420 425 430

Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys

435 440 445

Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys

450 455 460

Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr

465 470 475 480

Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile

485 490 495

Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr

500 505 510

Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro

515 520 525

Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala

530 535 540

Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys

545 550 555 560

Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly

565 570 575

Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met

580 585 590

Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro

595 600 605

Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly

610 615 620

Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys

625 630 635 640

Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn

645 650 655

Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu

660 665 670

Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys

675 680 685

Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His

705 710 715 720

Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile

725 730 735

Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys

740 745 750

Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys

755 760 765

Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr

770 775 780

Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile

785 790 795 800

Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val

805 810 815

Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp

820 825 830

Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly

835 840 845

Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn

850 855 860

Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu

865 870 875 880

Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile

885 890 895

Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys

900 905 910

Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn

915 920 925

Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln

930 935 940

Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys

945 950 955 960

Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile

965 970 975

Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe

980 985 990

Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr

995 1000 1005

Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp

1010 1015 1020

Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro

1025 1030 1035

Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser

1040 1045 1050

Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr

1055 1060 1065

Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val

1070 1075 1080

Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu

1085 1090 1095

Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala

1100 1105 1110

Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met

1115 1120 1125

Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly

1130 1135 1140

Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp

1145 1150 1155

Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala

1160 1165 1170

Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala

1175 1180 1185

Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp

1190 1195 1200

Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp

1205 1210 1215

Leu Glu Tyr Ala Gln Thr Ser Val Lys His

1220 1225

<210> 3

<211> 1300

<212> PRT

<213> Francisella tularensis

<220>

<223> Francisella tularensis subsp. novicida (strain U112)

<220>

<223> Cas12a

<400> 3

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Met

1295 1300

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> Nuclear localization sequence (NLS)

<400> 4

Pro Lys Lys Lys Arg Lys Val

1 5

<210> 5

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> Nuclear localization sequence (NLS)

<400> 5

Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1 5 10 15

<210> 6

<211> 20

<212> RNA

<213> Acidaminococcus sp.

<220>

<223> Acidaminococcus sp. (strain BV3L6)

<220>

<223> AsCas12 CRISPR repeat

<400> 6

uaauuucuac ucuuguagau 20

<210> 7

<211> 40

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> AsCas12 crRNA

<220>

<221>modified_base

<222> (21)..(40)

<223> a, c, g, or u

<400> 7

uaauuucuac ucuuguagau nnnnnnnnnn nnnnnnnnnn 40

<210> 8

<211> 21

<212> RNA

<213> Unknown

<220>

<223> Description of Unknown:

Lachnospiraceae bacterium ND2006

<220>

<223> LbaCas12 CRISPR repeat

<400> 8

uaauuucuac uaaguguaga u 21

<210> 9

<211> 41

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> LbaCas12 crRNA

<220>

<221>modified_base

<222> (22)..(41)

<223> a, c, g, or u

<400> 9

uaauuucuac uaaguguaga unnnnnnnnn nnnnnnnnnn n 41

<210> 10

<211> 20

<212> RNA

<213> Francisella tularensis

<220>

<223> Francisella tularensis subsp. novicida (strain U112)

<220>

<223> FnoCas12 CRISPR repeat

<400> 10

uaauuucuac uguuguagau 20

<210> 11

<211> 40

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> FnoCas12 crRNA

<220>

<221>modified_base

<222> (21)..(40)

<223> a, c, g, or u

<400> 11

uaauuucuac uguuguagau nnnnnnnnnn nnnnnnnnnn 40

<210> 12

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt1

<400> 12

tttctctcct ctttcccaca gaat 24

<210> 13

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt2

<400> 13

tttcccacag aatttagcat ggct 24

<210> 14

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt3

<400> 14

tttagcatgg ctcagacagt cact 24

<210> 15

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt4

<400> 15

tttattctgg tacaagcagc ctcc 24

<210> 16

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt5

<400> 16

tttctggaag ttcacagaga aacg 24

<210> 17

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt6

<400> 17

tttctctgtg aacttccaga aagc 24

<210> 18

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt7

<400> 18

tttggctgct ttctggaagt tcac 24

<210> 19

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt8

<400> 19

tttatcaacc tcaccaactc acac 24

<210> 20

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt9

<400> 20

tttgctgtca gtccaagctc catg 24

<210> 21

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt10

<400> 21

tttccaaaga caagaggtgt gttt 24

<210> 22

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt11

<400> 22

tttggaaagg gcacaagact ttct 24

<210> 23

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt12

<400> 23

tttagagtct ctcagctggt acac 24

<210> 24

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt13

<400> 24

tttgagaatc aaaatcggtg aata 24

<210> 25

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt14

<400> 25

tttgtttgag aatcaaaatc ggtg 24

<210> 26

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt15

<400> 26

tttgattctc aaacaaatgt gtca 24

<210> 27

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt16

<400> 27

tttgtgacac atttgtttga gaat 24

<210> 28

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt17

<400> 28

tttgtctgtg atatacacat caga 24

<210> 29

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt18

<400> 29

tttgttgctc caggccacag cact 24

<210> 30

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt19

<400> 30

tttgcacatg caaagtcaga tttg 24

<210> 31

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt20

<400> 31

tttgcatgtg caaacgcctt caac 24

<210> 32

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt21

<400> 32

tttcctttta gaaagttcct gtga 24

<210> 33

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt22

<400> 33

tttagaaagt tcctgtgatg tcaa 24

<210> 34

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt23

<400> 34

tttctcgacc agcttgacat caca 24

<210> 35

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt24

<400> 35

tttcaaagct tttctcgacc agct 24

<210> 36

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt25

<400> 36

tttacagata cgaacctaaa cttt 24

<210> 37

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt26

<400> 37

tttgaaagtt taggttcgta tctg 24

<210> 38

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt27

<400> 38

tttcaaaacc tgtcagtgat tggg 24

<210> 39

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt28

<400> 39

tttcaggagg aggattcgga accc 24

<210> 40

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt29

<400> 40

tttaatctgc tcatgacgct gcgg 24

<210> 41

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt30

<400> 41

tttggagagg gagaagaggg gcaa 24

<210> 42

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt31

<400> 42

tttcacctcc ttggggggtag gaga 24

<210> 43

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt32

<400> 43

tttatgatta agattgctga agag 24

<210> 44

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt33

<400> 44

tttggcagct cttcagcaat ctta 24

<210> 45

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt34

<400> 45

tttatttttt tttaatagtg ttca 24

<210> 46

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt35

<400> 46

tttatgaaca ctattaaaaa aaaa 24

<210> 47

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt36

<400> 47

tttctttatg aacactatta aaaa 24

<210> 48

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt37

<400> 48

tttaatagtg ttcataaaga aata 24

<210> 49

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt38

<400> 49

tttcccccca cgtcttgaga agaa 24

<210> 50

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> TRAC-tgt39

<400> 50

tttaatgaag gcatcggcag cagg 24

<210> 51

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt1

<400> 51

tttaatataa gtggaggcgt cgcg 24

<210> 52

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt2

<400> 52

tttctggcct ggaggctatc cagc 24

<210> 53

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt3

<400> 53

tttcccgata ttcctcaggt actc 24

<210> 54

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt4

<400> 54

tttactcacg tcatccagca gaga 24

<210> 55

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt5

<400> 55

tttccattct ctgctggatg acgt 24

<210> 56

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt6

<400> 56

tttgactttc cattctctgc tgga 24

<210> 57

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt7

<400> 57

tttcctgaat tgctatgtgt ctgg 24

<210> 58

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt8

<400> 58

tttcatccat ccgacattga agtt 24

<210> 59

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt9

<400> 59

tttcaattct ctctccatc ttca 24

<210> 60

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt10

<400> 60

tttcagcaag gactggtctt tcta 24

<210> 61

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt11

<400> 61

tttctatctc ttgtactaca ctga 24

<210> 62

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt12

<400> 62

tttcagtggg ggtgaattca gtgt 24

<210> 63

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt13

<400> 63

tttgtcacag cccaagatag ttaa 24

<210> 64

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt14

<400> 64

tttctccact gtctttttca taga 24

<210> 65

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt15

<400> 65

tttcatagat cgagacatgt aagc 24

<210> 66

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt16

<400> 66

tttctcaagg tcaaaaactt acct 24

<210> 67

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt17

<400> 67

tttcttttct tttcaggttt gaag 24

<210> 68

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt18

<400> 68

tttcttttca ggtttgaaga tgcc 24

<210> 69

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt19

<400> 69

tttcaggttt gaagatgccg catt 24

<210> 70

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt20

<400> 70

tttgaagatg ccgcatttgg attg 24

<210> 71

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt21

<400> 71

tttggaattc atccaatcca aatg 24

<210> 72

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt22

<400> 72

tttggattgg atgaattcca aatt 24

<210> 73

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt23

<400> 73

tttaatattg atatgcttat acac 24

<210> 74

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt24

<400> 74

tttgtgcata aagtgtaagt gtat 24

<210> 75

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt25

<400> 75

tttatgcaca aaatgtaggg ttat 24

<210> 76

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt26

<400> 76

tttataattc tactttgagt gctg 24

<210> 77

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt27

<400> 77

tttgagtgct gtctccatgt ttga 24

<210> 78

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt28

<400> 78

tttgatgtat ctgagcaggt tgct 24

<210> 79

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt29

<400> 79

tttgaactct tcaatctctt gcac 24

<210> 80

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt30

<400> 80

tttgagtgca agagattgaa gagt 24

<210> 81

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt31

<400> 81

tttacatact ctgcttagaa tttg 24

<210> 82

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt32

<400> 82

tttcccccaa attctaagca gagt 24

<210> 83

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt33

<400> 83

tttctaaatt ttcccccaaa ttct 24

<210> 84

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt34

<400> 84

tttgggggaa aatttagaaa tata 24

<210> 85

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt35

<400> 85

tttagaaata taattgacag gatt 24

<210> 86

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt36

<400> 86

tttccaataa tcctgtcaat tata 24

<210> 87

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt37

<400> 87

tttcattcat tataacaaat ttcc 24

<210> 88

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt38

<400> 88

tttgttataa tgaatgaaac attt 24

<210> 89

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt39

<400> 89

tttgtcatat aagattcata ttta 24

<210> 90

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt40

<400> 90

tttacttctt atacatttga taaa 24

<210> 91

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt41

<400> 91

tttatcaaat gtataagaag taaa 24

<210> 92

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt42

<400> 92

tttgataaag taaggcatgg ttgt 24

<210> 93

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt43

<400> 93

tttatttttg ttccacaagt taaa 24

<210> 94

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt44

<400> 94

tttaacttgt ggaacaaaaa taaa 24

<210> 95

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt45

<400> 95

tttatttaac ttgtggaaca aaaa 24

<210> 96

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt46

<400> 96

tttgttccac aagttaaata aatc 24

<210> 97

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt47

<400> 97

tttatgattt atttaacttg tgga 24

<210> 98

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt48

<400> 98

tttgaaaata aaggggtaat agtg 24

<210> 99

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt49

<400> 99

tttccctgtt tgaaaataaa gggg 24

<210> 100

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt50

<400> 100

tttattttca aacagggaaa cagt 24

<210> 101

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt51

<400> 101

tttcaaacag ggaaacagtc ttca 24

<210> 102

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt52

<400> 102

tttaccaagt ggaacttgaa gact 24

<210> 103

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt53

<400> 103

tttacactgt gagccaaact ctat 24

<210> 104

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt54

<400> 104

tttggctcac agtgtaaagg gcct 24

<210> 105

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt55

<400> 105

tttccagatt aggaatctga tgct 24

<210> 106

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt56

<400> 106

tttgagcatc agattcctaa tctg 24

<210> 107

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt57

<400> 107

tttaacttct ttgagcatca gatt 24

<210> 108

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt58

<400> 108

tttagccagc gttctttcct gcgg 24

<210> 109

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt59

<400> 109

tttcctgcgg gccaggtcat gagg 24

<210> 110

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt60

<400> 110

tttgctccct cttagagtct gcat 24

<210> 111

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt61

<400> 111

tttaatattt tagcaaggaa taga 24

<210> 112

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt62

<400> 112

tttagcaagg aatagatata caat 24

<210> 113

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt63

<400> 113

tttcaggacc ccttcttgga cacc 24

<210> 114

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt64

<400> 114

tttggtgtcc aagaaggggt cctg 24

<210> 115

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt65

<400> 115

tttatagaag ggacttaaat atcc 24

<210> 116

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt66

<400> 116

tttaagtccc ttctataaaa tggt 24

<210> 117

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt67

<400> 117

tttgcatata acctatccac atcc 24

<210> 118

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt68

<400> 118

tttaaagtat acaggaggat gtgg 24

<210> 119

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt69

<400> 119

tttaaatcat ttctagatta cttg 24

<210> 120

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt70

<400> 120

tttctagatt acttgtaata ccta 24

<210> 121

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt71

<400> 121

tttacattgt attaggtatt acaa 24

<210> 122

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt72

<400> 122

tttgcatagc atttacattg tatt 24

<210> 123

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt73

<400> 123

tttcttgtca ttattcctta aaca 24

<210> 124

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt74

<400> 124

tttaaggaat aatgacaaga aaaa 24

<210> 125

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt75

<400> 125

tttactgagc atgtacagac tttt 24

<210> 126

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt76

<400> 126

tttccccagt gtttttgatc catg 24

<210> 127

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt77

<400> 127

tttgatccat ggtttgctga atcc 24

<210> 128

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt78

<400> 128

tttgctgaat ccacagatgt ggag 24

<210> 129

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt79

<400> 129

tttgtcattc aaagtacagc gggc 24

<210> 130

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt80

<400> 130

tttgaatgac aaataacaga ttta 24

<210> 131

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt81

<400> 131

tttaaatctg ttatttgtca ttca 24

<210> 132

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt82

<400> 132

tttaaaattt tcaaggcata gttt 24

<210> 133

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> B2M-tgt83

<400> 133

tttcaaggca tagttttata cctg 24

<210> 134

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt1

<400> 134

tttaattata attataatat aata 24

<210> 135

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt2

<400> 135

tttaaagggg ttggccggggc tccc 24

<210> 136

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt3

<400> 136

tttaaataat ttcaggaatg ggtt 24

<210> 137

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt4

<400> 137

tttcaggaat gggttccaag gaga 24

<210> 138

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt5

<400> 138

tttcaggccc agccagcact ctgg 24

<210> 139

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt6

<400> 139

tttgtggggc agggaagctg aggc 24

<210> 140

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt7

<400> 140

tttacacatg cccaggcagc acct 24

<210> 141

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt8

<400> 141

tttcctgccc tgcccaccac agcc 24

<210> 142

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt9

<400> 142

tttcagggaa ggtcagaaga gctc 24

<210> 143

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt10

<400> 143

tttcgggatg ccactgccag gggc 24

<210> 144

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt11

<400> 144

tttcctcagg agaagcaggc aggg 24

<210> 145

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt12

<400> 145

tttcctagcg gaatggggcac ctca 24

<210> 146

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt13

<400> 146

tttccagtgg cgagagaaga cccc 24

<210> 147

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt14

<400> 147

tttctcctca aagaaggagg accc 24

<210> 148

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt15

<400> 148

tttctctgca gggacaatag gagc 24

<210> 149

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt16

<400> 149

tttggaactg gccggctggc ctgg 24

<210> 150

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt17

<400> 150

tttgtgccct tccagagaga aggg 24

<210> 151

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt18

<400> 151

tttgatctgc gccttggggg ccag 24

<210> 152

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt19

<400> 152

tttagcacga agctctccga tgtg 24

<210> 153

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt20

<400> 153

tttcccttcc gctcacctcc gcct 24

<210> 154

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt21

<400> 154

cttactgcct cagcttccct gccc 24

<210> 155

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>PDCD1-tgt22

<400> 155

cttagcatgc tctcatattt aatt 24

<210> 156

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt1

<400> 156

tttaccctag gtgagcaccc cctt 24

<210> 157

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt2

<400> 157

tttgttccct gacacccaac agta 24

<210> 158

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt3

<400> 158

tttagactgc tgcgctccaa agcg 24

<210> 159

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt4

<400> 159

tttggagcgc agcagtctaa aact 24

<210> 160

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt5

<400> 160

tttgtacgca gaagcagtgc ccgc 24

<210> 161

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt6

<400> 161

tttaggtgta cagcagtggc tggt 24

<210> 162

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt7

<400> 162

tttccggatg tggtcagcct tgtg 24

<210> 163

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt8

<400> 163

tttcactgac agcgtgaaca ggta 24

<210> 164

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt9

<400> 164

tttggctcac tctctgtctg ggct 24

<210> 165

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CISH-tgt10

<400> 165

tttgctggct gtggagcgga ctgg 24

<210> 166

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt1

<400> 166

tttctgccat tcattgagtt tgcc 24

<210> 167

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt2

<400> 167

tttcctcctc gaccaccagg gttt 24

<210> 168

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>CBLB-tgt3

<400> 168

tttcggggat ttcctcctcg acca 24

<210> 169

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt4

<400> 169

tttgggtatt attgatgcta ttca 24

<210> 170

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt5

<400> 170

tttggggattt tggcacagtc ttac 24

<210> 171

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt6

<400> 171

tttgcctgat acatatcagc attt 24

<210> 172

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt7

<400> 172

tttaaagtac tcattctcac tgag 24

<210> 173

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt8

<400> 173

tttgactttt tcataaggct atca 24

<210> 174

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt9

<400> 174

tttaaagagt cttattgccc gttt 24

<210> 175

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>CBLB-tgt10

<400> 175

tttcttagct gtgacacatc cagg 24

<210> 176

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt11

<400> 176

tttctgtagt cgtgctttaa cttc 24

<210> 177

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt12

<400> 177

tttggtgcta tatttctgta gtcg 24

<210> 178

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt13

<400> 178

tttcacaata taattcatat tgtt 24

<210> 179

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt14

<400> 179

tttgtcattc tctgcacaaa tctt 24

<210> 180

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt15

<400> 180

tttcagctct gtaagatttg tgca 24

<210> 181

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>CBLB-tgt16

<400> 181

tttgtgcaga gaatgacaaa gatg 24

<210> 182

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt17

<400> 182

tttgatgtgc acctcttgcc ttac 24

<210> 183

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt17

<400> 183

tttgatgtgc acctcttgcc ttac 24

<210> 184

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt17

<400> 184

tttgatgtgc acctcttgcc ttac 24

<210> 185

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>CBLB-tgt20

<400> 185

tttctgtcgt tgtgaaataa aagg 24

<210> 186

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt21

<400> 186

tttcaggtgg cggtggagga ggat 24

<210> 187

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt22

<400> 187

tttccacatg atggatgtgt ctac 24

<210> 188

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt23

<400> 188

tttgggacta atcagcttgt ggga 24

<210> 189

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> CBLB-tgt24

<400> 189

tttgaactcg ctgtgattcc aggt 24

<210> 190

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> crRNA

<220>

<221>modified_base

<222> (1)..(20)

<223> a, c, g, or u

<400> 190

nnnnnnnnnn nnnnnnnnnn 20

<210> 191

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d1

<220>

<221>modified_base

<222> (1)..(1)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (2)..(20)

<223> a, c, g, or u

<400> 191

nnnnnnnnnn nnnnnnnnnn 20

<210> 192

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d2

<220>

<221>modified_base

<222> (1)..(1)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (2)..(2)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (3)..(20)

<223> a, c, g, or u

<400> 192

nnnnnnnnnn nnnnnnnnnn 20

<210> 193

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d3

<220>

<221>modified_base

<222> (1)..(2)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (3)..(3)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (4)..(20)

<223> a, c, g, or u

<400> 193

nnnnnnnnnn nnnnnnnnnn 20

<210> 194

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d4

<220>

<221>modified_base

<222> (1)..(3)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (4)..(4)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (5)..(20)

<223> a, c, g, or u

<400> 194

nnnnnnnnnn nnnnnnnnnn 20

<210> 195

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d5

<220>

<221>modified_base

<222> (1)..(4)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (5)..(5)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (6)..(20)

<223> a, c, g, or u

<400> 195

nnnnnnnnnn nnnnnnnnnn 20

<210> 196

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d6

<220>

<221>modified_base

<222> (1)..(5)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (6)..(6)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (7)..(20)

<223> a, c, g, or u

<400> 196

nnnnnnnnnn nnnnnnnnnn 20

<210> 197

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d7

<220>

<221>modified_base

<222> (1)..(6)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (7)..(7)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (8)..(20)

<223> a, c, g, or u

<400> 197

nnnnnnnnnn nnnnnnnnnn 20

<210> 198

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d8

<220>

<221>modified_base

<222> (1)..(7)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (8)..(8)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (9)..(20)

<223> a, c, g, or u

<400> 198

nnnnnnnnnn nnnnnnnnnn 20

<210> 199

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d9

<220>

<221>modified_base

<222> (1)..(8)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (9)..(9)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (10)..(20)

<223> a, c, g, or u

<400> 199

nnnnnnnnnn nnnnnnnnnn 20

<210> 200

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d10

<220>

<221>modified_base

<222> (1)..(9)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (10)..(10)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (11)..(20)

<223> a, c, g, or u

<400> 200

nnnnnnnnnn nnnnnnnnnn 20

<210> 201

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d11

<220>

<221>modified_base

<222> (1)..(10)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (11)..(11)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (12)..(20)

<223> a, c, g, or u

<400> 201

nnnnnnnnnn nnnnnnnnnn 20

<210> 202

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d12

<220>

<221>modified_base

<222> (1)..(11)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (12)..(12)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (13)..(20)

<223> a, c, g, or u

<400> 202

nnnnnnnnnn nnnnnnnnnn 20

<210> 203

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d13

<220>

<221>modified_base

<222> (1)..(12)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (13)..(13)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (14)..(20)

<223> a, c, g, or u

<400> 203

nnnnnnnnnn nnnnnnnnnn 20

<210> 204

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d14

<220>

<221>modified_base

<222> (1)..(13)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (14)..(14)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (15)..(20)

<223> a, c, g, or u

<400> 204

nnnnnnnnnn nnnnnnnnnn 20

<210> 205

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d15

<220>

<221>modified_base

<222> (1)..(14)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (15)..(15)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (16)..(20)

<223> a, c, g, or u

<400> 205

nnnnnnnnnn nnnnnnnnnn 20

<210> 206

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d16

<220>

<221>modified_base

<222> (1)..(15)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (16)..(16)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (17)..(20)

<223> a, c, g, or u

<400> 206

nnnnnnnnnn nnnnnnnnnn 20

<210> 207

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d17

<220>

<221>modified_base

<222> (1)..(16)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (17)..(17)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (18)..(20)

<223> a, c, g, or u

<400> 207

nnnnnnnnnn nnnnnnnnnn 20

<210> 208

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d18

<220>

<221>modified_base

<222> (1)..(17)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (18)..(18)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (19)..(20)

<223> a, c, g, or u

<400> 208

nnnnnnnnnn nnnnnnnnnn 20

<210> 209

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d19

<220>

<221>modified_base

<222> (1)..(18)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (19)..(19)

<223> a, c, g, or t

<220>

<221>modified_base

<222> (20)..(20)

<223> a, c, g, or u

<400> 209

nnnnnnnnnn nnnnnnnnnn 20

<210> 210

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> d20

<220>

<221>modified_base

<222> (1)..(19)

<223> a, c, g, or u

<220>

<221>modified_base

<222> (20)..(20)

<223> a, c, g, or t

<400> 210

nnnnnnnnnn nnnnnnnnnn 20

<210> 211

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223>BM.t12_cr

<400> 211

agugggggug aauucagugu 20

<210> 212

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d1

<400> 212

agugggggug aauucagugu 20

<210> 213

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d2

<400> 213

agugggggug aauucagugu 20

<210> 214

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d3

<400> 214

agggggggug aauucagugu 20

<210> 215

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d4

<400> 215

agugggggug aauucagugu 20

<210> 216

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d5

<400> 216

agugggggug aauucagugu 20

<210> 217

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d6

<400> 217

agugggggug aauucagugu 20

<210> 218

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d7

<400> 218

agugggggug aauucagugu 20

<210> 219

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d8

<400> 219

agugggggug aauucagugu 20

<210> 220

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d9

<400> 220

agugggggtg aauucagugu 20

<210> 221

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d10

<400> 221

agugggggug aauucagugu 20

<210> 222

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d11

<400> 222

agugggggug aauucagugu 20

<210> 223

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d12

<400> 223

agugggggug aauucagugu 20

<210> 224

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d13

<400> 224

agugggggug aatucagugu 20

<210> 225

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d14

<400> 225

agugggggug aautcagugu 20

<210> 226

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d15

<400> 226

agugggggug aauucagugu 20

<210> 227

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d16

<400> 227

agugggggug aauucagugu 20

<210> 228

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d17

<400> 228

agugggggug aauucagugu 20

<210> 229

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d18

<400> 229

agugggggug aauucagtgu 20

<210> 230

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d19

<400> 230

agugggggug aauucagugu 20

<210> 231

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d20

<400> 231

agugggggug aauucagugt 20

<210> 232

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> TR.t12_cr

<400> 232

gagucucuca gcugguacac 20

<210> 233

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d1

<400> 233

gagucucuca gcugguacac 20

<210> 234

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d2

<400> 234

gagucucuca gcugguacac 20

<210> 235

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d3

<400> 235

gagucucuca gcugguacac 20

<210> 236

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d4

<400> 236

gagtcucuca gcugguacac 20

<210> 237

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d5

<400> 237

gagucucuca gcugguacac 20

<210> 238

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d6

<400> 238

gaguctcuca gcugguacac 20

<210> 239

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d7

<400> 239

gagucucuca gcugguacac 20

<210> 240

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d8

<400> 240

gagucuctca gcugguacac 20

<210> 241

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d9

<400> 241

gagucucuca gcugguacac 20

<210> 242

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d10

<400> 242

gagucucuca gcugguacac 20

<210> 243

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d11

<400> 243

gagucucuca gcugguacac 20

<210> 244

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d12

<400> 244

gagucucuca gcugguacac 20

<210> 245

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d13

<400> 245

gagucucuca gctgguacac 20

<210> 246

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d14

<400> 246

gagucucuca gcugguacac 20

<210> 247

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d15

<400> 247

gagucucuca gcugguacac 20

<210> 248

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d16

<400> 248

gagucucuca gcuggtacac 20

<210> 249

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d17

<400> 249

gagucucuca gcugguacac 20

<210> 250

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d18

<400> 250

gagucucuca gcugguacac 20

<210> 251

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d19

<400> 251

gagucucuca gcugguacac 20

<210> 252

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d20

<400> 252

gagucucuca gcugguacac 20

<210> 253

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr

<400> 253

cugauggucc augucuguua 20

<210> 254

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d1

<400> 254

cugauggucc augucuguua 20

<210> 255

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d2

<400> 255

ctgauggucc augucuguua 20

<210> 256

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d3

<400> 256

cugauggucc augucuguua 20

<210> 257

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d4

<400> 257

cugauggucc augucuguua 20

<210> 258

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d5

<400> 258

cugatggucc augucuguua 20

<210> 259

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d6

<400> 259

cugauggucc augucuguua 20

<210> 260

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d7

<400> 260

cugauggucc augucuguua 20

<210> 261

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d8

<400> 261

cugauggtcc augucuguua 20

<210> 262

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d9

<400> 262

cugauggucc augucuguua 20

<210> 263

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d10

<400> 263

cugauggucc augucuguua 20

<210> 264

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d11

<400> 264

cugauggucc augucuguua 20

<210> 265

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d12

<400> 265

cugauggucc atgucuguua 20

<210> 266

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d13

<400> 266

cugauggucc augucuguua 20

<210> 267

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d14

<400> 267

cugauggucc augtcuguua 20

<210> 268

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d15

<400> 268

cugauggucc augucuguua 20

<210> 269

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d16

<400> 269

cugauggucc auguctguua 20

<210> 270

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d17

<400> 270

cugauggucc augucuguua 20

<210> 271

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d18

<400> 271

cugauggucc augucugtua 20

<210> 272

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d19

<400> 272

cugauggucc augucuguta 20

<210> 273

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d20

<400> 273

cugauggucc augucuguua 20

<210> 274

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_crRNA

<400> 274

auauaagugg aggcgucgcg 20

<210> 275

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM_tgt1_act.3'_d.1

<400> 275

auauaagugg aggcgucgcg 20

<210> 276

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM_tgt1_act.3'_d.2

<400> 276

atauaagugg aggcgucgcg 20

<210> 277

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM_tgt1_act.3'_d.3

<400> 277

auauaagugg aggcgucgcg 20

<210> 278

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.4

<400> 278

auataagugg aggcgucgcg 20

<210> 279

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.5

<400> 279

auauaagugg aggcgucgcg 20

<210> 280

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM_tgt1_act.3'_d.6

<400> 280

auauaagugg aggcgucgcg 20

<210> 281

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM_tgt1_act.3'_d.7

<400> 281

auauaagugg aggcgucgcg 20

<210> 282

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM_tgt1_act.3'_d.8

<400> 282

auauaagtgg aggcgucgcg 20

<210> 283

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.9

<400> 283

auauaagugg aggcgucgcg 20

<210> 284

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.10

<400> 284

auauaagugg aggcgucgcg 20

<210> 285

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.11

<400> 285

auauaagugg aggcgucgcg 20

<210> 286

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.12

<400> 286

auauaagugg aggcgucgcg 20

<210> 287

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.13

<400> 287

auauaagugg aggcgucgcg 20

<210> 288

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.14

<400> 288

auauaagugg aggcgucgcg 20

<210> 289

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.15

<400> 289

auauaagugg aggcgucgcg 20

<210> 290

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.16

<400> 290

auauaagugg aggcgtcgcg 20

<210> 291

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.17

<400> 291

auauaagugg aggcgucgcg 20

<210> 292

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.18

<400> 292

auauaagugg aggcgucgcg 20

<210> 293

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.19

<400> 293

auauaagugg aggcgucgcg 20

<210> 294

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_d.20

<400> 294

auauaagugg aggcgucgcg 20

<210> 295

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_act.3'_crRNA

<400> 295

ucuggaggcu cucaaggacu 20

<210> 296

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.1

<400> 296

tcuggaggcu cucaaggacu 20

<210> 297

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.in_t12_cr-3'_d.2

<400> 297

ucuggaggcu cucaaggacu 20

<210> 298

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.in_t12_cr-3'_d.3

<400> 298

uctggaggcu cucaaggacu 20

<210> 299

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.4

<400> 299

ucuggaggcu cucaaggacu 20

<210> 300

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.5

<400> 300

ucuggaggcu cucaaggacu 20

<210> 301

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.6

<400> 301

ucuggaggcu cucaaggacu 20

<210> 302

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.7

<400> 302

ucuggaggcu cucaaggacu 20

<210> 303

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.8

<400> 303

ucuggaggcu cucaaggacu 20

<210> 304

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.9

<400> 304

ucuggaggcu cucaaggacu 20

<210> 305

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.10

<400> 305

ucuggaggct cucaaggacu 20

<210> 306

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.11

<400> 306

ucuggaggcu cucaaggacu 20

<210> 307

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.12

<400> 307

ucuggaggcu ctcaaggacu 20

<210> 308

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.13

<400> 308

ucuggaggcu cucaaggacu 20

<210> 309

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.14

<400> 309

ucuggaggcu cucaaggacu 20

<210> 310

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.15

<400> 310

ucuggaggcu cucaaggacu 20

<210> 311

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.16

<400> 311

ucuggaggcu cucaaggacu 20

<210> 312

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.17

<400> 312

ucuggaggcu cucaaggacu 20

<210> 313

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.18

<400> 313

ucuggaggcu cucaaggacu 20

<210> 314

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.19

<400> 314

ucuggaggcu cucaaggacu 20

<210> 315

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.20

<400> 315

ucuggaggcu cucaaggact 20

<210> 316

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> TR.t12_cr

<400> 316

gagucucuca gcugguacac 20

<210> 317

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d20.19

<400> 317

gagucucuca gcugguacac 20

<210> 318

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d20.17

<400> 318

gagucucuca gcugguacac 20

<210> 319

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d19.15

<400> 319

gagucucuca gcugguacac 20

<210> 320

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d11.10

<400> 320

gagucucuca gcugguacac 20

<210> 321

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d20.11.1

<400> 321

gagucucuca gcugguacac 20

<210> 322

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d17.11

<400> 322

gagucucuca gcugguacac 20

<210> 323

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d2.1

<400> 323

gagucucuca gcugguacac 20

<210> 324

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d15.10.11

<400> 324

gagucucuca gcugguacac 20

<210> 325

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d20.17.10.1

<400> 325

gagucucuca gcugguacac 20

<210> 326

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d18.15.2

<400> 326

gagucucuca gcugguacac 20

<210> 327

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d19.15.11.10.3

<400> 327

gagucucuca gcugguacac 20

<210> 328

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d20.17.15.10.1

<400> 328

gagucucuca gcugguacac 20

<210> 329

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR.t12_d_7

<400> 329

gagucucuca gcugguacac 20

<210> 330

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_act.3'_crRNA

<400> 330

auauaagugg aggcgucgcg 20

<210> 331

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d20.17

<400> 331

agugggggug aauucagugt 20

<210> 332

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.t12_d15.11

<400> 332

agugggggug aauucagugu 20

<210> 333

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d15.14

<400> 333

agugggggug aautcagugu 20

<210> 334

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d11.10

<400> 334

agugggggug aauucagugu 20

<210> 335

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.t12_d20.11.1

<400> 335

agugggggug aauucagugt 20

<210> 336

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.t12_d17.11

<400> 336

agugggggug aauucagugu 20

<210> 337

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d2.1

<400> 337

agugggggug aauucagugu 20

<210> 338

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.t12_d20.17.15.2

<400> 338

agugggggug aauucagugt 20

<210> 339

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.t12_d15.14.11.10

<400> 339

agugggggug aautcagugu 20

<210> 340

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d_6a

<400> 340

agugggggug aauucagugt 20

<210> 341

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d_8

<400> 341

agtggggggtg aautcagugu 20

<210> 342

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d_6b

<400> 342

agugggggug aatucagugu 20

<210> 343

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.t12_d16.9.1-3

<400> 343

agtggggggtg aauucagugu 20

<210> 344

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>BM.t12_d_10

<400> 344

agugggggug aattcagtgt 20

<210> 345

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223>BM_tgt1_cr-3'

<400> 345

auauaagugg aggcgucgcg 20

<210> 346

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_cr-3'_d.20.11.1

<400> 346

auauaagugg aggcgucgcg 20

<210> 347

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_cr-3'_d.19.14.8

<400> 347

auauaagtgg aggcgucgcg 20

<210> 348

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_cr-3'_d.19.17.14.8

<400> 348

auauaagtgg aggcgucgcg 20

<210> 349

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_cr-3'_d.12.11.8

<400> 349

auauaagugg aggcgucgcg 20

<210> 350

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_cr-3'_d.18.16.10

<400> 350

auauaagugg aggcgtcgcg 20

<210> 351

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_cr-3'_d.19.17.15.1

<400> 351

auauaagugg aggcgucgcg 20

<210> 352

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM_tgt1_cr-3'_d.19-16

<400> 352

auauaagugg aggcgtcgcg 20

<210> 353

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223>BM.in_t12_cr-3'

<400> 353

ucuggaggcu cucaaggacu 20

<210> 354

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.01.11.20

<400> 354

tcuggaggcu cucaaggact 20

<210> 355

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.11.17.20

<400> 355

ucuggaggcu cucaaggact 20

<210> 356

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.08.09.10.11

<400> 356

ucuggaggct cucaaggacu 20

<210> 357

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.01.08.10.17

<400> 357

tcuggaggct cucaaggacu 20

<210> 358

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.17.19.20

<400> 358

ucuggaggcu cucaaggact 20

<210> 359

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d.08.09.19.20

<400> 359

ucuggaggcu cucaaggact 20

<210> 360

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> BM.in_t12_cr-3'_d01.10.11.17.19.20

<400> 360

tcuggaggct cucaaggact 20

<210> 361

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223>DNMt_crRNA

<400> 361

cugauggucc augucuguua 20

<210> 362

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d1.20

<400> 362

cugauggucc augucuguua 20

<210> 363

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d14.20

<400> 363

cugauggucc augtcuguua 20

<210> 364

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d8.19

<400> 364

cugaggtcc augucuguta 20

<210> 365

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d8.14

<400> 365

cugaggtcc augtcuguua 20

<210> 366

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d1.8.20

<400> 366

cugauggtcc augucuguua 20

<210> 367

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d8-10

<400> 367

cugauggtcc augucuguua 20

<210> 368

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d8.9.15

<400> 368

cugauggtcc augucuguua 20

<210> 369

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_d1.8.19

<400> 369

cugaggtcc augucuguta 20

<210> 370

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> RPL32_on

<400> 370

tttggggtga tcagacccaa cagc 24

<210> 371

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> RPL_32-off.1

<400> 371

tttggggtga tcagacccaa cacc 24

<210> 372

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223> RPL_32-off.2

<400> 372

tttggggtga tcagacccaa cacc 24

<210> 373

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>DNMT1_on

<400> 373

tttcctgatg gtccatgtct gtta 24

<210> 374

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<223>DNMT1_off-1

<400> 374

tttcctgatg gtccatgtct gaat 24

<210> 375

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223>RP_cr

<400> 375

gggugaucag acccaacagc 20

<210> 376

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_2_d.1

<400> 376

gggugaucag acccaacagc 20

<210> 377

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_2_d.8

<400> 377

gggugaucag acccaacagc 20

<210> 378

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_2_d.9

<400> 378

gggugaucag acccaacagc 20

<210> 379

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.10

<400> 379

gggugaucag acccaacagc 20

<210> 380

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.11

<400> 380

gggugaucag acccaacagc 20

<210> 381

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.12

<400> 381

gggugaucag acccaacagc 20

<210> 382

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.14

<400> 382

gggugaucag acccaacagc 20

<210> 383

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.15

<400> 383

gggugaucag acccaacagc 20

<210> 384

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.16

<400> 384

gggugaucag acccaacagc 20

<210> 385

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.17

<400> 385

gggugaucag acccaacagc 20

<210> 386

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.18

<400> 386

gggugaucag acccaacagc 20

<210> 387

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_2_d.19

<400> 387

gggugaucag acccaacagc 20

<210> 388

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_2_d.20

<400> 388

gggugaucag acccaacagc 20

<210> 389

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d.13

<400> 389

gggugaucag acccaacagc 20

<210> 390

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d11.12

<400> 390

gggugaucag acccaacagc 20

<210> 391

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d12.14

<400> 391

gggugaucag acccaacagc 20

<210> 392

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d14.15

<400> 392

gggugaucag acccaacagc 20

<210> 393

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d11.15

<400> 393

gggugaucag acccaacagc 20

<210> 394

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d11.12.14

<400> 394

gggugaucag acccaacagc 20

<210> 395

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d12.14.15

<400> 395

gggugaucag acccaacagc 20

<210> 396

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d11.14.15

<400> 396

gggugaucag acccaacagc 20

<210> 397

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d11.12.14.15

<400> 397

gggugaucag acccaacagc 20

<210> 398

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_3'_sp.20

<220>

<221>modified_base

<222> (20)..(20)

<223> an abasic site that lacks 2' - hydroxyl

<400> 398

gggugaucag acccaacagn t 21

<210> 399

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_3'_d14_sp20

<220>

<221>modified_base

<222> (20)..(20)

<223> an abasic site that lacks 2' - hydroxyl

<400> 399

gggugaucag acccaacagn t 21

<210> 400

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_3'_d15_sp20

<220>

<221>modified_base

<222> (20)..(20)

<223> an abasic site that lacks 2' - hydroxyl

<400> 400

gggugaucag acccaacagn t 21

<210> 401

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_3'_d12_sp20

<220>

<221>modified_base

<222> (20)..(20)

<223> an abasic site that lacks 2' - hydroxyl

<400> 401

gggugaucag acccaacagn t 21

<210> 402

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>RP_cr_3'_d11_sp20

<220>

<221>modified_base

<222> (20)..(20)

<223> an abasic site that lacks 2' - hydroxyl

<400> 402

gggugaucag acccaacagn t 21

<210> 403

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> RP_cr_3'_d14.15_sp20

<220>

<221>modified_base

<222> (20)..(20)

<223> an abasic site that lacks 2' - hydroxyl

<400> 403

gggugaucag acccaacagn t 21

<210> 404

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> DNMT1-crRNA

<400> 404

cugauggucc augucuguua 20

<210> 405

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d1.20

<400> 405

cugauggucc augucuguua 20

<210> 406

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d8.19

<400> 406

cugaggtcc augucuguta 20

<210> 407

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d9

<400> 407

cugauggucc augucuguua 20

<210> 408

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d8.14

<400> 408

cugaggtcc augtcuguua 20

<210> 409

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d10.17

<400> 409

cugauggucc augucuguua 20

<210> 410

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d1.8.20

<400> 410

cugauggtcc augucuguua 20

<210> 411

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d1.8.19

<400> 411

cugaggtcc augucuguta 20

<210> 412

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223>DNMT1-d8.9.15

<400> 412

cugauggtcc augucuguua 20

<210> 413

<211> 3147

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polynucleotide

<220>

<223> anti-BCMA CAR construct

<400> 413

cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc cagattccaa 60

gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct gccagagtta 120

tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta ttattaagta 180

gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac gttcactgaa 240

atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc agtccatcac 300

gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg acttgccagc 360

cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg gttggggcaa 420

agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata tccagaaccc 480

tgaccctgcc gtgtaccagc ttaattaatt agtccataga gcccaccgca tccccagcat 540

gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa 600

tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag 660

tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct 720

ggcaactaga aggcacagtt attacctcgg tggcagggcc tgcatatgga gggcatcgta 780

tgtatctttc gtggccgtac tcaggccttg gtagagaccg tcgtggcctt tccctctgcg 840

tctctctccc ttcatcccga tctcgctgta agcctccgcc atcttatctt tctgcagttc 900

attataaagc ccctcttgcg ggttttttct cctcggtttt cctcccatct ctgggtccct 960

ccctcggcgc ttatccaata catcatattc ctctcgcctc cccaggttaa gctcattgta 1020

aagttggttt tgcccttgct gataggccgg agcgtcagca gaccggctaa atttcactct 1080

cagttcgcaa ccaccctcct cttcttcggg gaatcggcag ctacatccat cctcctcttg 1140

cgtagtttgg accgggcgca taaatggttg cttaaaaata tacagcaact tttttcttcc 1200

tcttttgcag taaagagtaa taacgaggga cagcagcaag accccgcacg ttccggcaag 1260

cggtgcccag atgtagatgt cgcaagcaaa gtccagcccg cgagtatgaa ccgcgcctcc 1320

cgctgcgggg cgacaagcct ccggccgaag agagagtggc tgggaagcga tagtgggtgc 1380

aggcgtgggt ggccgagggg caggcgtggt tgtggcagcg gctttaattt ccactttggt 1440

gccgccgcca aaggtcggcg gaaagctatg gccgttctgg caataataca ccgcaaaatc 1500

ttccggttcc aggctgctaa tggtcagggt aaaatcggtg ccgctgccgc tgccgctaaa 1560

gcgcgccgga atgccggtaa tgctctggct cgcataataa atcagcaggc gcggcgcctg 1620

gcccggtttc tgctgatacc aatgcagata atcgctaatg ctctggctcg cgcggcagct 1680

cagggtcgcg cgttcgcccg ggctcaggct cagggtcgcc gggctctggg tcagcacaat 1740

ttcgctgccg ccgccgccgc tgccgccgcc gccgctgccg ccgccgccgc tgctcacggt 1800

caccagggtg ccctggcccc acacatcaaa atagccatca tagttatagc gcgcgcaata 1860

atacaccgcg gtatcttcgc tgcgcaggct gctcagttcc atatacgcgg tgctggtgct 1920

tttatccgcg gtaatggtca cgcggccttt aaatttttcg ttatatttgg tcgcatcgtt 1980

atacggaata atatagccca tccattccag gccctggccc ggcgcctggc gcacccaatg 2040

catcacatag ctggtaaagg tatagccgct cgctttgcag ctcactttca cgctgctgcc 2100

cggttttttc acttccgcgc cgctctgcac cagctgcacc tgcggtcttg ccgcgtggag 2160

caggagtgcc aatggaagca gaagtgcggt cacgggcaac gccatggtgg ctatgacgcg 2220

tcgcgccgag tgaggggttg tgggctcttt tattgagctc ggggagcaga agcgcgcgaa 2280

cagaagcgag aagcgaactg attggttagt tcaaataagg cacagggtca tttcaggtcc 2340

ttggggcacc ctggaaacat ctgatggttc tctagaaact gctgagggcg ggaccgcatc 2400

tggggaccat ctgttcttgg ccctgagccg gggcaggaac tgcttaccac agatatcctg 2460

tttggcccat attctgctgt tccaactgtt cttggccctg agccggggca ggaactgctt 2520

accacagata tcctgtttgg cccatattct gctgtctctc tgttcctaac cttgatccta 2580

gcttgccaaa cctacaggtg gggtctttca ttcccccctt tttctggaga ctaaataaag 2640

tttaaactga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 2700

tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 2760

ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 2820

gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 2880

agcccaggta agggcagctt tggtgccttc gcaggctgtt tccttgcttc aggaatggcc 2940

aggttctgcc cagagctctg gtcaatgatg tctaaaactc ctctgattgg tggtctcggc 3000

cttatccatt gccaccaaaa ccctcttttt actaagaaac agtgagcctt gttctggcag 3060

tccagagaat gacacgggaa aaaagcagat gaagagaagg tggcaggaga gggcacgtgg 3120

cccagcctca gtctctccaa ctgagtt 3147

<210> 414

<211> 3409

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polynucleotide

<220>

<223> B2M-HLA-e construct

<400> 414

tgagtgctga gagggcatca gaagtccttg agagcctcca gagaaaggct cttaaaaatg 60

cagcgcaatc tccagtgaca gaagatactg ctagaaatct gctagaaaaa aaacaaaaaa 120

ggcatgtata gaggaattat gagggaaaga taccaagtca cggtttattc ttcaaaatgg 180

aggtggcttg ttgggaaggt ggaagctcat ttggccagag tggaaatgga attggggagaa 240

atcgatgacc aaatgtaaac acttggtgcc tgatatagct tgacaccaag ttagccccaa 300

gtgaaatacc ctggcaatat taatgtgtct tttcccgata ttcctcaggt actccaaaga 360

ttcaggttta ctcacgtcat ccagcagaga atggaaagtc aaatttcctg aattgctatg 420

tgtctgggtt tcatccatcc gacattgaag ttgacttact gaagaatgga gagagaattg 480

aaaaagtgga gcattcagac ttgtctttca gcaaggactg gtctttctat ctcttgtact 540

acactgaatt tttaattaat tagtccatag agcccaccgc atccccagca tgcctgctat 600

tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac 660

acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg 720

caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag 780

aaggcacagt tattacaagc tgtgagactc agacccctgg gcactgtcgc tccactcagc 840

cttagagtag ctccctcctt ttccacctga gctcttcttc ctccatatca cagcagcaac 900

cacagctcca gagaccacag atccaaggag aaccaggcca gcaatgatgc ccacgatggg 960

gatggtgggc tgggaagccg gcttccatct cagggtgacg ggctcgggta gcccctcatg 1020

ctgcacatgg cacgtgtatc tctgctcctc tccagaaggc accaccacag ctgcccactt 1080

ctggaaggtt ccatcccctg caggcctggt ctccacgagc tccgtgtcct gggtatggcc 1140

ctccccatcc tgctgccagg tcagtgtgat ctccgcaggg tagaagccca gggcccagca 1200

cctcaggtg gcctcatggt cagagatggg gtggtgagtc acgtgtgtct ttgggggctc 1260

caggtgaagc agcgtctcct tccccttctc caggtatttg tggagccact ccacgcatgt 1320

gtcttccagg taggctctct ggtgctccgc ctcagaagca tcatttgact tttgctcgga 1380

gatctgagcc gccgtgtcca ccgcggtcca ggagcgcagg tcctcattca gggtgagata 1440

atccttgccg tcgtaggcga actgttcata cccgcggagg aagcgcccgt cgggccccag 1500

ctcgcagcca tgcatccact gcagggtgtg agacccggcc tcgctctgat tgtagtagcc 1560

gcgcagcgtc cgcagattca ctcggaaaat ctgtgcggtg tccctggcgc tccgtgtctc 1620

ccggtcccaa tactctgacc cctcctgctc catccacggc gcccgcggca ccatcctcgg 1680

actcgcggcg tcgttgtcga agcgcacgaa ctgggtgtcg tccacgtagc ccacagagat 1740

gaagcggggc tccccgcggc cgggccggga cacggaagtg tggaaatact tcaaggagtg 1800

ggatccagac cctccgccac cagatccccc tcctccagaa ccgcctccgc cagaccctcc 1860

gccacccatg tctcgatccc atttcacgat cttgggctgt gacaaagtca catggttcac 1920

acggcaggca tactcatctt tctccgtagg tgtaaactct gtatagtaca agagatagaa 1980

agaccagtcc ttgctgaaag acaagtctga atgctccact ttttcaattc tctctccatt 2040

cttcagtaag tcaacttcaa tgtcggatgg atgaaaccca gacacatagc aattcaggaa 2100

atttgacttt ccattctctg ctggatgacg tgagtaaacc tgaatctttg gagtacgctg 2160

gatggagccg ccgcctccgc taccgcctcc tccgctccca cctccaccca gaaagaggt 2220

ccgtggtgcc attacagcct ccaggccaga aagagagagt agcgcgagca cagctaaggc 2280

cacggagcga gacatggtgg ctctagagta ggcgccggtc acagcttgga tctgtaacgg 2340

cgcagaacag aaaacgaaac aaagacgtag agttgagcaa gcagggtcag gcaaagcgtg 2400

gagagccggc tgagtctagg taggctccaa gggagcgccg gacaaaggcc cggtctcgac 2460

ctgagcttta aacttaccta gacggcggac gcagttcagg aggcaccaca ggcgggaggc 2520

ggcagaacgc gactcaaccg gcgtggatgg cggcctcagg tagggcggcg ggcgcgtgaa 2580

ggagagatgc gagcccctcg aagcttcagc tgtgttctgg cggcaaaccc gttgcgaaaa 2640

agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg 2700

cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccc 2760

gttcaattgc cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc 2820

cactgacggg caccggagcg atcgcagatc cttgtcgacc acccccactg aaaaagatga 2880

gtatgcctgc cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtggggtaa 2940

gtcttacatt cttttgtaag ctgctgaaag ttgtgtatga gtagtcatat cataaagctg 3000

ctttgatata aaaaaggtct atggccatac taccctgaat gagtcccatc ccatctgata 3060

taaacaatct gcatattggg attgtcaggg aatgttctta aagatcagat tagtggcacc 3120

tgctgagata ctgatgcaca gcatggtttc tgaaccagta gtttccctgc agttgagcag 3180

ggagcagcag cagcacttgc acaaatacat atacactctt aacacttctt acctactggc 3240

ttcctctagc ttttgtggca gcttcaggta tatttagcac tgaacgaaca tctcaagaag 3300

gtataggcct ttgtttgtaa gtcctgctgt cctagcatcc tataatcctg gacttctcca 3360

gtactttctg gctggattgg tatctgaggc tagtaggaag ggcttgttc 3409

<210> 415

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TRAC_cr3'_tgt.12-d20.11.1

<400> 415

taauuucuac ucutguagau gagucucuca gcugguacac 40

<210> 416

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> B2M_cr3'_tgt.12-d20.11.1

<400> 416

taauuucuac ucutguagau agugggggug aauucagugt 40

<210> 417

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> crRNA

<400> 417

uaauuucuac ucuuguagau 20

<210> 418

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d1

<400> 418

taauuucuac ucuuguagau 20

<210> 419

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d2

<400> 419

uaauuucuac ucuuguagau 20

<210> 420

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d3

<400> 420

uaauuucuac ucuuguagau 20

<210> 421

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d4

<400> 421

uaatuucuac ucuuguagau 20

<210> 422

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d5

<400> 422

uaautucuac ucuugagau 20

<210> 423

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d6

<400> 423

uaauutcuac ucuugagau 20

<210> 424

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d7

<400> 424

uaauuucuac ucuuguagau 20

<210> 425

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d8

<400> 425

uaauuuctac ucuuguagau 20

<210> 426

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d9

<400> 426

uaauuucuac ucuuguagau 20

<210> 427

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d10

<400> 427

uaauuucuac ucuuguagau 20

<210> 428

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d11

<400> 428

uaauuucuac tcuuguagau 20

<210> 429

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d12

<400> 429

uaauuucuac ucuuguagau 20

<210> 430

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d13

<400> 430

uaauuucuac uctuguagau 20

<210> 431

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d14

<400> 431

uaauuucuac ucutguagau 20

<210> 432

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d15

<400> 432

uaauuucuac ucuuguagau 20

<210> 433

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d16

<400> 433

uaauuucuac ucuugtagau 20

<210> 434

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d17

<400> 434

uaauuucuac ucuuguagau 20

<210> 435

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d18

<400> 435

uaauuucuac ucuuguagau 20

<210> 436

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d19

<400> 436

uaauuucuac ucuuguagau 20

<210> 437

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISPR-d20

<400> 437

uaauuucuac ucuuguagat 20

<210> 438

<211> 40

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> DMT.t3_crRNA

<400> 438

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 439

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d1

<400> 439

taauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 440

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d2

<400> 440

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 441

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d3

<400> 441

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 442

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d4

<400> 442

uaatuucuac ucuuguagau cugauggucc augucuguua 40

<210> 443

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d5

<400> 443

uaautucuac ucuuguagau cugauggucc augucuguua 40

<210> 444

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d6

<400> 444

uaauutcuac ucuuguagau cugauggucc augucuguua 40

<210> 445

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d7

<400> 445

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 446

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d8

<400> 446

uaauuuctac ucuuguagau cugauggucc augucuguua 40

<210> 447

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d9

<400> 447

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 448

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d10

<400> 448

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 449

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d11

<400> 449

uaauuucuac tcuuguagau cugauggucc augucuguua 40

<210> 450

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d12

<400> 450

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 451

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d13

<400> 451

uaauuucuac uctuguagau cugauggucc augucuguua 40

<210> 452

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d14

<400> 452

uaauuucuac ucutguagau cugauggucc augucuguua 40

<210> 453

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d15

<400> 453

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 454

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d16

<400> 454

uaauuucuac ucuugtagau cugauggucc augucuguua 40

<210> 455

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d17

<400> 455

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 456

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d18

<400> 456

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 457

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d19

<400> 457

uaauuucuac ucuuguagau cugauggucc augucuguua 40

<210> 458

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> DMT.t3_cr-d20

<400> 458

uaauuucuac ucuuguagat cugauggucc augucuguua 40

<210> 459

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISRP-3'

<400> 459

taauuucuac ucutguagau 20

<210> 460

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISRP-2

<400> 460

taauuucuac ucuugtagau 20

<210> 461

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISRP-6

<400> 461

taauuucuac uctugtagau 20

<210> 462

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISRP-401

<400> 462

uaauuucuac ucutguagau 20

<210> 463

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISRP-402

<400> 463

taauuucuac ucutgtagau 20

<210> 464

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISRP-403

<400> 464

uaauuucuac ucutguagau 20

<210> 465

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CRISRP-404

<400> 465

taauuucuac ucutguagau 20

<210> 466

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr_tgt.12-d20.11.1

<400> 466

uaauuucuac ucuuguagau gagucucuca gcugguacac 40

<210> 467

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr3'_tgt.12-d20.11.1

<400> 467

taauuucuac ucutguagau gagucucuca gcugguacac 40

<210> 468

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr-2_tgt.12-d20.11.1

<400> 468

taauuucuac ucuugtagau gagucucuca gcugguacac 40

<210> 469

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr-6_tgt.12-d20.11.1

<400> 469

taauuucuac uctugtagau gagucucuca gcugguacac 40

<210> 470

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr-401_tgt.12-d20.11.1

<400> 470

uaauuucuac ucutguagau gagucucuca gcugguacac 40

<210> 471

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr-403_tgt.12-d20.11.1

<400> 471

taauuucuac ucutgtagau gagucucuca gcugguacac 40

<210> 472

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr-404_tgt.12-d20.11.1

<400> 472

uaauuucuac ucutguagau gagucucuca gcugguacac 40

<210> 473

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TR_cr-405_tgt.12-d20.11.1

<400> 473

taauuucuac ucutguagau gagucucuca gcugguacac 40

<210> 474

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223>BCMA scFv chain 1

<400> 474

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Ile Pro Tyr Asn Asp Ala Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asn Tyr Asp Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser

115

<210> 475

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223>BCMA scFv chain 2

<400> 475

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Gln Ser Ile Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 476

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> BCMA scFv linker

<400> 476

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 477

<211> 240

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223> BCMA scFv

<400> 477

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Ile Pro Tyr Asn Asp Ala Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asn Tyr Asp Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

130 135 140

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

145 150 155 160

Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

165 170 175

Pro Arg Leu Leu Ile Tyr Tyr Ala Ser Gln Ser Ile Thr Gly Ile Pro

180 185 190

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

195 200 205

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly

210 215 220

His Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

225 230 235 240

<210> 478

<211> 63

<212> DNA

<213> Homo sapiens

<220>

<223> CD8-alpha signal sequence

<400> 478

atggcgttgc ccgtgaccgc acttctgctt ccattggcac tcctgctcca cgcggcaaga 60

ccg 63

<210> 479

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>GS_SV40-NLS

<400> 479

Gly Ser Pro Lys Lys Lys Arg Lys Val

1 5

<210> 480

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>GS_SV40-NLS_GS_SV40-NLS

<400> 480

Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val

<210> 481

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>GS_NPL-NLS

<400> 481

Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys

1 5 10 15

Lys Lys

<210> 482

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223>GS_NPL-NLS_GS_NPL-NLS

<400> 482

Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys

1 5 10 15

Lys Lys Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala

20 25 30

Lys Lys Lys Lys

35

<210> 483

<211> 27

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>GS_SV40-NLS_GS_NPL-NLS

<400> 483

Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Ser Lys Arg Pro Ala Ala

1 5 10 15

Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

20 25

<210> 484

<211> 27

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>GS_NPL-NLS_GS_SV40-NLS

<400> 484

Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys

1 5 10 15

Lys Lys Gly Ser Pro Lys Lys Lys Arg Lys Val

20 25

<210> 485

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223> (GGGGS)2_SV40-NLS_GS_NPL-NLS

<400> 485

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys

1 5 10 15

Val Gly Ser Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys

20 25 30

Lys Lys Lys

35

<210> 486

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223> (GGGGS)2_NPL-NLS_GS_SV40-NLS

<400> 486

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Arg Pro Ala Ala Thr

1 5 10 15

Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Gly Ser Pro Lys Lys Lys

20 25 30

Arg Lys Val

35

<210> 487

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> (GGGGS)2_SV40-NLS

<400> 487

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys

1 5 10 15

Val

<210> 488

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> (GGGGS)2_SV40-NLS_GS_SV40-NLS

<400> 488

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys

1 5 10 15

Val Gly Ser Pro Lys Lys Lys Arg Lys Val

20 25

<210> 489

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> (GGGGS)2_NPL-NLS

<400> 489

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Arg Pro Ala Ala Thr

1 5 10 15

Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

20 25

<210> 490

<211> 44

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223> (GGGGS)2_NPL-NLS_GS_NPL-NLS

<400> 490

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Arg Pro Ala Ala Thr

1 5 10 15

Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Gly Ser Lys Arg Pro Ala

20 25 30

Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

35 40

<210> 491

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt25

<400> 491

cagauacgaa ccuaaacuuu 20

<210> 492

<211> 20

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt28

<400> 492

aggaggagga uucggaaccc 20

<210> 493

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> 53BP1

<400> 493

Gly Lys Arg Lys Leu Ile Thr Ser Glu Glu Glu Arg Ser Pro Ala Lys

1 5 10 15

ArgGlyArgLysSer

20

<210> 494

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> VACM-1/CUL5

<400> 494

Pro Lys Leu Lys Arg Gln

1 5

<210> 495

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>CXCR4

<400> 495

Arg Pro Arg Lys

1

<210> 496

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> VP1

<400> 496

Arg Arg Ala Arg Arg Pro Arg Gly

1 5

<210> 497

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>ING4

<400> 497

Lys Gly Lys Lys Gly Arg Thr Gln Lys Glu Lys Lys Ala Ala Arg Ala

1 5 10 15

Arg Ser Lys Gly Lys Asn

20

<210> 498

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> IER5

<400> 498

Arg Lys Arg Cys Ala Ala Gly Val Gly Gly Gly Pro Ala Gly Cys Pro

1 5 10 15

Ala Pro Gly Ser Thr Pro Leu Lys Lys Pro Arg Arg

20 25

<210> 499

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<220>

<223> ERK5

<400> 499

Arg Lys Pro Val Thr Ala Gln Glu Arg Gln Arg Glu Arg Glu Glu Lys

1 5 10 15

Arg Arg Arg Arg Gln Glu Arg Ala Lys Glu Arg Glu Lys Arg Arg Gln

20 25 30

Glu Arg Glu Arg

35

<210> 500

<211> 27

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223>UL79

<400> 500

Thr Leu Leu Leu Arg Glu Thr Met Asn Asn Leu Gly Val Ser Asp His

1 5 10 15

Ala Val Leu Ser Arg Lys Thr Pro Gln Pro Tyr

20 25

<210> 501

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> EWS

<400> 501

Pro Gly Lys Met Asp Lys Gly Glu His Arg Gln Glu Arg Arg Asp Arg

1 5 10 15

Pro Tyr

<210> 502

<211> 27

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> Hrp1

<400> 502

Arg Ser Gly Gly Asn His Arg Arg Asn Gly Arg Gly Gly Arg Gly Gly

1 5 10 15

Tyr Asn Arg Arg Asn Asn Gly Tyr His Pro Tyr

20 25

<210> 503

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> cMyc (1)

<400> 503

Pro Ala Ala Lys Arg Cys Lys Leu Asp

1 5

<210> 504

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> cMyc (2)

<400> 504

Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro

1 5 10

<210> 505

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> mouse c-able IV

<400> 505

Ser Ala Leu Ile Lys Lys Lys Lys Lys Asn Ala Pro

1 5 10

<210> 506

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> Matalpha2

<400> 506

Lys Ile Pro Ile Lys

1 5

<210> 507

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<220>

<223> MINIYO

<400> 507

Lys Ser Gly Ser Arg Lys

1 5

<210> 508

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt25 chRDNA

<400> 508

taauuucuac ucutguagau cagauacgaa ccuaaacuut 40

<210> 509

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CISH-tgt3 chRDNA

<400> 509

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 510

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CBLB-tgt3 chRDNA

<400> 510

taauuucuac ucutguagau ccugauacau aucagcauuu 40

<210> 511

<211> 40

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt12 crRNA

<400> 511

uaauuucuac ucuuguagau gagucucuca gcugguacac 40

<210> 512

<211> 40

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt12 ps-r1.2_s19.20

<220>

<221> misc_feature

<222> (1)..(3)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (38)..(40)

<223> Phosphorothioate linkage

<400> 512

uaauuucuac ucuuguagau gagucucuca gcugguacac 40

<210> 513

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt12 ps-r2_r-d1

<220>

<221> misc_feature

<222> (1)..(3)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (38)..(40)

<223> Phosphorothioate linkage

<400> 513

taauuucuac ucuuguagau gagucucuca gcugguacac 40

<210> 514

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt12 ps-r1.2_r-d7

<220>

<221> misc_feature

<222> (1)..(3)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (7)..(8)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (38)..(40)

<223> Phosphorothioate linkage

<400> 514

uaauuucuac ucuuguagau gagucucuca gcugguacac 40

<210> 515

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt12 ps-r1.2_r-d10

<220>

<221> misc_feature

<222> (1)..(3)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (10)..(11)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (38)..(40)

<223> Phosphorothioate linkage

<400> 515

uaauuucuac ucuuguagau gagucucuca gcugguacac 40

<210> 516

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt12 ps-r1.2_r-d12

<220>

<221> misc_feature

<222> (1)..(3)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (12)..(13)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (38)..(40)

<223> Phosphorothioate linkage

<400> 516

uaauuucuac ucuuguagau gagucucuca gcugguacac 40

<210> 517

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> TRAC-tgt12 ps-r1.2_r-d14

<220>

<221> misc_feature

<222> (1)..(3)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (14)..(15)

<223> Phosphorothioate linkage

<220>

<221> misc_feature

<222> (38)..(40)

<223> Phosphorothioate linkage

<400> 517

uaauuucuac ucutguagau gagucucuca gcugguacac 40

<210> 518

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d1

<400> 518

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 519

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d8

<400> 519

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 520

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d9

<400> 520

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 521

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d10

<400> 521

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 522

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d11

<400> 522

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 523

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d12

<400> 523

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 524

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d14

<400> 524

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 525

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d15

<400> 525

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 526

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d17

<400> 526

taauuucuac ucutguagau gguguacagc aguggctggu 40

<210> 527

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d18

<400> 527

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 528

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d19

<400> 528

taauuucuac ucutguagau gguguacagc aguggcuggu 40

<210> 529

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

oligonucleotide

<220>

<223> Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide

<220>

<223> CSH-tgt3_3'_d20

<400> 529

taauuucuac ucutguagau gguguacagc aguggcuggt 40

<---

Claims (26)

1. A method for producing a cell expressing a chimeric antigen receptor (CAR), said method comprising: a) contacting a first target nucleic acid comprising a TRAC sequence in a cell with a first nucleic acid/CRISPR protein composition comprising a CRISPR guide molecule and a Cas12 protein, wherein the CRISPR guide molecule comprises a targeting region capable of binding to a target nucleic acid sequence and an activating region comprising an RNA sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 6) comprising at least one deoxyribonucleotide instead of a ribonucleotide, wherein the activating region is capable of forming a nucleoprotein complex with a Cas12 protein, wherein said first nucleic acid/CRISPR protein composition is capable of cleaving the first target nucleic acid sequence; b) bringing a second target nucleic acid sequence comprising a B2M sequence into contact in the same cell with a second nucleic acid/CRISPR protein composition comprising a CRISPR guide molecule and a Cas12 protein, wherein the CRISPR guide molecule comprises a targeting region capable of binding to a target nucleic acid sequence and an activating region comprising the RNA sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 6), comprising at least one deoxyribonucleotide instead of a ribonucleotide, wherein the activating region is capable of forming a nucleoprotein complex with the Cas12 protein, wherein said second nucleic acid/CRISPR protein composition is capable of cleaving the second target nucleic acid sequence; c) obtaining a first donor polynucleotide encoding a CAR comprising an scFv, a transmembrane domain, a costimulatory domain and an activating domain, wherein the CAR is capable of inserting into a cleavage site in a first target nucleic acid sequence; d) obtaining a second donor polynucleotide encoding a B2M-HLA-E fusion construct comprising a B2M secretion signal, an HLA-G peptide signal sequence, a first linker sequence, a B2M sequence, a second linker sequence, and an HLA-E sequence, wherein the B2M-HLA-E fusion construct is capable of inserting into a cleavage site in a second target nucleic acid sequence; e) cleaving a first target nucleic acid sequence and incorporating at least a portion of a first donor polynucleotide into the cleavage site; and f) cleaving a second target nucleic acid sequence and incorporating at least a portion of a second donor polynucleotide into the cleavage site. 2. The method according to claim 1, wherein one or more positions 1, 3, 7, 10, 12, 14, 15, 16, 17, 18 and 19 in the activating region contain a deoxyribonucleotide base. 3. The method according to claim 1, wherein the activating region comprises a sequence selected from SEQ ID NO: 459-465. 4. The method of claim 1, wherein the CRISPR guide molecule comprises one or more phosphorothioate chemical modifications between the last 3-5 nucleotides at the 5' end or 3' end of the polynucleotide sequence. 5. The method according to claim 4, wherein the CRISPR guide molecule comprises a sequence selected from SEQ ID NO: 512-517. 6. The method of claim 1, wherein the targeting region targets the B2M gene and comprises the RNA sequence AGUGGGGGUGAAUUCAGUGU (SEQ ID NO: 211), wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 and 20 in the targeting region comprise a deoxyribonucleotide base. 7. The method according to claim 6, wherein the CRISPR guide molecule comprises a sequence selected from SEQ ID NO: 212-231, 275-294, 296-315, 331-334 and 346-350. 8. The method according to claim 6, wherein the CRISPR guide molecule comprises the sequence SEQ ID NO: 416. 9. The method of claim 1, wherein the targeting region targets the TRAC gene and comprises the RNA sequence GAGUCUCUCAGCUGGUACAC (SEQ ID NO: 232), wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 and 20 in the targeting region comprise a deoxyribonucleotide base. 10. The method of claim 9, wherein the CRISPR guide molecule comprises a sequence selected from SEQ ID NO: 233-252, 317-329, 331-334, 491-492 and 508. 11. The method according to claim 9, wherein the CRISPR guide molecule comprises the sequence SEQ ID NO: 415. 12. The method according to claim 1, wherein the targeting region targets the CISH gene and comprises an RNA sequence capable of hybridizing with a human genomic sequence selected from SEQ ID NO: 156-165, wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 and 20 in the targeting region comprise a deoxyribonucleotide base. 13. The method according to claim 1, wherein the targeting region targets the PDCD1 gene and comprises an RNA sequence capable of hybridizing with a human genomic sequence selected from SEQ ID NO: 134-155, wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 and 20 in the targeting region comprise a deoxyribonucleotide base. 14. The method according to claim 1, wherein the targeting region targets the CBLB gene and comprises an RNA sequence capable of hybridizing with a human genomic sequence selected from SEQ ID NO: 166-189, wherein one or more positions 1, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 and 20 in the targeting region comprise a deoxyribonucleotide base. 15. The method according to claim 1, wherein the Casl2 protein is a Cas12a protein comprising at the C-terminus a sequence that comprises a linker and a nuclear localization signal (NLS) and that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 479-490. 16. The method according to claim 15, wherein the Cas12a protein comprises the amino acid sequence SEQ ID NO: 1. 17. The method according to claim 1, wherein the second donor polynucleotide comprises SEQ ID NO: 414. 18. The method of claim 1, wherein the CAR comprises a transmembrane domain derived from CD8, a 4-1BB costimulatory domain or a CD28 costimulatory domain, and a CD3ζ activation domain. 19. A CAR-expressing cell obtained by the method of claim 1, comprising the sequence of SEQ ID NO: 414, wherein the cell is selected from a lymphocyte, a CAR-T cell, a TCR cell, a CAR-T cell engineered based on a TCR, a TIL, a CAR-TIL, a dendritic cell, a CAR-DC, a macrophage, a CAR-M, an iPSC cell, a cell differentiated from an iPSC, an NK cell, or a CAR-NK cell. 20. A method of adoptive cell therapy comprising administering to an individual in need thereof a CAR-expressing cell according to claim 19.