CPF1-related methods and compositions for gene editing

Abstract

This invention provides CRISPR / Cpf1-related methods and components for editing target nucleic acid sequences and / or modulating the expression of target nucleic acid sequences, as well as methods and compositions for evaluating such editing and / or expression modification. [Solution] Isolated cells containing modifications to the HBG gene sequence or the BCL11a gene sequence are provided, which are generated by delivery of an RNP complex comprising a CRISPR 1 (Cpf1) RNA-induced nuclease from the genera Prevotella and Francisella, and a gRNA molecule targeting the HBG gene sequence or the BCL11a gene sequence.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 597,118 filed on 11 December 2017, U.S. Provisional Patent Application No. 62 / 623,501 filed on 29 January 2018, U.S. Provisional Patent Application No. 62 / 664,905 filed on 30 April 2018, and U.S. Provisional Patent Application No. 62 / 746,494 filed on 16 October 2018, each claiming priority and the contents of each of them being incorporated herein by reference as a whole.

[0002] Array List This specification further incorporates, by reference, the sequence listing submitted via EFS on December 11, 2018. The sequence listing text file, identified as 0841770210SL.txt in accordance with 37C.FR§1.52(e)(5), is 444,032 bytes in size and was created on December 11, 2018. The entire contents of the sequence listing are incorporated herein by reference. The sequence listing is not extended beyond the scope of this specification and therefore does not contain any new information.

[0003] This disclosure relates to CRISPR / Cpf1-related methods and components for editing and / or modulating the expression of target nucleic acid sequences, as well as methods and compositions for evaluating such editing and / or expression modification. [Background technology]

[0004] CRISPR (Crystal Repeat Palindromic Sequence), a cluster of short repeats arranged at regular intervals, has developed in bacteria and archaea as an adaptive immune system to defend against viral attacks. Upon exposure to a virus, a short segment of viral DNA is incorporated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus containing the viral sequence. This RNA, containing a sequence complementary to the viral genome, mediates the targeting of the Cpf1 protein to a target sequence in the viral genome. The Cpf1 protein, also known as Cas12a ("CRISPR1 from Prevotella and Francisella"), then cleaves, thereby arresting the expression of the viral target.

[0005] Recently, the CRISPR / Cpf1 system has been applied to genome editing in eukaryotic cells. The introduction of site-directed double-strand breaks (DSBs) enables target sequence modification through endogenous DNA repair mechanisms such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). [Overview of the project]

[0006] This disclosure provides improvements to CRISPR / Cpf1-related methods and components for editing and / or modulating the expression of target nucleic acid sequences, for example, in therapy-related cell lines and with respect to therapy-related target sequences, as well as strategies for evaluating the efficiency of such target editing and / or expression modification.

[0007] In one embodiment, the disclosure relates to the use of CRISPR / Cpf1-mediated editing of a therapy-related target site in a therapy-related cell population. For example, but not limited to, the disclosure provides isolated cells that have been modified to a therapy-related target site. In certain embodiments, the cells are, for example, CD8 + T cells, CD8 + Naive T cells, CD4 + central memory T cells, CD8 + central memory T cells, CD4 + Effector memory T cells, CD4+ Effector memory T cells, CD4 + T cells, CD4 + Stem cell memory T cells, CD8 + Stem cell memory T cells, CD4 + Helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, CD4+ naive T cells, TH17 CD4 + T cells, TH1 CD4 + T cells, TH2 CD4 + T cells, TH9 CD4 + T cells, CD4 + Foxp3 + T cells, CD4 + CD25 + CD127 - T cells or CD4 + CD25 + CD127 - Foxp3 + T cells such as T cells. In certain embodiments, the cells are lymphoid progenitor cells, hematopoietic stem cells (HSCs), human umbilical cord blood-derived erythroid progenitor (HUDEP) cells, natural killer cells or dendritic cells. In certain embodiments, the cells are HSCs or HUDEP cells.

[0008] In certain embodiments, the disclosure provides isolated cells or cell populations that include modifications such as disruption of the HBG locus, generated by delivery of an RNP complex comprising, for example, a Cpf1 RNA-induced nuclease and a gRNA molecule that targets the HBG locus, including, for example, the regulatory region of the HBG gene. In certain embodiments, the RNP complex comprises a complex between the Cpf1 RNA-induced nuclease and the gRNA molecule. In certain embodiments, any region of the HBG locus may be targeted. In certain embodiments, the cis-regulatory region of the HBG gene is targeted. In certain embodiments, the disclosure relates to the use of CRISPR / Cpf1-mediated editing, such as disruption of the promoter region of the HBG locus. In certain embodiments, the disclosure relates to the use of CRISPR / Cpf1-mediated editing of the -800 to -60nt promoter region of the HBG locus, such as the -110nt promoter region. In certain embodiments, the cis-regulatory region of the HBG locus may be edited, such as by disruption. For example, but not limited to, CRISPR / Cpf1-mediated editing can disrupt the CAAT box located in the cis-regulatory region of the HBG locus. In general, disruption of the HBG promoter region and the CAAT box can be achieved through the delivery of a CRISPR / Cpf1 editing system targeting these sequences. Non-exclusive examples of gRNA molecules for use in such CRISPR / Cpf1 editing systems targeting these sequences of the HBG locus are specified in Figures 6, 9, and 11 and Table 19. In certain embodiments, the gRNA molecule targeting the HBG gene sequence includes the sequence of a gRNA molecule referred to as HBG1-1.

[0009] In certain embodiments, the disclosure relates to isolated CRISPR / Cpf1 edited cells in which the -110nt promoter region of the HBG locus is disrupted using a complex comprising a CRISPR / Cpf1 RNA-induced nuclease and a guide RNA that targets the -110nt promoter region of the HBG locus. In certain embodiments, such CRISPR / Cpf1 edited cells may comprise one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, such CRISPR / Cpf1 edited cells may not comprise one or more components of the CRISPR / Cpf1 editing system, as determined by a suitable method used to detect such components. In certain embodiments, the disclosure relates to a population of CRISPR / Cpf1 edited cells in which the -110nt promoter region of the HBG locus is disrupted using a complex comprising a CRISPR / Cpf1 RNA-induced nuclease and a guide RNA that targets the -110nt promoter region of the HBG locus. In certain embodiments, such a population of CRISPR / Cpf1-edited cells may include cells containing one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, such a population of CRISPR / Cpf1-edited cells does not include one or more components of the CRISPR / Cpf1 editing system, as determined by a suitable method used to detect such components. In certain embodiments, the disclosure relates to CRISPR / Cpf1-edited cells in which a CAAT box present in the HBG promoter region is disrupted using a complex comprising a CRISPR / Cpf1 RNA-induced nuclease and a guide RNA that targets the CAAT box present in the promoter region of the HBG locus. In certain embodiments, such cells contain one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, such CRISPR / Cpf1-edited cells do not include one or more components of the CRISPR / Cpf1 editing system, as determined by a suitable method used to detect such components.In certain embodiments, the disclosure relates to a population of CRISPR / Cpf1-edited cells in which a CAAT box located in the HBG promoter region is disrupted using a complex comprising a CRISPR / Cpf1 RNA-induced nuclease and a guide RNA that targets the CAAT box located in the promoter region of the HBG locus. In certain embodiments, such a population of CRISPR / Cpf1-edited cells may comprise cells containing one or more components of the CRISPR / Cpf1 editing system.

[0010] In certain embodiments, the disclosure provides CRISPR / Cpf1-edited cells or cell populations edited using CRISPR / Cpf1, including modifications such as interference with the specific expression of the transcriptional repressor BCL11a, which are generated by the delivery of a complex comprising, for example, a Cpf1 RNA-induced nuclease and a gRNA molecule targeting the BCL11a gene sequence. In certain embodiments, any region of the BCL11a gene sequence may be targeted. For example, but not limited to, the erythroid enhancer region of the BCL11a gene, such as the erythroid enhancer region +55kb to +62kb from the transcription start site (TSS), may be targeted. In certain embodiments, the GATA1 binding motif of BCL11a located in the +58DHS region of intron 2 of the BCL11a gene may be disrupted using CRISPR / Cpf1-mediated editing. Disruption of the GATA1 binding motif of BCL11a can be achieved via delivery of a CRISPR / Cpf1 editing system that targets that motif. Non-limiting examples of gRNA molecules for use with such a CRISPR / Cpf1 editing system that targets the GATA1 motif of BCL11a are identified in Figures 7, 10, and 12.

[0011] In certain embodiments, this disclosure relates to CRISPR / Cpf1 edited cells in which the +58DHS region of intron 2 of the BCL11a gene is disrupted. In certain embodiments, such CRISPR / Cpf1 edited cells may include one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, this disclosure relates to a population of CRISPR / Cpf1 edited cells in which the +58DHS region of intron 2 of the BCL11a gene is disrupted. In certain embodiments, such a population of CRISPR / Cpf1 edited cells may include cells containing one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, this disclosure relates to CRISPR / Cpf1 edited cells in which the GATA1 motif of the BCL11a gene is disrupted. In certain embodiments, such CRISPR / Cpf1 edited cells may include one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, the disclosure relates to a population of CRISPR / Cpf1-edited cells in which the GATA1 motif of the BCL11a gene is disrupted. In certain embodiments, such a population of CRISPR / Cpf1-edited cells may include cells containing one or more components of a CRISPR / Cpf1 editing system. In certain embodiments, one or more components of the CRISPR / Cpf1 system used to modify or disrupt the BCL11a gene within an intracellular or cell population are undetectable using appropriate means used to detect such components.

[0012] In certain embodiments, the disclosure provides isolated CRISPR / Cpf1-edited T cells or populations of CRISPR / Cpf1-edited T cells, including modifications such as disruption of one or more endogenous genes of T cells. In certain embodiments, the disclosure relates to the use of CRISPR / Cpf1-mediated editing of endogenous genes of T cells, selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, TRBC, and any combination thereof. For example, but not limited to, modifications are generated by the delivery of one or more complexes comprising a Cpf1 RNA-induced nuclease, such as an RNP complex, and a gRNA molecule, targeting, for example, a portion of the FAS gene sequence, a portion of the BID gene sequence, a portion of the CTLA4 gene sequence, a portion of the PDCD1 gene sequence, a portion of the CBLB gene sequence, a portion of the PTPN6 gene sequence, a portion of the B2M gene sequence, a portion of the TRAC gene sequence, a portion of the CIITA gene sequence, a portion of the TRBC gene sequence, or a combination thereof. For example, but not limited to, two or more complexes, such as RNP complexes, may be delivered, each complex targeting a different gene. In certain embodiments, the gRNA may be complementary to any strand of the gene being targeted. In certain embodiments, the gRNA molecule may target a regulatory region, intron, or exon of the gene being targeted.

[0013] In certain embodiments, the CRISPR / Cpf1 system contained herein targets the TRAC gene, generating, for example, isolated CRISPR / Cpf1-edited T cells or a population of CRISPR / Cpf1-edited T cells, which include modifications such as disruption of the TRAC gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the TRAC gene sequence. In certain embodiments, the gRNA may be complementary to either strand of the TRAC gene. In certain embodiments, the targeted portion of the TRAC gene sequence is within the coding sequence of the TRAC gene. In certain embodiments, the targeted portion of the TRAC gene sequence is within an exon. In certain embodiments, the targeted portion of the TRAC gene sequence is within an intron. In certain embodiments, the targeted portion of the TRAC gene sequence is within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the TRAC gene sequence is within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, gRNA molecular targeting domains for use in such CRISPR / Cpf1 systems targeting TRAC include the targeting domain sequences listed in Tables 2 and 3.

[0014] In certain embodiments, the CRISPR / Cpf1 system contained herein targets the TRBC gene, generating, for example, isolated CRISPR / Cpf1-edited T cells or a population of CRISPR / Cpf1-edited T cells, which include modifications such as disruption of the TRBC gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the TRBC gene sequence. In certain embodiments, the gRNA may be complementary to either strand of the TRBC gene. In certain embodiments, the targeted portion of the TRBC gene sequence is within the coding sequence of the TRBC gene. In certain embodiments, the targeted portion of the TRBC gene sequence is within an exon. In certain embodiments, the targeted portion of the TRBC gene sequence is within an intron. In certain embodiments, the targeted portion of the TRBC gene sequence is within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the TRBC gene sequence is within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, gRNA molecular targeting domains for use in such CRISPR / Cpf1 systems targeting TRBCs include the targeting domain sequences listed in Tables 4 and 5.

[0015] In certain embodiments, the CRISPR / Cpf1 system contained herein targets the B2M gene, generating, for example, isolated CRISPR / Cpf1-edited T cells or a population of CRISPR / Cpf1-edited T cells that include modifications such as disruption of the B2M gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the B2M gene sequence. In certain embodiments, the gRNA may be complementary to either strand of the B2M gene. In certain embodiments, the targeted portion of the B2M gene sequence is located within the coding sequence of the B2M gene. In certain embodiments, the targeted portion of the B2M gene sequence is located within an exon. In certain embodiments, the targeted portion of the B2M gene sequence is located within an intron. In certain embodiments, the targeted portion of the B2M gene sequence is located within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the B2M gene sequence is located within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, the gRNA molecular targeting domain for use in such a CRISPR / Cpf1 system targeting B2M includes the targeting domain sequences listed in Tables 6, 7, and 8. In certain embodiments, the gRNA molecular targeting domain for use in such a CRISPR / Cpf1 system targeting B2M includes the nucleic acid sequence AGUGGGGGUGAAUUCAGUGU.

[0016] In certain embodiments, the CRISPR / Cpf1 system contained herein targets the CIITA gene, generating, for example, isolated CRISPR / Cpf1-edited T cells or a population of CRISPR / Cpf1-edited T cells that include modifications such as disruption of the CIITA gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the CIITA gene sequence. In certain embodiments, the gRNA may be complementary to any strand of the CIITA gene. In certain embodiments, the targeted portion of the CIITA gene sequence is located within the coding sequence of the CIITA gene. In certain embodiments, the targeted portion of the CIITA gene sequence is located within an exon. In certain embodiments, the targeted portion of the CIITA gene sequence is located within an intron. In certain embodiments, the targeted portion of the CIITA gene sequence is located within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the CIITA gene sequence is located within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, a gRNA molecular targeting domain for use in such a CRISPR / Cpf1 system targeting CIITA includes the targeting domain sequences listed in Table 9.

[0017] In certain embodiments, the CRISPR / Cpf1 systems contained herein use gRNAs that target two or more combinations of the TRAC, CIITA, TRBC, and B2M genes, thereby generating isolated CRISPR / Cpf1-edited T cells or populations of CRISPR / Cpf1-edited T cells that include modifications such as the disruption of two or more TRAC, CIITA, TRBC, and B2M genes, for example, by targeting one or more exons, one or more introns, or one or more regulatory regions of these genes. In certain embodiments, the CRISPR / Cpf1 systems of this disclosure may include one or more complexes comprising a Cpf1 RNA-induced nuclease and a gRNA molecule that target one or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC, for example. For example, but not limited to, the CRISPR / Cpf1 system of this disclosure may include (a) a first RNP complex comprising a first gRNA containing a first target domain complementary to the target sequence of a first gene, and a first Cpf1 RNA-induced nuclease; and (b) a second RNP complex comprising a second gRNA molecule containing a second targeting domain complementary to the target sequence of a second gene, and a second Cpf1 RNA-induced nuclease. In certain embodiments, the first and second genes are selected from the group consisting of B2M, TRAC, CIITA, and TRBC. The CRISPR / Cpf1 system may further include additional RNP complexes targeting one or more additional genes. For example, but not limited to, in the case of multiplexing, each RNP complex may contain the same Cpf1 protein, or each RNP complex may contain different Cpf1 proteins, such as Cpf1 protein mutants.

[0018] In certain embodiments, isolated cells, such as isolated CRISPR / Cpf1-edited HSCs or CRISPR / Cpf1-edited T cells or a population of such CRISPR / Cpf1-edited cells, do not contain one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, determination using appropriate means for detecting such components reveals that less than 10%, less than 5%, or less than 1% of the CRISPR / Cpf1-edited cells in the cell population contain one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in a cell population are edited and / or modified, for example, having disruption of the BCL11a gene, disruption of the HBG locus, and / or disruption of one or more genes selected from FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC. In certain embodiments, the cell population has more than about 15% editing, more than about 20% editing, more than about 25% editing, more than about 30% editing, more than about 35% editing, more than about 40% editing, more than about 45% editing, more than about 50% editing, more than about 55% editing, or more than about 60% editing. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in a cell population have productive indels.

[0019] In another aspect, the disclosure relates to modified Cpf1 proteins and their use in CRISPR / Cpf1-related methods for editing and / or modulating the expression of target nucleic acid sequences. The disclosure further provides nucleic acids encoding modified Cpf1 proteins.

[0020] In certain embodiments, the modified Cpf1 protein is the Cpf1 protein of the Acidaminococcus species BV3L6 (AsCpf1), Francisella novicida U112 (FnCpf1), Moraxella bovoculi 237 (MbCpf1), Candidatus Methanomethylphilus alvus Mx1201 (CMaCpf1), Sneathia amnii (SaCpfq), Moraxella lacunata (MlCpf1), Moraxella bovoculi (Moraxella Moraxella bovoculi AAX08_00205 (Mb2Cpf1), Moraxella bovoculi AAX11_00205 (Mb3Cpf1), Lachnospiraceae bacterium ND2006 Cpf1 protein (LbCpf1), Lachnospiraceae bacterium MA2020 (Lb5Cpf1), Lachnospiraceae bacterium MC2017 (Lb4Cpf1), Flavobacterium branchiofilm Branchiophilum FL-15 (FbCpf1), Thiomicrospira species XS5 (TsCpf1), Parcubacteria group bacteria GW2011 (PgCpf1), Candidatus Roizmanbacteria bacteria GW2011 (CRbCpf1), Candidatus Peregrinbacteria bacteria GW2011 (CPbCpf1), Butyrivibrio species NC3005 (BsCpf1), Butyrivibrio fibrisolvens (BfCpf1), Prevotella bryanchiiIt is derived from a Cpf1 protein selected from the group consisting of bryantii)B14(Pb2Cpf1) and Bacteroidetes oral taxa 274(BoCpf1) (see, for example, Zetsche et al., bioRxiv 134015;doi:https: / / doi.org / 10.1101 / 134015, the entire content of which is incorporated herein by reference).

[0021] In certain embodiments, the modified Cpf1 protein includes a nuclear localization signal (NLS). For example, but not limited to, such NLS sequences are selected from the group consisting of nucleoplasmin NLS (nNLS) (SEQ ID NO: 1) and Simianvirus 40 "SV40" NLS (sNLS) (SEQ ID NO: 2).

[0022] In certain embodiments, the NLS sequence of the modified Cpf1 protein is located at or near the C-terminus of the Cpf1 protein sequence. For example, but are not limited to, the modified Cpf1 protein may be selected from His-AsCpf1-nNLS (SEQ ID NO: 3); His-AsCpf1-sNstaneyLS (SEQ ID NO: 4); and His-AsCpf1-sNLS-sNLS (SEQ ID NO: 5). In certain embodiments, the NLS sequence of the modified Cpf1 protein is located at or near the N-terminus of the Cpf1 protein sequence. For example, but are not limited to, the modified Cpf1 protein may be selected from His-sNLS-AsCpf1 (SEQ ID NO: 6), His-sNLS-sNLS-AsCpf1 (SEQ ID NO: 7), and sNLS-sNLS-AsCpf1 (SEQ ID NO: 8). In certain embodiments, the modified Cpf1 protein includes an NLS sequence located at or near both the N-terminus and the C-terminus of the Cpf1 protein sequence. For example, but not limited to, modified Cpf1 proteins may be selected from His-sNLS-AsCpf1-sNLS (SEQ ID NO: 9) and His-sNLS-sNLS-AsCpf1-sNLS-sNLS (SEQ ID NO: 10). For example, combinations of two or more nNLS sequences or nNLS and sNLS sequences (or other NLS sequences), as well as the identity of NLS sequences and additional permutations of N-terminal / C-terminal positions, such as the addition of sequences with or without purified sequences, such as 6-histidine sequences, are within the scope of the subject matter currently disclosed.

[0023] In certain embodiments, the modified Cpf1 protein includes modifications (e.g., deletions or substitutions) to one or more cysteine ​​residues in the Cpf1 protein sequence. For example, but not limited to, the modified Cpf1 protein includes modifications at positions selected from the group consisting of C65, C205, C334, C379, C608, C674, C1025, and C1248. In certain embodiments, the modified Cpf1 protein includes substitutions of one or more serine or alanine cysteine ​​residues. In certain embodiments, the modified Cpf1 protein includes modifications selected from the group consisting of C65S, C205S, C334S, C379S, C608S, C674S, C1025S, and C1248S. In certain embodiments, the modified Cpf1 protein includes modifications selected from the group consisting of C65A, C205A, C334A, C379A, C608A, C674A, C1025A, and C1248A. In certain embodiments, the modified Cpf1 protein includes modifications at positions C334 and C674 or C334, C379, and C674. In certain embodiments, the modified Cpf1 protein includes modifications at C334S and C674S or C334S, C379S, and C674S. In certain embodiments, the modified Cpf1 protein includes modifications at C334A and C674A or C334A, C379A, and C674A. In certain embodiments, modified Cpf1 proteins include both one or more cysteine ​​residue modifications, such as His-AsCpf1-nNLS Cys-less (SEQ ID NO: 11) or His-AsCpf1-nNLS Cys-low (SEQ ID NO: 12), and the introduction of one or more NLS sequences. In certain embodiments, Cpf1 proteins including one or more cysteine ​​residue deletions or substitutions exhibit reduced aggregation.

[0024] In further embodiments, the disclosure provides methods for modifying one or more target sequences within a cell. In certain embodiments, such methods include contacting a cell or cell population with (a) a gRNA molecule complementary to a target sequence of interest; and (b) a Cpf1 RNA-induced nuclease. In certain embodiments, the Cpf1 RNA-induced nuclease modifies the target sequence of interest in the cell or cell population. In certain embodiments, the cell is a T cell, a hematopoietic stem cell (HSC), or a human umbilical cord blood-induced erythroid progenitor (HUDEP) cell. In certain embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population are modified. In certain embodiments, the target sequence of interest is, for example, an HBG1 gene sequence such as a promoter region, and the gRNA molecule includes the sequence of the gRNA molecule HBG1-1. In certain embodiments, the target sequence of interest is the BCL11a gene sequence. Alternatively, the target nucleic acid sequence is selected from the group consisting of parts of the FAS gene sequence, parts of the BID gene sequence, parts of the CTLA4 gene sequence, parts of the PDCD1 gene sequence, parts of the CBLB gene sequence, parts of the PTPN6 gene sequence, parts of the B2M gene sequence, parts of the TRAC gene sequence, parts of the CIITA gene sequence, parts of the TRBC gene sequence, and combinations thereof.

[0025] The disclosure further provides a method for modifying one or more genes, such as two or more, three or more, or four or more, within a cell, for example, by contacting a cell with a first RNP complex comprising (a) a first gRNA containing a first target domain complementary to the target sequence of a first gene and a first Cpf1 RNA-induced nuclease; and (b) a second RNP complex comprising a second gRNA molecule containing a second targeting domain complementary to the target sequence of a second gene and a second Cpf1 RNA-induced nuclease. In certain embodiments, the method may further comprise a third RNP complex comprising (c) a third gRNA molecule containing a third targeting domain complementary to the target sequence of a third gene and a third Cpf1 RNA-induced nuclease, and / or (d) a fourth RNP complex comprising a fourth gRNA molecule containing a fourth targeting domain complementary to the target sequence of a fourth gene and a fourth Cpf1 RNA-induced nuclease. In certain embodiments, each RNP complex may comprise the same Cpf1 protein, or each RNP complex may comprise different Cpf1 proteins, such as Cpf1 protein mutants. In certain embodiments, a method for modifying one or more genes, such as two or more, three or four or more, within a cell may include contacting a cell with (a) a first gRNA containing a first targeting domain complementary to the target sequence of a first gene; (b) a second gRNA molecule containing a second targeting domain complementary to the target sequence of a second gene; and (c) a Cpf1 RNA-inducible nuclease encoded by a Cpf1 RNA-inducible nuclease disclosed herein or a nucleic acid encoding a disclosed Cpf1 RNA-inducible nuclease. In certain embodiments, the method may further include (d) a third gRNA molecule containing a third targeting domain complementary to the target sequence of a third gene, and / or (e) a fourth gRNA molecule containing a fourth targeting domain complementary to the target sequence of a fourth gene. The Cpf1 RNA-inducible nuclease modifies the first gene, the second gene, the third gene, and / or the fourth gene. In certain embodiments, the first, second, third, and fourth genes are selected from the group consisting of the B2M, TRAC, CIITA, and TRBC genes. In certain embodiments, the cells are T cells.

[0026] In another embodiment, the Disclosure relates to a method for treating a subject by administering to the subject one or more cells modified using the CRISPR / Cpf1 system contained herein. In certain embodiments, one or more cells are modified ex vivo or in vitro and then administered to the subject. In certain embodiments, the method for treating the subject includes contacting cells obtained from a subject having a CRISPR / Cpf1 system comprising (a) a gRNA molecule complementary to the target sequence of a target nucleic acid; and (b) a Cpf1 RNA-induced nuclease disclosed herein. In certain embodiments, the Disclosure relates to a method for treating a subject in need by administering to the subject one or more cells obtained from a donor and genetically modified ex vivo or in vitro using the CRISPR / Cpf1 system of the Disclosure prior to administration to the subject. In certain embodiments, the subject suffers from, for example, an abnormal hemoglobinopathy such as sickle cell disease or β-thalassemia. In certain embodiments, the subject suffers from cancer or an autoimmune disorder.

[0027] In certain embodiments, the disclosure further provides a method for administering a cell population to a subject suffering from an abnormal hemoglobinopathy, wherein the cell population includes modifications to the HBG gene sequence or the BCL11a gene sequence, generated by delivery of a complex comprising a Cpf1 RNA-induced nuclease and a gRNA molecule targeting the HBG gene sequence or the BCL11a gene sequence. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population are modified. In certain embodiments, the cells are hematopoietic stem cells (HSCs) or human umbilical cord blood-induced erythroid progenitor (HUDEP) cells.

[0028] In further embodiments, the Disclosure provides gRNA molecules for targeting nucleic acid sequences of interest to generate modified cells, such as CRISPR / Cpf1 edited cells. In certain embodiments, the gRNA molecule comprises a first targeting domain complementary to the target sequence, where the target sequence is the HBG gene sequence or the BCL11a gene sequence. Non-limiting examples of such gRNAs are provided in Figures 6-12 and 46 and Table 19. In certain embodiments, the Disclosure provides a CRISPR / Cpf1 system comprising a gRNA molecule, which, upon introduction into cells, forms an indel at or near the target sequence complementary to the first targeting domain of the gRNA molecule, and / or upon introduction into cells, results in a deletion at a sequence complementary to the first targeting domain of the gRNA within the HBG1 or HBG2 promoter region. In certain embodiments, the CRISPR / Cpf1 system comprising the gRNA molecule of the Disclosure, upon introduction into cells, results in increased expression of fetal hemoglobin. In certain embodiments, a CRISPR / Cpf1 system comprising the gRNA molecule of the Disclosure results in an increase in the expression of fetal hemoglobin in an amount suitable for partially or completely alleviating the symptoms of an abnormal hemoglobin disorder, such as sickle cell disease or β-thalassemia. For example, but not limited to, the expression of fetal hemoglobin may increase by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% compared to the level of fetal hemoglobin expression in cells or cell populations without disruption of the BCL11a gene or HBG gene locus and / or the gene. In certain embodiments, the increase in fetal hemoglobin expression may exceed about 1 picogram (pg), about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, or about 10 pg.

[0029] This disclosure further provides gRNA molecules containing a first targeting domain complementary to a target sequence, the target sequence being selected from the group consisting of a portion of the B2M gene sequence, a portion of the TRAC gene sequence, a portion of the CIITA gene sequence, a portion of the TRBC gene sequence, and combinations thereof. Non-limiting examples of such gRNAs are provided in Tables 2-9.

[0030] This disclosure provides compositions comprising gRNA molecules disclosed herein. In certain embodiments, the gRNA molecules comprise the gRNAs disclosed in Tables 2-9 and 19 and Figures 6-12. In certain embodiments, the gRNAs target chromosomal locations (e.g., genomic coordinates) provided in Table 18. In certain embodiments, the compositions may further comprise, for example, the Cpf1 protein to generate RNP complexes. In certain embodiments, this disclosure provides compositions comprising one or more RNP complexes, such as an RNP complex population, where each RNP complex targets a different gene or gene region. In certain embodiments, the compositions may be used to treat subjects in need, such as those suffering from cancer, autoimmune disorders, or hemoglobin disorders.

[0031] In another embodiment, the disclosure relates to genome editing systems for modifying target nucleic acid sequences. In certain embodiments, the genome editing system may include a gRNA molecule and a Cpf1 RNA-inducible nuclease disclosed herein. The disclosure further provides, for example, a multiple genome editing system for editing two or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC.

[0032] In a further aspect, the disclosure relates to a method for evaluating CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or the modification of the expression of a target nucleic acid sequence, as well as components for achieving this.

[0033] In certain embodiments, a method for evaluating CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of a target nucleic acid sequence includes comparing the activity of a test Cpf1 protein with respect to the target nucleic acid sequence to a control Cpf1 protein. In certain embodiments, the test Cpf1 protein includes one or more modifications compared to a control, such as a wild-type Cpf1 protein. Examples of such modifications include, but are not limited to, the incorporation of one or more NLS sequences, the incorporation of a 6-histidine purified sequence, and modifications of Cpf1 protein cysteine ​​amino acids and combinations thereof.

[0034] In certain embodiments, a method for evaluating CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of a target nucleic acid sequence includes comparing the activity of a test Cpf1 protein with respect to a “matching site” target nucleic acid sequence to a control Cas9 protein. In the use herein, a matching site target nucleic acid sequence incorporates both the requirement of being edited by Cpf1 and Cas9, such as, for example, the TTTV AsCpf1 wild-type protospacer adjacent motif ("PAM") and the NGG SpCas9 wild-type PAM. As described above, a test Cpf1 protein may contain one or more modifications compared to a wild-type Cpf1 protein. Examples of such modifications include, but are not limited to, the aforementioned modifications for incorporating one or more NLS sequences, the aforementioned modifications for incorporating 6-histidine purified sequences, and modifications of Cpf1 protein cysteine ​​amino acids and combinations thereof.

[0035] In certain embodiments, this disclosure relates to assays for comparing CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of the target nucleic acid sequence by a test CRISPR / Cpf1 genome editing system with a control RNA-induced nuclease genome editing system. For example, but not limited to, the test and control genome editing systems may differ in one or more aspects: the sequence of the RNA-induced nuclease; the source of the components of the genome editing system, such as a method for manufacturing the components of the genome editing system; the formulation of one or more components of the genome editing system; and the identity of the cells into which the genome editing system is introduced, such as the cell type or method of cell preparation. In certain embodiments, the assays described herein enable quality control analysis of the test genome editing system. In certain embodiments, the assays of this disclosure evaluate CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of the target nucleic acid sequence, where the target includes a matching site sequence.

[0036] In certain embodiments, the use of matched site-targeted nucleic acids enables assays and / or evaluations of CRISPR / Cas9-mediated editing (or editing by another CRISPR-based system) of the target nucleic acid sequence and / or CRISPR / Cpf1-mediated editing in contrast to the modification of the expression of the target nucleic acid sequence.

[0037] In certain embodiments, the use of matched site-targeted nucleic acids enables assays and / or evaluations of CRISPR / Cas9-mediated editing (or editing by another CRISPR-based system) of a target nucleic acid sequence and / or CRISPR / Cpf1-mediated editing in contrast to the modification of the expression of the target nucleic acid sequence in specific cell types. For example, but not limited to, such methods can be used to analyze several cell types, particularly T cells and hematopoietic stem cells (CD34). + CRISPR / Cpf1-mediated editing can be evaluated in comparison to CRISPR / Cas9-mediated editing and / or regulation of target nucleic acid sequence expression in HSCs (including, but not limited to, HSCs) and human umbilical cord blood-induced erythroid progenitor (HUDEP) cells.

[0038] In certain embodiments, the use of matched site target nucleic acids enables assays and / or evaluations of CRISPR / Cpf1-mediated editing in comparison to CRISPR / Cas9-mediated editing (or editing by another CRISPR-based system) of the target nucleic acid sequence and / or modification of the expression of the target nucleic acid sequence, with respect to specific attributes of the CRISPR / Cpf1-mediated editing system used. For example, but not limited to, such methods can be used to evaluate CRISPR / Cpf1-mediated editing in comparison to CRISPR / Cas9-mediated editing of the target nucleic acid sequence and / or modification of the expression of the target nucleic acid sequence, and to identify differences in the activity of Cpf1 RNA-induced nucleases and / or gRNAs prepared by different manufacturing processes. Such methods can also identify differences in the activity of Cpf1 RNA-induced nucleases and / or gRNAs present in different formulations and using different delivery strategies.

[0039] In a particular embodiment, the matching site target nucleic acid sequence is selected from the group consisting of matching site 1 ("MS1"; SEQ ID NO: 13), matching site 5 ("MS5"; SEQ ID NO: 14), matching site 11 ("MS11"; SEQ ID NO: 15), and matching site 18 ("MS18"; SEQ ID NO: 16). In a particular embodiment, the matching site target nucleic acid sequence is MS5.

[0040] The CRISPR / Cpf1 editing system of this disclosure can be delivered to cells using a variety of strategies. For example, the expression of components of the CRISPR / Cpf1 editing system can be induced in cells using a vector, such as AAV or other viral vectors, which encode components of the CRISPR / Cpf1 editing system, but is not limited to these methods. Alternatively, the RNP complex containing various components of the CRISPR / Cpf1 editing system can be delivered to cells by electroporation or any other suitable method, which can be used to deliver the RNP complex to cells. In certain embodiments, the RNP complex can be delivered to cells using lipid nanoparticles.

[0041] The accompanying drawings are intended to provide illustrative and schematic examples, rather than comprehensive, of any particular aspects and embodiments of the present disclosure. The drawings are not intended to limit or be bound by any particular theory or model, and are not necessarily drawn to scale. Without limiting the foregoing, nucleic acids and polypeptides may be depicted as linear sequences or as schematic two- or three-dimensional structures; these depictions are intended to be illustrative, rather than limiting or being bound by any particular model or theory relating to their structures. [Brief explanation of the drawing]

[0042] [Figure 1] This provides an overview of how manipulated Cpf1 mutants expand the PAM targeting space. [Figure 2] This paper provides an overview of four matching site sequences (MS1, MS5, MS11, and MS18) from Kleinstiver et al., Nature Biotechnology, 34(8):869-74 Aug.2016, and the cell types used to evaluate the performance of Cpf1 and Cas9 in relation to these matching site target sequences. [Figure 3A] Figure 3A depicts the results of dose-response experiments comparing the increased concentration of Cpf1 / gRNA RNP at two matching site loci (MS1 and MS5) with that of Cas9 / gRNA RNP, as well as assay results comparing the activity of AsCpf1 and SpCas9 against matching site targets MS1, MS5, MS11, and MS18, showing that Cpf1 edits specific target sites more efficiently than Cas9. [Figure 3B] Figure 3A depicts the results of dose-response experiments comparing the increased concentration of Cpf1 / gRNA RNP at two matching site loci (MS1 and MS5) with that of Cas9 / gRNA RNP, as well as assay results comparing the activity of AsCpf1 and SpCas9 against matching site targets MS1, MS5, MS11, and MS18, showing that Cpf1 edits specific target sites more efficiently than Cas9. [Figure 4]This report compares various AsCpf1 NLS mutants across multiple cell types at a fixed 4.4 μM RNP dose using a five-site matching guide. Data are normalized to the mutant presenting maximum editing for each cell type. [Figure 5A] Figure 5A compares two optimal AsCpf1 NLS mutants at a 4.4 μM RNP dose using the guide RNA GWED545, which targets the TRAC locus in primary T cells, and Figure 5B compares the His-AsCpf1-sNLS-sNLS mutant at a 4.4 μM RNP dose using the guide RNA B2M-12, which targets the TRAC locus in primary T cells. In both cases, the data are normalized to the mutant that presents the greatest editing. [Figure 5B] Figure 5A compares two optimal AsCpf1 NLS mutants at a 4.4 μM RNP dose using the guide RNA GWED545, which targets the TRAC locus in primary T cells, and Figure 5B compares the His-AsCpf1-sNLS-sNLS mutant at a 4.4 μM RNP dose using the guide RNA B2M-12, which targets the TRAC locus in primary T cells. In both cases, the data are normalized to the mutant that presents the greatest editing. [Figure 6] This shows the gRNA sequences used in the HBG1 assay in HSC and HUDEP. [Figure 7] This shows the gRNA sequences used in the BCL11a assay in HSC and HUDEP. [Figure 8] This document shows specific sequences of HBG1 or BCL11a in either HSC or HUDEP, and their corresponding % edits. gRNAs proposed to target the HBB are also provided. [Figure 9] This shows the HBG1 promoter region where gRNA AsCpf1 WT HBG1-1 is bound to the CAAT box motif. [Figure 10] This shows a portion of the BCL11a enhancer region where gRNA BCL11a AsCpf1 RR-8 is bound to the GATA1 motif. [Figure 11] Figure 6 shows the HBG1 promoter region screened using the gRNA identified. This region extends for approximately 150 bp. HBG1-1 is shown overlapping with the CAAT box motif. [Figure 12] Figure 7 shows the region of the BCL11a erythrocyte enhancer screened using the gRNA identified. This region extends for approximately 600 base pairs, and BCL11a RR-8 is shown overlapping with the GATA1 motif. [Figure 13] This shows cysteine ​​variants in which the AsCpf1 low-cysteine ​​construct has been identified. [Figure 14] The results of the AlexaFluor maleimide assay demonstrate significantly reduced accessibility to cysteine ​​residues in AsCpf1 C334S, C379S, and C674S. [Figure 15] This paper describes the demonstration of equivalent endonuclease activity against MS5 substrate DNA in WT AsCpf1, AsCpf1 cysteine-free, and two low-cysteine ​​mutants. [Figure 16] This study demonstrates the targeting of the HBG1 promoter region by AsCpf1 WT and RR PAM mutants in HUDEP and HSCs. HUDEP experiments were performed using an optimal CA-137 pulse program and Lonza Solution SE. HSC screening was performed using pulse code EO-100 and Lonza Solution P3, as recommended by the manufacturer. The dose was 4.4 μM RNP for all guides, with a guide:protein ratio of 2:1. 50,000 HSCs were processed per condition. AsCpf1 WT and RR proteins had endotoxin levels of <5 EU / mL. [Figure 17]Screening of the BCL11a enhancer region with AsCpf1 WT, RR, and RVR PAM mutants, along with one WT FnCpf1 target in HUDEP and HSCs, is presented. HUDEP screening was performed using the optimal CA-137 pulse program with Lonza Solution SE. HSC screening was performed using pulse code EO-100 and Lonza Solution P3, as recommended by the manufacturer. A control guide for BCL11a (named KOBEH) is also shown. The dose for all guides was 4.4 μM RNP at a 2:1 guide:protein ratio. 50,000 HSCs were processed per condition. AsCpf1 WT, RR, and RVR proteins had endotoxin levels of <5 EU / mL. [Figure 18] This report describes nucleofection screening of AsCpf1 in HUDEP. The dose was 2.2 μM AsCpf1 RNP using matching site 5 (MS5) guide RNA in a 2:1 guide:protein ratio. AsCpf1 WT protein had endotoxin levels of <5 EU / mL. Lonza solutions SE, SF, and SG were tested with 50,000 HUDEPs per condition using different pulse programs. Pulse codes CA-137 and CA-138 in solution SE demonstrated optimal editing. [Figure 19] This report describes nucleofection screening of AsCpf1 in HSCs. The dose was 2.2 μM AsCpf1 RNP using matching site 5 (MS5) guide RNA in a 2:1 guide:protein ratio. AsCpf1 WT protein had endotoxin levels of <5 EU / mL. Lonza solutions P1, P2, P3, P4, and P5 were tested with 50,000 HSCs per condition using different pulse programs. Pulse codes CA-137 and CA-138 in solution P2 demonstrated optimal editing, as did FF-100 and FF-104. [Figure 20]The use of a specific pulse code in Lonza Amaxa is shown to increase HSC editing across target and PAM variants. The dose was 4.4 μM RNP at a 2:1 guide:protein ratio for all guides. 50,000 HSCs were treated per condition. AsCpf1 WT, RR, and RVR proteins had endotoxin levels of <5 EU / mL. [Figure 21] This study presents a screening of T cell therapeutic targets using AsCpf1 and its RR and RVR PAM variants at the TRBC, TRAC, and B2M loci. Preliminary screening showed that approximately 30% of gRNAs exhibited editing exceeding 50%, which is comparable to commonly observed SpCas9 hit rates, demonstrating the potential use of Cpf1 for gene editing of patient T cells at therapeutic loci, including but not limited to TRAC, TRBC, and / or B2M. [Figure 22] This study demonstrates that modifying the electroporation pulse code significantly improves the maximum editing of T cells at multiple therapeutic target gene loci. [Figure 23A] This demonstrates efficient knockout editing of primary T cells at disease-related loci using Cpf1 RNPs. Figure 23A shows the RNP workflow for ex vivo cell therapy. Figure 23B shows efficient single knockout at multiple therapeutically relevant T cell loci using AsCpf1 or engineered PAM mutants. [Figure 23B] This demonstrates efficient knockout editing of primary T cells at disease-related loci using Cpf1 RNPs. Figure 23A shows the RNP workflow for ex vivo cell therapy. Figure 23B shows efficient single knockout at multiple therapeutically relevant T cell loci using AsCpf1 or engineered PAM mutants. [Figure 24] This demonstrates highly efficient dual knockout of two therapeutic targets in Cpf1 RNP-treated T cells, as measured by flow cytometry. [Figure 25]This report presents a screening of T cell therapeutic targets using AsCpf1 and its RR and RVR PAM variants at the TRBC, TRAC, and B2M gene loci. [Figure 26] This paper summarizes the high editing efficiency of AsCpf1 WT, RR, and RVR in T cells on three allogeneic T cell targets. [Figure 27] This diagram illustrates dual knockout of two T cell targets in human primary T cells using either Cpf1 or Cas9. [Figure 28] This shows screening of T cell therapeutic targets by Cpf1 at the CIITA locus. [Figure 29] This paper summarizes the high editing efficiency of Cpf1 on T cells on three allogeneic T cell targets: TRAC, CIITA, and B2M, compared to SpCas9. [Figure 30] The efficiency of triple knockout of three T cell targets by Cpf1 RNP in T cells is illustrated. [Figure 31A] Figure 31A summarizes the specificity of the top Cpf1 candidate guides for the three T cell targets, CIITA, TRAC, and B2M, and shows the number of off-targets detected. Figure 31B shows that no detectable off-targets were found by targeted amplicon sequencing. [Figure 31B] Figure 31A summarizes the specificity of the top Cpf1 candidate guides for the three T cell targets, CIITA, TRAC, and B2M, and shows the number of off-targets detected. Figure 31B shows that no detectable off-targets were found by targeted amplicon sequencing. [Figure 32] This report identifies electroporation conditions that improve maximal editing in T cells. Condition 1 was DS-130, and Condition 2 was CA-137. [Figure 33] This paper identifies an NLS configuration that improves the efficacy of gene editing in T cells. NLS v1 represents the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 1), and NLS v2 represents the sequence 2xPKKKRKV (SEQ ID NO: 2). [Figure 34]This study demonstrates the editing efficiency at the HBG-1 gene locus in HSCs using AsCpf1 and an HBG1-1 guide. [Figure 35] This demonstrates the editing efficiency of NLS mutations in T cells at matching site 5 using MS5 guide RNA. [Figure 36] This shows a reduction in MHC II in T cells edited at the CIITA locus, as measured by flow cytometry. [Figure 37A] This shows the editing efficiency in T cells edited at the CIITA gene locus. [Figure 37B] This shows the genomic locations targeted by CIITA gRNA, CIITA-34, CIITA-41, CIITA-45, and CIITA-10. [Figure 38] This paper summarizes the percentage reduction in MHC II in T cells edited at the CIITA locus. [Figure 39] This shows the editing efficiency of Cpf1 CIITA gRNA and the number of off-targets detected by the gRNA. [Figure 40] This shows the editing efficiency of AspCpf1 RR, WT TRAC, CIITA, and B2M gRNA. [Figure 41] This shows the editing efficiency of AspCpf1 RR and WT B2M gRNA of different lengths. [Figure 42] This shows the editing efficiency of AspCpf1 RR and WT TRAC gRNA of different lengths. [Figure 43] This shows the editing efficiency of AspCpf1 RR and WT CIITA gRNA of different lengths. [Figure 44A]This is a schematic diagram of three potential PCR amplicons obtained from the use of an unedited genomic DNA targeting site, an exemplary DNA donor template for targeted incorporation, potential insertion results (i.e., untargeted incorporation at the cleavage site or targeted incorporation at the cleavage site), and a primer pair targeting the P1 priming site and P2 primer site (amplicon X), a primer pair targeting the P1 primer site and P2' priming site (amplicon Y), or a primer pair targeting the P1' primer site and P2 primer site (amplicon Z). The exemplary DNA donor template depicted contains the incorporated primer sites (P1' and P2') and stuffer sequences (S1 and S2). A1 / A2: donor homology arms, S1 / S2: donor stuffer sequences, P1 / P2: genomic primer sites, P1' / P2': incorporated primer sites, H1 / H2: genomic homology arms, N: cargo, X: cleavage site. [Figure 44B] This is a schematic diagram of two potential PCR amplicons obtained from the use of an unedited genomic DNA targeting site, an exemplary DNA donor template for targeted integration, potential insertion results (i.e., untargeted integration at the cleavage site or targeted integration at the cleavage site), and a primer pair targeting the P1 primer site and the P2 primer site (amplicon X) or the P1' primer site and the P2 primer site (amplicon Y). The exemplary DNA donor template contains the integrated primer site (P1') and the stuffer sequence (S2). A1 / A2: donor homology arm, S1 / S2: donor stuffer sequence, P1 / P2: genomic primer site, P1': integrated primer site, H1 / H2: genomic homology arm, N: cargo, X: cleavage site. [Figure 44C]This diagram shows two potential PCR amplicons obtained from the use of an unedited genomic DNA targeting site, an exemplary DNA donor template for targeted integration, potential insertion results (i.e., untargeted integration at the cleavage site or targeted integration at the cleavage site), and a primer pair targeting the P1 and P2 primer sites (amplicon X) or the P1 and P2' primer sites (amplicon Y). The exemplary DNA donor template contains the integrated primer site (P2') and stuffer sequence (S1). A1 / A2: donor homology arms, S1 / S2: donor stuffer sequence, P1 / P2: genomic primer sites, P2': integrated primer site, H1 / H2: genomic homology arms, N: cargo, X: cleavage site. [Figure 45] This shows an exemplary DNA donor template designed for gRNA targeting of the T cell receptor alpha constant (TRAC) gene locus. [Figure 46] This shows the gRNAs identified from screening the promoter regions of HBG1 and HBG2. [Modes for carrying out the invention]

[0043] Definitions and Abbreviations Unless otherwise specified, the following terms have the meanings associated with them in this section.

[0044] The indefinite articles "a" and "an" refer to at least one of the related nouns and are used interchangeably with the terms "at least one" and "one or more." For example, "module" means at least one module or one or more modules.

[0045] The conjunctions "or" and "and / or" are used synonymously as non-exclusive disjunctions.

[0046] The terms “approximately” or “about” as used herein may mean within the tolerance range of a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, such as limitations of the measuring system. For example, “approximately” may mean within or exceeding one standard deviation according to the convention of a given value. Where a particular value is described in this application and claims, unless otherwise stated, the term “approximately” may mean within the tolerance range of that particular value, such as ±10% of the value modified by the term “approximately.”

[0047] The phrase "essentially composed of" means that the listed chemical species are dominant, but other chemical species may be present in trace or small amounts that do not affect the structure, function, or behavior of the composition in question. For example, a composition essentially composed of a particular chemical species generally contains that chemical species in 90%, 95%, 96%, or more.

[0048] The term "domain" is used to represent a segment of a protein or nucleic acid. Unless otherwise specified, a domain does not need to possess any specific functional properties.

[0049] An "indel" is an insertion and / or deletion in a nucleic acid sequence. Indels may be products of DNA double-strand break repair, such as double-strand breaks formed by the genome editing systems of this disclosure. Indels are most commonly formed when breaks are repaired by "erroneous" repair pathways, such as the NHEJ pathway described below.

[0050] In relation to HSCs, a “productive indel” refers to an indel (deletion and / or insertion) that results in HbF expression. In certain embodiments, a productive indel in an HSC may induce HbF expression. In certain embodiments, a productive indel in an HSC may result in an increase in the level of HbF expression. In relation to T cells, a “productive indel” refers to an indel (deletion and / or insertion) that reduces the expression of a target gene in a T cell, such as an endogenous T cell gene. In certain embodiments, a “productive indel” in a T cell may result in a decrease or removal of the expression of a cell surface protein or marker on the T cell.

[0051] "Genetic transformation" refers to the modification of a DNA sequence by incorporating endogenous homologous sequences (e.g., homologous sequences within a gene array). "Genetic modification" refers to the modification of a DNA sequence by incorporating exogenous homologous sequences, such as exogenous single-stranded or double-stranded donor template DNA. Genetic transformation and genetic modification are products of DNA double-strand break repair via HDR pathways, such as those described below.

[0052] Indel, gene transformation, gene modification, and other genome editing results are typically evaluated by sequencing (most commonly by "next-generation" or "synthetic sequencing" methods, although Sanger sequencing may still be used) and quantified by the relative frequency of numerical changes in all sequencing reads (e.g., ±1, ±2 or more bases). DNA samples for sequencing may be prepared by a variety of methods known in the art, including amplification of the target site by polymerase chain reaction (PCR), capture of DNA ends resulting from double-strand breaks, such as in the GUIDEseq process described by Tsai et al. (Nat. Biotechnol. 34(5):483 (2016), incorporated herein by reference), or by other means known in the art. Genome editing results may also be evaluated by in-situ hybridization methods such as the FiberComb® system commercialized by Genomic Vision (Bagneux, France) and any other suitable methods known in the art.

[0053] In the use of this specification, the term “modification of a target sequence” and its equivalents include, but are not limited to, deletions, insertions, gene transformations, gene modifications, and / or the introduction of indels into a target sequence. Modifications of a target sequence may result in changes in the expression of the target sequence; for example, modifications of a coding sequence may interfere with the expression of the protein encoded by that sequence, while modifications of a regulatory sequence may result in an increase or decrease in the expression of proteins under the control of that regulatory sequence, depending on whether the regulatory sequence activates or inhibits protein expression.

[0054] "Alt-HDR," "alternative homology-directed repair," or "alternative HDR" are used synonymously to refer to the process of repairing DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences such as sister chromatids or exogenous nucleic acids such as template nucleic acids). Alt-HDR differs from standard HDR in that the process utilizes a different pathway and can be inhibited by standard HDR mediators, RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of single-stranded or nicked homologous nucleic acid templates, whereas standard HDR generally involves double-stranded homologous templates.

[0055] Standard HDR, standard homology-directed repair, or cHDR refers to the process of repairing NA damage using homologous nucleic acids (e.g., endogenous homologous sequences such as sister chromatids or exogenous nucleic acids such as template nucleic acids). Standard HDR typically functions when there is significant excision of a double-strand break, forming a single-stranded portion of at least one DNA molecule. In normal cells, cHDR typically involves a series of steps including break recognition, break stabilization, excision, single-stranded DNA stabilization, DNA crossover intermediate formation, crossover intermediate degradation, and ligation. The process requires RAD51 and BRCA2, and the homologous nucleic acid is typically double-stranded.

[0056] Unless otherwise specified, the term "HDR" in this specification encompasses both standard HDR and alt-HDR.

[0057] "Non-homologous end joining" or "NHEJ" refers to ligation-mediated repairs and / or non-template-mediated repairs such as standard NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), which then include microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

[0058] When used in relation to the modification of a molecule (e.g., nucleic acid or protein), "substitution" or "substituted" simply indicates the presence of a substituted entity, without requiring any process restrictions.

[0059] "Subject" means human or non-human animal. Human subjects can be of any age (e.g., infants, children, young adults, or adults) and may be susceptible to disease or require genetic modification. Alternatively, subjects can be animals, and the above terms include, but are not limited to, mammals, birds, fish, reptiles, amphibians, and more particularly non-human primates, rodents (mice, rats, hamsters, etc.), rabbits, guinea pigs, dogs, cats, etc. In certain embodiments of this disclosure, subjects are livestock such as cattle, horses, sheep, or goats. In certain embodiments, subjects are poultry. In certain embodiments, subjects are plants.

[0060] "To treat," "to treat," and "treatment" mean the treatment of a disease in an object (e.g., a human object), including suppressing the disease, i.e., preventing or stopping its onset or progression; alleviating the disease, i.e., causing regression of the diseased state; alleviating one or more symptoms of the disease; and curing the disease.

[0061] "Preventing," "preventing," and "prevention" refer to the prevention of disease in mammals such as humans, including (a) avoiding or eliminating disease; (b) influencing predisposition to disease; or (c) preventing or delaying the onset of at least one symptom of disease.

[0062] A “kit” means any set of two or more components that together constitute a functional unit that can be used for a particular purpose. For illustrative purposes (and not limited to), a kit according to this disclosure may include a guide RNA that is or can be complexed with an RNA-inducing nuclease and accompanied by a pharmaceutically acceptable carrier (e.g., suspended or suspendable therein). For example, the kit may be used to introduce the complex into cells or subjects for the purpose of causing a desired genomic modification in such cells or subjects. The components of the kit may be packaged together or they may be packaged separately. A kit according to this disclosure may optionally include, for example, instructions for use (DFU) describing the use of the kit according to the methods of this disclosure. The DFU may be physically packaged with the kit or it may be provided to the kit user by, for example, electronic means.

[0063] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and mean any chain of two or more nucleotides. Polynucleotides, nucleotide sequences, nucleic acids, etc., may be single-stranded or double-stranded chimeric mixtures or derivatives or modified versions thereof. They may be modified, for example, with base moieties, sugar moieties, or phosphate backbone to improve molecular stability, their hybridization parameters, etc. Nucleic acid sequences typically carry genetic information, including, but not limited to, information used by cellular mechanisms to produce proteins and enzymes. These terms include double-stranded or single-stranded genomic DNA, RNA, any synthetic and genetically engineered polynucleotides, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

[0064] As shown in Table 1 below, the conventional IUPAC notation is used in the nucleotide sequences presented herein (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10;13(9):3021-30, which is incorporated herein by reference). However, it should be noted that if the sequence can be encoded by either DNA or RNA, for example in a gRNA targeting domain, “T” indicates “thymine or uracil”.

[0065] TIFF2026048661000001.tif113170

[0066] The terms “protein,” “peptide,” and “polypeptide” are used synonymously and refer to a continuous chain of amino acids linked together via peptide bonds. This term includes individual proteins, groups or complexes of proteins linked together, and fragments or parts, variants, derivatives, and analogues of such proteins. Peptide sequences are presented herein using conventional notation, starting from the amino or N-terminus on the left and progressing to the carboxyl or C-terminus on the right. Standard one- or three-letter abbreviations may be used.

[0067] The term "mutant" refers to an entity such as a polypeptide, polynucleotide, or small molecule that exhibits significant structural identity with a reference entity (e.g., a wild-type or naturally occurring entity), but is structurally different from the reference entity in the presence or level of one or more chemical parts, such as amino acids in relation to polypeptides or nucleotides in relation to polynucleotides, compared to the reference entity. In the usage herein, the term "mutant" also includes entities such as polypeptides, polynucleotides, or small molecules that are functionally better or superior to the reference entity in one or more properties associated with such entity. In many embodiments, mutants are also functionally different from their reference entity. For example, without limitation, "mutant Cpf1 polypeptide" includes the AsCpf1 mutant containing the S542R / K607R substitution and recognizing TYCV PAM, and the AsCpf1 mutant containing the S542R / K548V / N552R substitution and recognizing TATV PAM.

[0068] As used herein, the term “cleavage event” refers to the cleavage of a nucleic acid molecule. A cleavage event may be a single-strand break or a double-strand break. A single-strand break may result in a 5' overhang or a 3' overhang. A double-strand break may result in a blunt end, two 5' overhangs, or two 3' overhangs.

[0069] As used herein with respect to a site on a target nucleic acid sequence, the term “cleavage site” refers to a target location between two nucleotide residues of the target nucleic acid where a double-strand break occurs, or a target location within a span of several nucleotide residues of the target nucleic acid where two single-strand breaks mediated by an RNA-induced nuclease-dependent process occur. A cleavage site may, for example, be the target location for a blunt double-strand break. Alternatively, a cleavage site may be a target location within a span of several nucleotide residues of the target nucleic acid for two single-strand breaks or nicks that form a double-strand break and are separated, for example, by about 10 base pairs. The closer of the double-strand break or two single-strand nicks of a pair is ideally located within 0 to 500 bp of the target location (e.g., 450, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bp or less from the target location). When dual nickase is used, the two nicks within a pair should be within 25-55 bp of each other (e.g., 25-50, 25-45, 25-40, 25-35, 25-30, 50-55, 45-55, 40-55, 35-55, 30-55, 30-50, 35-50, 40-50, 45-50, 35-45, or 40-45 bp) and not more than 100 bp apart (e.g., 90, 80, 70, 60, 50, 40, 30, 20, or 10 bp or less).

[0070] This disclosure provides CRISPR / Cpf1-related methods and components for editing and / or modulating the expression of target nucleic acid sequences. For example, this disclosure provides CRISPR / Cpf1-related methods for targeting nucleic acid sequences that affect the proliferation, survival, persistence and / or function of hematopoietic stem cells (HSCs). In certain non-limiting embodiments, this disclosure provides a CRISPR / Cpf1 RNA-induced nuclease for CD34 +This disclosure provides the first evidence of efficient editing of target nucleic acid sequences in cells. Furthermore, this disclosure provides the first evidence of efficient editing of BCL11a and HBG1, genes associated with the heritability persistence of fetal hemoglobin (hereinafter referred to as "HPFH"), by Cpf1 RNA-induced nucleases. This disclosure also provides CRISPR / Cpf1-related methods for targeting nucleic acid sequences that affect T cell proliferation, survival, persistence, and / or function. This disclosure further provides modified Cpf1 proteins that exhibit significant editing efficiency and improved properties, and strategies for evaluating the efficiency of such modified Cpf1 proteins.

[0071] Modified Cpf1 protein In one aspect, the disclosure relates to modified Cpf1 proteins and their use in CRISPR / Cpf1-related methods for editing and / or modulating the expression of target nucleic acid sequences.

[0072] In certain embodiments, the modified Cpf1 protein is the Cpf1 protein of the Acidaminococcus species BV3L6 (AsCpf1), Francisella novicida U112 (FnCpf1), Moraxella bovoculi 237 (MbCpf1), Candidatus Methanomethylphilus alvus Mx1201 (CMaCpf1), Sneathia amnii (SaCpfq), Moraxella lacunata (MlCpf1), Moraxella bovoculi (Moraxella Moraxella bovoculi AAX08_00205 (Mb2Cpf1), Moraxella bovoculi AAX11_00205 (Mb3Cpf1), Lachnospiraceae bacterium ND2006 Cpf1 protein (LbCpf1), Lachnospiraceae bacterium MA2020 (Lb5Cpf1), Lachnospiraceae bacterium MC2017 (Lb4Cpf1), Flavobacterium branchiofilm Branchiophilum FL-15 (FbCpf1), Thiomicrospira species XS5 (TsCpf1), Parcubacteria group bacteria GW2011 (PgCpf1), Candidatus Roizmanbacteria bacteria GW2011 (CRbCpf1), Candidatus Peregrinbacteria bacteria GW2011 (CPbCpf1), Butyrivibrio species NC3005 (BsCpf1), Butyrivibrio fibrisolvens (BfCpf1), Prevotella bryanchiiThe Cpf1 protein is derived from the group consisting of bryantii)B14(Pb2Cpf1) and Bacteroidetes oral taxa 274(BoCpf1) (see, for example, Zetsche et al., bioRxiv 134015; doi:https: / / doi.org / 10.1101 / 134015, the entire content of which is incorporated herein by reference). In certain embodiments, the Cpf1 protein contains a sequence selected from the group consisting of SEQ ID NOs. 17-19, each having a codon-optimized nucleic acid sequence of SEQ ID NOs. 20-22.

[0073] Cpf1 nuclear localization signal (NLS) variant In certain embodiments, the modified Cpf1 protein contains a nuclear localization signal (NLS) (also referred to herein as the "Cpf1NLS variant"). For example, but not limited to, NLS sequences useful in relation to the methods and compositions disclosed herein would include amino acid sequences that can facilitate protein translocation into the cell nucleus. NLS sequences useful in relation to the methods and compositions disclosed herein are known in the art. Non-limiting examples of such NLS sequences include the nucleoplasmin NLS having the amino acid sequence: KRPAATKKAGQAKKKK (SEQ ID NO: 1) and the Simian virus 40 "SV40" NLS having the amino acid sequence PKKKRKV (SEQ ID NO: 2).

[0074] In certain embodiments, the modified Cpf1 protein may have one or more NLS sequences, such as two or more, three or more, or four or more. For example, but not limited to, the modified Cpf1 protein may have two NLS sequences, three NLS sequences, or four NLS sequences. In certain embodiments, the modified Cpf1 protein may have two NLS sequences. In certain embodiments, the NLS sequences of the modified Cpf1 protein are located at or near the C-terminus of the Cpf1 protein sequence. In certain embodiments, the NLS sequences of the modified Cpf1 protein are located at or near the N-terminus of the Cpf1 protein sequence. In certain embodiments, the modified Cpf1 protein of the Disclosure may have one or more NLS sequences located at or near the N-terminus of the Cpf1 protein sequence and one or more NLS sequences located at or near the C-terminus of the Cpf1 protein sequence, for example, the modified Cpf1 protein includes NLS sequences located at or near both the N-terminus and C-terminus of the Cpf1 protein sequence.

[0075] In certain embodiments, modified Cpf1 proteins having an NLS sequence located at or near the C-terminus of the Cpf1 protein sequence may be selected from His-AsCpf1-nNLS (also referred herein as "Asp Cpf1 NLS v1") (SEQ ID NO: 3); His-AsCpf1-sNLS (SEQ ID NO: 4); and His-AsCpf1-sNLS-sNLS (also referred herein as "Asp Cpf1 NLS v2") (SEQ ID NO: 5) (wherein "His" refers to a purified 6-histidine sequence, "AsCpf1" refers to an Acidaminococcus species Cpf1 protein sequence, "nNLS" refers to a nucleoplasmin NLS, and "sNLS" refers to an SV40 NLS). For example, additional permutations of NLS sequence identity and C-terminal position, such as the addition of two or more nNLS sequences or a combination of nNLS and sNLS sequences (or other NLS sequences), and the addition of sequences that include or do not include purified sequences, such as 6-histidine sequences, are within the scope of the currently disclosed subject matter.

[0076] In certain embodiments, modified Cpf1 proteins having an NLS sequence located at or near the N-terminus of the Cpf1 protein sequence may be selected from His-sNLS-AsCpf1 (SEQ ID NO: 6), His-sNLS-sNLS-AsCpf1 (SEQ ID NO: 7), and sNLS-sNLS-AsCpf1 (SEQ ID NO: 8). Additional permutations of NLS sequence identity and N-terminal position, such as the addition of two or more nNLS sequences or a combination of nNLS and sNLS sequences (or other NLS sequences), and the addition of sequences that include or do not include purified sequences, such as a 6-histidine sequence, are within the scope of the subject matter currently disclosed.

[0077] In certain embodiments, modified Cpf1 proteins having NLS sequences located at or near both the N-terminus and C-terminus of the Cpf1 protein sequence may be selected from His-sNLS-AsCpf1-sNLS (SEQ ID NO: 9) and His-sNLS-sNLS-AsCpf1-sNLS-sNLS (SEQ ID NO: 10). For example, additional permutations of NLS sequence identity and N-terminus / C-terminus positions, such as the addition of two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences) to either the N-terminus or C-terminus position, and the addition of sequences that include or do not include purified sequences such as 6-histidine sequences, are within the scope of the subject matter currently disclosed.

[0078] CD34 + To determine Cpf1 protein modifications, such as NLS modifications favorable for editing in cells and T cells, AsCpf1 proteins containing NLS sequences at different locations and types were synthesized. The protein mutants were complexed at matching site 5 targeting gRNA, and CD34 + Electroporation was performed within cells, T cells, and HUDEP (4.4 μM RNP). In Figure 4, the results are depicted as % editing normalized to the mutant that presents the maximum editing for each cell type. Data were obtained for different species of nucleases in CD34 + It exhibits various activities at the same target site in cells and T cells (especially among other cells), and efficient editing by AsCpf1 is possible for CD34 +This demonstrates that this can be achieved in cells and T cells (in particular among other cells).

[0079] Cysteine-modified Cpf1 protein and RNP Disulfide bond formation is known to promote protein aggregation. Therefore, as part of efforts to identify cysteine ​​that can be modified to reduce the likelihood of such disulfide bond formation, the crystal structure of Cpf1 and known primary amino acid sequences of Cpf1 were analyzed (Figure 13).

[0080] The modified Cpf1 proteins of this disclosure may include modifications (e.g., deletions or substitutions) at one or more cysteine ​​residues in the Cpf1 protein sequence. Such modified Cpf1 proteins exhibit reduced aggregation, which is particularly useful when scaling up protein production. For example, but not limited to, modified Cpf1 proteins include modifications at one or more positions, such as two or more, three or more, four or more, five or more, six or more, seven or more, or eight positions, selected from the group consisting of C65, C205, C334, C379, C608, C674, C1025, and C1248. In certain embodiments, modified Cpf1 proteins include substitutions of one or more serine or alanine cysteine ​​residues. In certain embodiments, the modified Cpf1 protein includes one or more modifications, such as substitutions selected from the group consisting of C65S, C205S, C334S, C379S, C608S, C674S, C1025S, and C1248S. In certain embodiments, the modified Cpf1 protein includes one or more modifications selected from the group consisting of C65A, C205A, C334A, C379A, C608A, C674A, C1025A, and C1248A. In certain embodiments, the modified Cpf1 protein includes modifications at positions C334 and C674 or C334, C379, and C674. In certain embodiments, the modified Cpf1 protein includes modifications at C334S and C674S or C334S, C379S, and C674S. In certain embodiments, the modified Cpf1 protein includes modifications to C334A and C674A or C334A, C379A and C674A. In certain embodiments, the modified Cpf1 protein includes both one or more cysteine ​​residue modifications, such as His-AsCpf1-nNLS Cys-less (SEQ ID NO: 11) or His-AsCpf1-nNLS Cys-low (SEQ ID NO: 12) as described herein, and the introduction of one or more NLS sequences.

[0081] CD34 in target sites associated with abnormal hemoglobin disorders + Editing Cpf1 for HSC This disclosure further provides CRISPR / Cpf1-related methods for editing targeted nucleic acid sequences to treat, for example, abnormal hemoglobin disorders such as β-thalassemia and sickle cell disease. For example, but not limited to, CRISPR / Cpf1-related methods modify the expression of fetal hemoglobin (HbF) CD34 + This results in the disruption of one or more genes in a cell.

[0082] One therapeutic strategy for treating hemoglobin disorders involves increasing HbF expression. HbF expression can be induced via targeted disruption of erythroid-specific expression of the transcriptional repressor BCL11a (Canvers et al., Nature, 527(12):192-197). One strategy to increase HbF expression is to interfere with BCL11a expression using gene editing. RNA-inducible nucleases, such as Cpf1 RNA-inducible nuclease, for example, can target specific target sequences that affect BCL11a gene expression. In certain embodiments, any region of the BCL11a gene may be targeted.

[0083] This disclosure provides cells or cell populations containing modifications to the BCL11a gene, for example, to interfere with, knock down, or knock out BCL11a expression. For example, but not limited to, cells or cell populations may be generated by delivery of a complex comprising a Cpf1 RNA-induced nuclease and a gRNA molecule, such as an RNP complex targeting the BCL11a gene sequence. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population are modified. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population have productive indels.

[0084] In certain embodiments, the Cpf1 RNA-induced nuclease may target intron 2 of the BCL11a gene. In certain embodiments, the Cpf1 RNA-induced nuclease would be targeted to disrupt the GATA1 binding motif of the erythrocyte-specific enhancer of BCL11a located in the +58DHS region of intron 2 of the BCL11a gene. Exemplary gRNA molecules for use in such a CRISPR / Cpf1 editing system targeting BCL11a are identified in Figures 7, 10, and 12.

[0085] In certain embodiments, this disclosure relates to cells in which the BCL11a gene is disrupted. In certain embodiments, the erythrocyte enhancer region of the BCL11a gene may be targeted, for example, the erythrocyte enhancer region +55kb to +62kb from the transcription start site (TSS). For example, but not limited to, this disclosure relates to cells in which the +58DHS region of intron 2 of the BCL11a gene is disrupted. In certain embodiments, such cells may include one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, this disclosure relates to a population of cells in which the BCL11a gene is disrupted, for example, the +58DHS region of intron 2 of the BCL11a gene is disrupted. In certain embodiments, such a population of cells includes cells containing one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, this disclosure relates to cells in which the GATA1 motif of the BCL11a gene is disrupted. In certain embodiments, such cells may include one or more components of a CRISPR / Cpf1 editing system. In certain embodiments, the disclosure relates to a population of cells in which the GATA1 motif of the BCL11a gene is disrupted. In certain embodiments, such a population of cells may include cells containing one or more components of a CRISPR / Cpf1 editing system.

[0086] As outlined in Example 3 below, AsCpf1 successfully mediated editing of the target site within the +58DHS region of intron 2 of the BCL11a gene. First, several AsCpf1 mutant guide RNAs with different PAMs (Figure 1) were screened in HUDEP2 cells, and then the most efficient guide RNA and nuclease mutant was identified as mPB CD34. + The tests were performed intracellularly (Figure 17). In particular, Figure 17 shows screening of the BCL11a enhancer region with AsCpf1 WT and RR and RVR PAM mutants, along with one WT FnCpf1 target in HUDEP and HSCs.

[0087] For example, in connection with the treatment of abnormal hemoglobin disorders such as β-thalassemia and sickle cell disease, another strategy to induce the expression of fetal hemoglobin is to interfere with the expression of HBG gene loci, particularly HGB1 and / or HGB2.

[0088] In certain embodiments, this disclosure relates to the use of CRISPR / Cpf1-mediated editing of the HBG locus. In certain embodiments, any region of the HBG locus may be targeted. In certain embodiments, non-coding regions of the HBG locus may be disrupted using CRISPR / Cpf1-mediated editing as described herein (see, for example, Table 18). In certain embodiments, introns of the HBG locus may be disrupted using CRISPR / Cpf1-mediated editing as described herein. In certain embodiments, cis-regulatory regions of the targeted HBG gene may be disrupted using CRISPR / Cpf1-mediated editing as described herein. For example, but not limited to, cis-regulatory regions may include promoters and / or enhancers. In certain embodiments, this disclosure relates to the use of CRISPR / Cpf1-mediated editing of the promoter region of the HBG locus. In certain embodiments, the -800 to -60nt region of the promoter region of the HBG locus may be disrupted using CRISPR / Cpf1-mediated editing as described herein. For example, but not limited to, CRISPR / Cpf1-mediated editing can disrupt the -110nt promoter region of the HBG promoter region and / or the CAAT box located within the HBG promoter region. In general, disruption of the HBG promoter region and the CAAT box can be achieved through the delivery of a CRISPR / Cpf1 editing system that targets these sequences. Exemplary gRNA molecules for use in such CRISPR / Cpf1 editing systems that target these sequences of the HBG locus are identified in Figures 6, 9, and 11 and Table 19. Regions of chromosomes (e.g., genomic coordinates) that can be targeted to disrupt the HBG locus are provided in Table 18. In a particular embodiment, the gRNA molecule used to disrupt the HBG1 locus is HBG1-1.

[0089] This disclosure provides cells or cell populations containing modifications to the HBG locus, for example, to interfere with, knock down, or knock out HBG expression. For example, but not limited to, cells or cell populations may be generated by delivery of a complex containing a Cpf1 RNA-induced nuclease and a gRNA molecule, such as an RNP complex targeting the HBG locus. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population are modified. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population have productive indels.

[0090] In certain embodiments, the disclosure relates to cells such as CD34+ hematopoietic stem and progenitor cells in which the HBG locus is disrupted. For example, but not limited to, the disclosure relates to cells in which the promoter region of the HBG locus is disrupted. In certain embodiments, the -110nt promoter region of the HBG locus is disrupted. In certain embodiments, such cells may contain one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, the disclosure relates to a cell population in which the -110nt promoter region of the HBG locus is disrupted. In certain embodiments, such a cell population may contain cells containing one or more components of the CRISPR / Cpf1 editing system, determined by a suitable method for detecting such components. In certain embodiments, the disclosure relates to cells in which the CAAT box present in the HBG promoter region is disrupted. In certain embodiments, such cells may contain one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, the disclosure relates to a cell population in which the CAAT box present in the HBG promoter region is disrupted. In certain embodiments, such a cell population may include cells containing one or more components of a CRISPR / Cpf1 editing system, determined by a suitable method for detecting such components. In certain embodiments, the disclosure provides a cell population in which the HBG1 locus is disrupted by the use of a CRISPR / Cpf1 editing system containing gRNA HBG1-1.

[0091] In certain embodiments, CRISPR / Cpf1 edited cells or populations of CRISPR / Cpf1 edited cells that include modifications to the HBG locus or the BCL11a gene do not contain one or more components as determined by a suitable method for detecting such components of the CRISPR / Cpf1 editing system. In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the CRISPR / Cpf1 edited cell population contains one or more components of the CRISPR / Cpf1 editing system as determined by a suitable method for detecting such components. In certain embodiments, the Disclosure provides a population of CRISPR / Cpf1-edited cells administered to a subject requiring it, wherein less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the CRISPR / Cpf1-edited cell population contains one or more components of a CRISPR / Cpf1 editing system.

[0092] In certain embodiments, disruption of the intracellular BCL11a gene or HBG gene by the CRISPR / Cpf1 editing system of the present disclosure may result in increased expression of fetal hemoglobin in cells compared to cells without disruption of the BCL11a gene or HBG gene. For example, but not limited to, compared to the expression level of fetal hemoglobin in cells without disruption of the BCL11a gene or HBG locus and / or gene, the expression of fetal hemoglobin may increase by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0093] In certain embodiments, disruption of the BCL11a gene or HBG gene in cells by the CRISPR / Cpf1 editing system of the present disclosure may result in an increase in the expression of fetal hemoglobin in an amount suitable for partially or completely alleviating the symptoms of abnormal hemoglobin disorders such as sickle cell disease or β-thalassemia. For example, but not limited to, the increase in the expression of fetal hemoglobin may be greater than about 1 picogram (pg), greater than about 2 pg, greater than about 3 pg, greater than about 4 pg, greater than about 5 pg, greater than about 6 pg, greater than about 7 pg, greater than about 8 pg, greater than about 9 pg, greater than about 10 pg, greater than about 11 pg, greater than about 12 pg, greater than about 13 pg, greater than about 14 pg, or greater than about 15 pg.

[0094] In certain embodiments, disruption of the BCL11a gene or HBG gene in cells by the CRISPR / Cpf1 editing system of the present disclosure may result in the production of at least about 1 picogram, at least about 2 picograms, at least about 3 picograms, at least about 4 picograms, at least about 5 picograms, at least about 6 picograms, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms, or about 8 to about 9 picograms or about 9 to about 10 picograms of fetal hemoglobin per cell.

[0095] This disclosure also relates to a cell population modified by the genome editing system described above, wherein a higher percentage of the cell population can differentiate into a population of erythrocyte lineages expressing HbF compared to a cell population not modified by the genome editing system. In certain embodiments, the higher percentage can be at least about 15%, at least about 20%, at least about 25%, at least about 30%, or at least about 40% higher. In certain embodiments, the cells may be hematopoietic stem cells. In certain embodiments, the cells may differentiate into erythroblasts, erythrocytes, or erythrocyte precursors or erythroblasts.

[0096] In certain embodiments, expression levels, such as the relative expression level of HbF (e.g., relative to the entire β-like globin chain), can be measured by ultra-high-performance liquid chromatography (UPLC).

[0097] The CRISPR / Cpf1 editing system of this disclosure can be delivered to cells using a variety of strategies. For example, the expression of components of the CRISPR / Cpf1 editing system can be induced in cells using a vector, such as AAV or other viral vectors, that encodes components of the CRISPR / Cpf1 editing system, although this is not limited to these methods. Alternatively, the RNP complex containing components of the CRISPR / Cpf1 editing system can be introduced into cells, for example, by electroporation. In certain embodiments, the RNP complex can be delivered to cells by lipid nanoparticles.

[0098] As outlined in Example 3 below, Figure 16 shows the successful targeting of the HBG1 promoter region by AsCpf1 WT and RR PAM mutants in HUDEP and HSC.

[0099] In addition, these data regarding disruption of the BCL11a gene and HBG gene loci are relevant to the CD34 loci of clinically relevant loci (i.e., known HPFH target sites). + This demonstrates efficient editing in cells by the AsCpf1 mutant.

[0100] Cpf1 editing of T cells at target sites related to T cell proliferation, survival, and / or function. One proposed therapeutic strategy for treating cancer involves adoptive T cell transfer. Factors limiting the effectiveness of genetically modified T cells as cancer therapies include (1) T cell proliferation, such as limited T cell proliferation following adoptive immune transfer; (2) T cell survival, such as induction of T cell apoptosis by tumor environmental factors; and (3) T cell function, such as inhibition of cytotoxic T cell function by inhibitors secreted by host immune cells and cancer cells. One strategy to enhance effectiveness is to modify or disrupt T cell genes related to T cell proliferation, survival, and / or function using gene editing. For example, but not limited to, RNA-induced nucleases such as Cpf1 RNA-induced nuclease can target specific sequences that affect T cell gene expression.

[0101] Methods and compositions contained herein may be used to modify one or more T cell expression genes, such as one or more of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes, thereby affecting T cell proliferation, survival, persistence, and / or function. In certain embodiments, methods and compositions disclosed herein may be used to modify one or more T cell expression genes, such as the CBLB and / or PTPN6 gene, thereby affecting T cell proliferation. In certain embodiments, methods and compositions disclosed herein may be used to modify one or more T cell expression genes, such as the FAS and / or BID gene, thereby affecting T cell survival. In certain embodiments, methods and compositions disclosed herein may be used to modify one or more T cell expression genes, such as the CTLA4, PDCD1, TRAC, CIITA, and / or TRBC gene, thereby affecting T cell function. In certain embodiments, T cell persistence may be improved by modifying the B2M gene using the methods and compositions disclosed herein.

[0102] In certain embodiments, one or more T cell expression genes, including but not limited to the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes, are independently targeted as targeted knockouts to affect, for example, T cell proliferation, survival, persistence, and / or function. In certain embodiments, the currently disclosed method includes knocking out one T cell expression gene (e.g., one selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes). In certain embodiments, the currently disclosed method includes independently knocking out two T cell expression genes (e.g., two selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes). In certain embodiments, the currently disclosed method includes independently knocking out three T cell expression genes, such as three selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes. In certain embodiments, the currently disclosed method includes independently knocking out four T cell expression genes, such as four selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes. In certain embodiments, the currently disclosed method includes independently knocking out five T cell expression genes, such as five selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes. In certain embodiments, the currently disclosed method includes independently knocking out six T cell expression genes, such as six selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes.In certain embodiments, the currently disclosed method includes independently knocking out seven T cell expression genes, such as seven selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes. In certain embodiments, the currently disclosed method includes independently knocking out eight T cell expression genes selected from the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes. In certain embodiments, the currently disclosed method includes independently knocking out nine T cell expression genes selected from the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes. In certain embodiments, the currently disclosed method includes independently knocking out nine T cell expression genes, such as FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes.

[0103] In addition to the genes described above, several other T cell expression genes may be targeted to affect the efficacy of genetically modified T cells. These genes include, but are not limited to, TGFBRI, TGFBRII, and TGFBRIII (Kershaw et al. 2013 Nat. Rev. Cancer 13, 525-541). In certain embodiments, one or more of the TGFBRI, TGFBRII, and TGFBRIII genes may be modified individually or in combination using the methods disclosed herein. In certain embodiments, one or more of the TGFBRI, TGFBRII, and TGFBRIII genes may be modified individually or in combination with one or more of the eight genes mentioned above (i.e., FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes) using the methods disclosed herein.

[0104] In certain embodiments, the methods and compositions disclosed herein modify the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC genes by targeting a location in the gene (e.g., a knockout site), such as a location within a non-coding region (e.g., a promoter region or regulatory region) or a location within a coding region, or by targeting a transcriptional sequence of the gene, such as an intron sequence or an exon sequence. In certain embodiments, coding sequences, such as coding regions, such as the initial coding regions of genes (e.g., FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC genes), are targets for expression modification and knockout. In certain embodiments, locations within the non-coding region of a T cell expression gene (e.g., promoter region or regulatory region) (e.g., FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC genes) become targets for modification and knockout of T cell expression gene expression.

[0105] In certain embodiments, the methods and compositions disclosed herein modify the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC genes by targeting their coding sequences. In certain embodiments, the coding sequence is the initial coding sequence. In certain embodiments, the coding sequence targets the knockout of T cell expression genes.

[0106] In certain embodiments, the methods and compositions disclosed herein modify the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC genes by targeting non-coding sequences of the genes. In certain embodiments, the non-coding sequences include sequences within the promoter region, enhancer sequences, intron sequences, sequences within the 3'UTR, polyadenylation signal sequences, or combinations thereof. In certain embodiments, the non-coding sequences of the genes are targeted for knockout of gene expression.

[0107] In certain embodiments, the currently disclosed method includes, for example, knocking out one or two alleles of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC genes by inducing gene modification. In certain embodiments, the modification includes insertions, deletions, mutations, or combinations thereof.

[0108] In certain embodiments, the targeted knockout approach is mediated by non-homologous end joining (NHEJ) using a CRISPR / Cpf1 system containing the Cpf1 enzyme.

[0109] In certain embodiments, the CRISPR / Cpf1 system disclosed herein targets the TRAC gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the TRAC gene sequence. In certain embodiments, the gRNA may be complementary to any strand of the TRAC gene. In certain embodiments, the targeted portion of the TRAC gene sequence is within the coding sequence of the TRAC gene. In certain embodiments, the targeted portion of the TRAC gene sequence is within an exon. In certain embodiments, the targeted portion of the TRAC gene sequence is within an intron. In certain embodiments, the targeted portion of the TRAC gene sequence is within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the TRAC gene sequence is within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, a portion of the TRAC gene sequence is within the first 500 bp of the coding sequence of the TRAC gene. In certain embodiments, gRNA molecular targeting domains for use in such CRISPR / Cpf1 systems targeting TRAC include targeting domain sequences listed in Tables 2 and 3. This disclosure provides compositions comprising one or more of the gRNAs provided in Tables 2 and 3. This disclosure further provides compositions comprising one or more RNP complexes comprising one or more of the gRNAs provided in Tables 2 and 3.

[0110] In certain embodiments, the CRISPR / Cpf1 system disclosed herein targets the TRBC gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the TRBC gene sequence. In certain embodiments, the gRNA may be complementary to either strand of the TRBC gene. In certain embodiments, the targeted portion of the TRBC gene sequence is located within the coding sequence of the TRBC gene. In certain embodiments, the targeted portion of the TRBC gene sequence is located within an exon. In certain embodiments, the targeted portion of the TRBC gene sequence is located within an intron. In certain embodiments, the targeted portion of the TRBC gene sequence is located within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the TRBC gene sequence is located within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, a portion of the TRBC gene sequence is located within the first 500 bp of the coding sequence of the TRBC gene. In certain embodiments, gRNA molecular targeting domains for use in such CRISPR / Cpf1 systems targeting TRBCs include targeting domain sequences listed in Tables 4 and 5. This disclosure provides compositions comprising one or more of the gRNAs provided in Tables 4 and 5. This disclosure further provides compositions comprising one or more RNP complexes comprising one or more of the gRNAs provided in Tables 4 and 5.

[0111] In certain embodiments, the CRISPR / Cpf1 system disclosed herein targets the B2M gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the B2M gene sequence. In certain embodiments, the gRNA may be complementary to any strand of the B2M gene. In certain embodiments, the targeted portion of the B2M gene sequence is within the coding sequence of the B2M gene. In certain embodiments, the targeted portion of the B2M gene sequence is within an exon. In certain embodiments, the targeted portion of the B2M gene sequence is within an intron. In certain embodiments, the targeted portion of the B2M gene sequence is within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the B2M gene sequence is within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, a portion of the B2M gene sequence is within the first 500 bp of the coding sequence of the B2M gene. In certain embodiments, a portion of the B2M gene sequence is located between the 501st and last nucleotide of the coding sequence of the B2M gene. In certain embodiments, the gRNA molecular targeting domain for use in such a CRISPR / Cpf1 system targeting B2M comprises the targeting domain sequences listed in Tables 6, 7, and 8. In certain embodiments, the targeting domain of the gRNA molecule for use in such a CRISPR / Cpf1 system targeting B2M comprises AGUGGGGGUGAAUUCAGUGU. This disclosure provides compositions comprising one or more of the gRNAs provided in Tables 6, 7, and 8. This disclosure further provides compositions comprising one or more RNP complexes comprising one or more of the gRNAs provided in Tables 6, 7, and 8.

[0112] In certain embodiments, the CRISPR / Cpf1 system disclosed herein targets the CIITA gene. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the CIITA gene sequence. In certain embodiments, the CRISPR system includes a gRNA complementary to a portion of the CIITA gene sequence. In certain embodiments, the gRNA may be complementary to any strand of the CIITA gene. In certain embodiments, the targeted portion of the CIITA gene sequence is within the coding sequence of the CIITA gene. In certain embodiments, the targeted portion of the CIITA gene sequence is within an exon. In certain embodiments, the targeted portion of the CIITA gene sequence is within an intron. In certain embodiments, the targeted portion of the CIITA gene sequence is within a regulatory region of the gene. In certain embodiments, two or more sequences are targeted, and the targeted portion of the CIITA gene sequence is within one or more exons, one or more introns, one or more regulatory regions, or one or more exons, one or more introns and one or more regulatory regions. In certain embodiments, a portion of the CIITA gene sequence is located within the first 500 bp of the coding sequence of the CIITA gene. In certain embodiments, a gRNA molecular targeting domain for use in such a CRISPR / Cpf1 system targeting CIITA comprises the targeting domain sequences listed in Table 9. This disclosure provides compositions comprising one or more of the gRNAs provided in Table 9. This disclosure further provides compositions comprising one or more RNP complexes comprising one or more of the gRNAs provided in Table 9.

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[0138] Knockout and / or knockdown of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC may be useful in a variety of situations, including, but not limited to, those related to adoptive immunotherapy for treating cancer and non-cancerous diseases such as autoimmune disorders. According to certain embodiments of this disclosure, FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC are knocked out in immune cells, such as T cells, used in therapy. As a non-limiting example, T cells may express engineered receptors, such as chimeric antigen receptors (CARs) or heterologous T cell receptors (TCRs), which may be configured to recognize antigens on cells or tissues that are thought to be involved in pathology, such as tumor cells. Whether or not they express engineered receptors, TCR, MHC I, and / or MHC II knockout T cells according to this disclosure may be used to target tissues or organs in which GvH or HvG responses may raise safety or efficacy concerns.

[0139] TCR, MHC I and / or MHC II knockout and / or knockdown cells may be used in "allogeneic" cell therapy, in which cells are harvested from a subject, modified, and knocked out or knocked down, for example, by interfering with the expression of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC, and then returned to a different subject. In either approach, between harvesting and administration, the TCR, MHC I and / or MHC II knockout and / or knockdown cells of this disclosure may be manipulated in various ways, such as proliferation, stimulation, purification or sorting, transgene transduction, freezing and / or thawing.

[0140] Knocking out or knocking down the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC genes as described herein may (1) prevent the GvH response; (2) prevent the HvG response; and / or (3) improve the safety and efficacy of T cells. Similarly, knocking down the expression of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC proteins as described herein may (1) prevent the GvH response; (2) prevent the HvG response; and / or (3) improve the safety and efficacy of T cells.

[0141] In certain embodiments, the currently disclosed method includes independently knocking out and / or knocking down one or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC in T cells. In certain embodiments, the currently disclosed method includes independently knocking out and / or knocking down two genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC in T cells. In certain embodiments, the currently disclosed method includes independently knocking out and / or knocking down three genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC in T cells. In certain embodiments, the currently disclosed method includes independently knocking out and / or knocking down all four genes of B2M, TRAC, CIITA, and TRBC in T cells.

[0142] In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M gene in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the TRAC gene in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the CIITA gene in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the TRBC gene in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M and TRAC genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M and CIITA genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M and TRBC genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the TRAC and CIITA genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the TRAC and TRBC genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the CIITA and TRBC genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M, TRAC and CIITA genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M, TRAC and TRBC genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M, CIITA and TRBC genes in T cells. In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the TRAC, CIITA and TRBC genes in T cells.In certain embodiments, the currently disclosed method includes knocking out and / or knocking down the B2M, TRAC, CIITA, and TRBC genes in T cells.

[0143] In certain embodiments, knockout and / or knockdown of one or more genes, two or more genes, three or more genes, or four or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC in T cells may (1) prevent the GvH response; (2) prevent the HvG response; and / or (3) improve the safety and efficacy of T cells. For example, but not limited to, knockout and / or knockdown of one or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC in T cells may be used to generate "allogeneic" cells, such as allogeneic T cells. In certain embodiments, knockout and / or knockdown of one or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC may be used in "allogeneic" cell therapy, in which cells are harvested from a subject, modified, knocked out or knocked down, for example by interfering with the expression of B2M, TRAC, CIITA, and / or TRBC, and then returned to a different subject.

[0144] In certain embodiments, knockout and / or knockdown of one or more genes, two or more genes, three or more genes, or four or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC in T cells results in a decrease in MHC II receptor expression in T cells compared to unmodified T cells. In certain embodiments, a cell population that is modified and has one or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC knocked out and / or knocked down shows a decrease of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in MHC II receptor, TCR, or B2M expression compared to the amount of MHC II receptor, TCR, or B2M expression in an unmodified cell population.

[0145] In certain embodiments, knockout and / or knockdown of two or more genes may involve the use of different nucleases for editing each target gene. For example, but not limited to, one target gene may be knocked out and / or knocked down using a CRISPR / Cpf1 editing system, and a second target gene may be knocked out and / or knocked down using a CRISPR / Cas9 editing system.

[0146] This disclosure provides isolated CRISPR / Cpf1-edited T cells or populations of CRISPR / Cpf1-edited T cells containing one or more modifications to one or more endogenous genes of the T cells disclosed herein. In certain embodiments, the CRISPR / Cpf1-edited T cells or populations of CRISPR / Cpf1-edited T cells contain one or more components of the CRISPR / Cpf1 editing system. Alternatively, the CRISPR / Cpf1-edited T cells or populations of CRISPR / Cpf1-edited T cells do not contain one or more components of the CRISPR / Cpf1 editing system. In certain embodiments, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than 2%, or less than 1% of the CRISPR / Cpf1-edited cell population contains one or more components of the CRISPR / Cpf1 editing system.

[0147] In certain embodiments, T cells are CD8 + T cells, CD8 + Naive T cells, CD4 + central memory T cells, CD8 + central memory T cells, CD4 + Effector memory T cells, CD4 + Effector memory T cells, CD4 + T cells, CD4 + Stem cell memory T cell, CD8 + Stem cell memory T cell, CD4 + Helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, CD4+ naive T cells, TH17CD4 + T cells, TH1CD4 + T cells, TH2CD4 + T cells, TH9CD4 + T cells, CD4 + Foxp3 + T cells, CD4 + CD25 + CD127 - T cells or CD4 + CD25 + CD127 - Foxp3 + These are T cells.

[0148] In certain embodiments, this disclosure relates to the use of CRISPR / Cpf1-mediated editing of endogenous T cell genes selected from the group consisting of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, TRBC, and any combination thereof. For example, but not limited to, modifications are generated by the delivery of one or more complexes comprising Cpf1 RNA-induced nucleases and gRNA molecules, such as the RNP complex, targeting, for example, a portion of the FAS gene sequence, a portion of the BID gene sequence, a portion of the CTLA4 gene sequence, a portion of the PDCD1 gene sequence, a portion of the CBLB gene sequence, a portion of the PTPN6 gene sequence, a portion of the B2M gene sequence, a portion of the TRAC gene sequence, a portion of the CIITA gene sequence, a portion of the TRBC gene sequence, or a combination thereof. In certain embodiments, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten complexes, such as the RNP complex, may be delivered, each complex targeting a different gene. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the T cell population are edited and / or modified. In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the T cell population have a productive indel in at least one endogenous T cell gene selected from the group consisting of, for example, FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC.

[0149] Benchmarking assays for Cpf1 variants, different cell types, and formulations. CRISPR / Cpf1-mediated editing of target nucleic acid sequences and / or regulation of target nucleic acid sequence expression can be evaluated, for example, by comparing the activity of a control CRISPR / RNA-induced nuclease editing system with that of a test CRISPR / Cpf1 editing system with respect to target nucleic acid sequences such as "matching site" target nucleic acid sequences.

[0150] The matching site target nucleic acid sequence incorporates both the requirement that it be edited by Cpf1 and a second RNA-induced nuclease, such as Cas9. For example, the TTTV AsCpf1 wild-type protospacer adjacent motif ("PAM") and the NGGSpCas9 wild-type PAM may be used in this example. As described above, the test Cpf1 protein may contain one or more modifications compared to the wild-type Cpf1 protein. Examples of such modifications include, but are not limited to, the aforementioned modifications for incorporating one or more NLS sequences, the aforementioned modifications for incorporating 6-histidine purified sequences, and modifications of Cpf1 protein cysteine ​​amino acids, as well as combinations thereof.

[0151] Examples of matching site target nucleic acid sequences that may be used in this example include matching site 1 ("MS1"; SEQ ID NO: 13), matching site 5 ("MS5"; SEQ ID NO: 14), matching site 11 ("MS11"; SEQ ID NO: 15), and matching site 18 ("MS18"; SEQ ID NO: 16).

[0152] For example, CD34 +To evaluate CRISPR / Cpf1-mediated editing of target nucleic acid sequences and / or modification of target nucleic acid sequence expression in specific cell types such as HSCs, in comparison to CRISPR / Cas9-mediated editing, a CRISPR / Cpf1 genome editing system, i.e., a system comprising a Cpf1 RNA-induced nuclease and a gRNA complementary to at least a portion of the target nucleic acid containing the matching site target, is introduced into cells of the cell type of interest, for example, as an RNP or through the use of a vector encoding a component of the system. The editing of the target nucleic acid sequence and / or modification of target nucleic acid sequence expression can be detected as disclosed herein. The detected editing of the target nucleic acid sequence and / or modification of target nucleic acid sequence expression can then be compared to the editing of the target nucleic acid sequence and / or modification of target nucleic acid sequence expression detected using a CRISPR / Cas9 genome editing system for the same matching site target and the same cell type.

[0153] The above method of comparing CRISPR / Cpf1-mediated editing in contrast to CRISPR / Cas9-mediated editing (or editing by another CRISPR-based system) of a target nucleic acid sequence and / or modification of the expression of the target nucleic acid sequence allows for the evaluation of specific attributes of the CRISPR / Cpf1-mediated editing system used. For example, but not limited to, such a method can be used to evaluate CRISPR / Cpf1-mediated editing in contrast to CRISPR / Cas9-mediated editing of a target nucleic acid sequence and / or modification of the expression of the target nucleic acid sequence, and differences in the activity of Cpf1 RNA-induced nucleases and / or gRNAs prepared by different manufacturing processes can be identified. Such a method can also identify differences in the activity of Cpf1 RNA-induced nucleases and / or gRNAs present in different formulations and using different delivery strategies.

[0154] In certain embodiments, the disclosure relates to an assay for comparing CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of a target nucleic acid sequence by a test CRISPR / Cpf1 genome editing system with a control RNA-induced nuclease genome editing system. More specifically, the disclosure provides an assay using a matching site (e.g., a cell containing matching site 5) to which a gene editing system (e.g., CRISPR / Cas9 or CRISPR / Cpf1 or a variant thereof and a gRNA complementary to the matching site) is targeted, such that the level or efficiency of editing at the matching site serves as an indicator of how efficient the gene editing system is at editing at any other site. In other words, editing efficiency can be evaluated by varying various components of the gene editing system and measuring the level or efficiency of editing achieved at the matching site (e.g., matching site 5).

[0155] For example, but not limited to, test and control genes or genome editing systems may differ in one or more of the following aspects: the sequence of the RNA-induced nuclease; the source of the components of the genome editing system, such as the method of manufacturing the components; the formulation of one or more components of the genome editing system; and the identity of the cells into which the genome editing system is introduced or the method of preparing the cells, such as the cell type. In certain embodiments, the assays described herein enable quality control analysis of a test genome editing system. In certain embodiments, the assays of the present disclosure evaluate CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of a target nucleic acid sequence, the target including a matching site sequence.

[0156] Electroporation pulse code screening This disclosure further provides electropermeable pulse codes that result in more advanced editing at target sites. As shown in the examples, screening of electropermeable pulse codes allows for the identification of codes that result in more efficient editing by the Cpf1 RNA-induced nucleases of this disclosure. For example, but not limited to, Figure 18 shows nucleofection screening of AsCpf1 in HUDEP using a series of specific pulse codes and solutions. Similarly, Figure 19 shows exemplary nucleofection screening of AsCpf1 in HSC. In certain embodiments, pulse codes CA-137 and CA-138 may be used to facilitate more efficient editing by the Cpf1 RNA-induced nucleases. For example, but not limited to, Figures 20 and 23C demonstrate the improved efficiency of the CA-137 pulse code.

[0157] Treatment method This disclosure further provides methods for treating diseases and / or disorders by administering cells edited using the disclosed genome editing methods. In certain embodiments, this disclosure relates to methods for treating a subject by modifying one or more cells of the subject. In certain embodiments, one or more cells are modified ex vivo and then administered to the subject. For example, but not limited to, methods for treating a subject may include contacting cells from the subject with (a) a gRNA molecule complementary to the target sequence of the target nucleic acid; and (b) a Cpf1 RNA-induced nuclease disclosed herein, for example, ex vivo. In certain embodiments, this disclosure provides methods for treating a subject, including administering one or more cells modified by the CRISPR / Cpf1 system of this disclosure to the subject. In certain embodiments, one or more cells are obtained from a donor, genetically modified using the CRISPR / Cpf1 system of this disclosure, and then administered to the subject.

[0158] In certain embodiments, the methods of the present disclosure may include administering T cells edited using the disclosed genome editing method to a subject in need, for example, to produce allogeneic T cells. For example, but not limited to, the methods of the present disclosure may include administering one or more T cells that have been edited to have knockout or knockdown of the expression of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and / or TRBC. In certain embodiments, the T cells are edited to have knockout or knockdown of the expression of B2M, TRAC, CIITA, and / or TRBC. In certain embodiments, one or more T cells are edited ex vivo and then administered to a subject. In certain embodiments, one or more cells are obtained from a donor. In certain embodiments, such T cells may be used to treat a subject having cancer or an autoimmune disorder. In certain embodiments, in a population of CRISPR / Cpf1-edited T cells administered to a subject, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the cells in the CRISPR / Cpf1-edited cell population contain one or more components of the CRISPR / Cpf1 editing system.

[0159] In certain embodiments, the methods of the present disclosure may include administering CD34+ hematopoietic stem and progenitor cells (HSPCs) edited using the disclosed genome editing methods to a subject requiring such editing. In certain embodiments, CD34+ cells may be edited to knock out or knock down BCL11a or HBG expression. For example, but not limited to, CD34+ hematopoietic stem and progenitor cells (HSPCs) edited using the genome editing methods disclosed herein may be used for the treatment of abnormal hemoglobinopathy in a subject requiring such editing. In certain embodiments, abnormal hemoglobinopathy may be severe sickle cell disease (SCD) or thalassemia such as β-thalassemia, δ-thalassemia, or β / δ-thalassemia. In certain embodiments, exemplary protocols for the treatment of abnormal hemoglobinopathy may include harvesting CD34+HSPCs from subjects in need, ex vivo editing autologous CD34+HSPCs using genome editing methods disclosed herein, and subsequently reinfusion of the edited autologous CD34+HSPCs into the subjects. In certain embodiments, treatment with edited autologous CD34+HSPCs may result in increased HbF induction.

[0160] In certain embodiments, prior to the collection of CD34+HSPCs, the subject may discontinue hydroxyurea treatment if applicable and receive blood transfusions to maintain adequate hemoglobin (Hb) levels. In certain embodiments, the subject may be intravenously administered prelixafor (e.g., 0.24 mg / kg) to mobilize CD34+HSPCs from the bone marrow into the peripheral blood. In certain embodiments, the subject may undergo one or more leukocyte apheresis cycles (e.g., with approximately one month between cycles, and one cycle defined as two prelixafor-mobilized leukocyte apheresis collections performed on consecutive days). In certain embodiments, the number of leukocyte apheresis cycles performed on the subject is determined by the number of doses (e.g., ≥1.5 × 10⁶) for reinfusion of the subject, along with a dose of unedited autologous CD34+HSPC / kg for backup storage. 6 (cells / kg), dose of edited autologous CD34+HSPC (e.g., ≥2 × 10) 6cells / kg, ≧3×10 6 cells / kg, ≧4×10 6 cells / kg, ≧5×10 6 cells / kg, 2×10 6 cells / kg~3×10 6 cells / kg, 3×10 6 cells / kg~4×10 6 cells / kg, 4×10 6 cells / kg~5×10 6 This may be the number of times required to achieve cells / kg. In certain embodiments, CD34+HSPCs extracted from a subject may be edited using any of the genome editing methods discussed herein. In certain embodiments, any one or more gRNAs and one or more RNA-inducing nucleases disclosed herein may be used in the genome editing method.

[0161] In certain embodiments, the treatment may include autologous stem cell transplantation. In certain embodiments, the subject may undergo myeloablative adaptation by busulfan adaptation (e.g., at a test dose of 1 mg / kg, with dose adjustment based on initial dose pharmacokinetic analysis). In certain embodiments, adaptation may be carried out for 4 consecutive days. In certain embodiments, after a 3-day busulfan-free period, edited autologous CD34+HSPCs (e.g., ≥2 × 10⁻¹⁰) are transplanted. 6 cells / kg, ≧3×10 6 cells / kg, ≧4×10 6 cells / kg, ≧5×10 6 cells / kg, 2×10 6 cells / kg~3×10 6 cells / kg, 3×10 6 cells / kg~4×10 6 cells / kg, 4×10 6 cells / kg~5×10 6 Cells / kg can be reinfused into the subject (e.g., into peripheral blood). In certain embodiments, edited autologous CD34+ HSPCs can be prepared for a specific subject and cryopreserved. In certain embodiments, the subject can achieve neutrophil transplantation following a sequential myeloablative adaptation regimen and infusion of edited autologous CD34+ cells. Neutrophil transplantation is performed when ≥0.5 × 10 9It can be defined as three consecutive measurements of ANC at / L. In certain embodiments, in a CRISPR / Cpf1-edited CD34+HSPC population administered to a subject, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the cells in the CRISPR / Cpf1-edited CD34+HSPC population contain one or more components of the CRISPR / Cpf1 editing system.

[0162] In certain embodiments, the CRISPR / Cpf1-mediated editing systems of the Disclosure may yield a clinically relevant or therapeutically relevant editing efficiency of about 10% or more. For example, but not limited to, the CRISPR / Cpf1-mediated editing systems of the Disclosure may yield a clinically relevant or therapeutically relevant editing efficiency of about 5% or more, about 10% or more, 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more.

[0163] In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population administered by the therapeutic method disclosed herein are modified.

[0164] In certain embodiments, less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.25%, or less than 0.1% of the cells in a CRISPR / Cpf1-edited cell population contain one or more components of the CRISPR / Cpf1 editing system.

[0165] genome editing system The terms “genome editing system” or “gene editing system” refer to any system having RNA-induced DNA editing activity. The genome editing systems of this disclosure include at least two components, a guide RNA (gRNA) and an RNA-induced nuclease, adapted from naturally occurring CRISPR systems. These two components bind to a specific nucleic acid sequence to form a complex capable of editing DNA in or around the nucleic acid sequence by, for example, generating one or more single-strand breaks (SSBs or nicks), double-strand breaks (DSCs), and / or point mutations.

[0166] Naturally occurring CRISPR systems are evolutionarily organized into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun;9(6):467-477 (Makarova), incorporated herein by reference). While the genome editing systems of this disclosure may be adapted from components of any type or class of naturally occurring CRISPR system, the embodiments presented herein are generally adapted from Class 2 and Type II or V CRISPR systems. Class 2 systems, encompassing Types II and V, are characterized by a relatively large multidomain RNA-induced nuclease protein (e.g., Cas9 or Cpf1) and one or more guide RNAs (e.g., crRNA, optionally tracrRNA) that form a ribonucleoprotein (RNP) complex that associates with (targets) and cleaves a specific locus complementary to the target (or spacer) sequence of the crRNA. The genome editing systems of this disclosure similarly target and edit cellular DNA sequences, but differ significantly from naturally occurring CRISPR systems. For example, the single-molecule guide RNAs described herein do not exist in nature, and both the guide RNAs and RNA-inducing nucleases disclosed herein may incorporate any number of modifications that do not exist in nature.

[0167] Genome editing systems can be implemented in various ways (e.g., they can be administered or delivered to cells or subjects), and different implementations may be suitable for different applications. For example, in certain embodiments, a genome editing system may be implemented as a protein / RNA complex (ribonucleoprotein or RNP), which may be included in a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier and / or encapsulant such as lipid or polymer microparticles or nanoparticles, micelles, or liposomes. In certain embodiments, a genome editing system may be implemented as one or more nucleic acids (optionally together with one or more additional components) encoding the RNA-inducing nuclease and guide RNA components described above; in certain embodiments, a genome editing system may be implemented as one or more vectors containing such nucleic acids, such as a viral vector such as an adeno-associated virus; and in certain embodiments, a genome editing system may be implemented as any combination of the foregoing. Additional or modified implementations operating according to the principles described herein will be apparent to those skilled in the art and are within the scope of this disclosure.

[0168] It should be noted that the genome editing systems of this disclosure can or may target a single specific nucleotide sequence, and that by using two or more guide RNAs, two or more specific nucleotide sequences can be edited in parallel. The use of multiple gRNAs is referred to as “multiplexing” throughout this disclosure and may be used to target multiple unrelated target sequences of interest or to form multiple SSBs or DSBs within a single target domain, and, if applicable, to perform specific edits within such target domains. For example, Maeder et al. (Maeder), International Publication No. 2015 / 138510, incorporated herein by reference, describes a genome editing system for correcting a point mutation in the human CEP290 gene (C.2991+1655A to G) that results in the generation of a potential splice site, thereby reducing or eliminating gene function. Maeder’s genome editing system utilizes two guide RNAs that target sequences on both sides (i.e., flanking) the point mutation to form a DSB located on the mutated side. This then promotes the deletion of the intervening sequence containing the mutation, thereby removing the potential splice site and restoring normal gene function.

[0169] As another example, Cotta-Ramusino et al. ("Cotta-Ramusino et al."), whose entirety is incorporated herein by reference, describes a genome editing system that utilizes two gRNAs in combination with Cas9 nickase (Cas9 that produces single-stranded nicks such as S. pyogenes D10A), in a configuration referred to as the "double nickase system." The Cotta-Ramusino double nickase system is configured to produce two nicks on the reverse strand of a target sequence offset by one or more nucleotides, and these nicks combine to create a double-strand break with an overhang (a 5' overhang in the case of Cotta-Ramusino et al., but a 3' overhang is also possible). The overhang can then facilitate homology-directed repair events in several situations. As another example, Palestrant et al., in international publication No. 2015 / 070083 ("Palestrant," the entire text of which is incorporated herein by reference), describe a gRNA (referred to as "control RNA") that targets a nucleotide sequence encoding Cas9, which may be included in a genome editing system comprising one or more additional gRNAs to enable transient expression of Cas9, which would otherwise be constitutively expressed, in some transgenic cells. These multiplexing applications are intended to be illustrative rather than limiting, and those skilled in the art will understand that other multiplexing applications are generally compatible with the genome editing systems described herein.

[0170] Genome editing systems may, in some cases, create double-strand breaks that are repaired by cellular DNA double-strand break mechanisms such as NHEJ or HDR. These mechanisms have been described in various publications, for example, Davis & Maizels, PNAS, 111(10):E924-932, March 11, 2014 (Davis) (described on Alt-HDR); Frit et al., DNA Repair 17(2014)81-97 (Frit) (described on Alt-NHEJ); and Iyama and Wilson III, DNA Repair (Amst.) 2013-Aug;12(8):620-636 (Iyama) (general description of standard HDR and NHEJ pathways).

[0171] When a genome editing system functions by forming double-strand breaks (DSBs), such a system may optionally include one or more components that promote or facilitate a particular type of double-strand break repair or a specific repair outcome. For example, Cotta-Ramusino et al. have described a genome editing system in which a single-strand oligonucleotide "donor template" is attached; the donor template can be incorporated into a target region of cellular DNA cleaved by the genome editing system, resulting in a change in the target sequence.

[0172] In certain embodiments, genome editing systems modify a target sequence or alter the expression of a gene in or near a target sequence without causing single-strand or double-strand breaks. For example, a genome editing system may include an RNA-inducible nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression. As an example, an RNA-inducible nuclease may bind (e.g., fuse) to a cytidine deaminase functional domain and function by generating a targeted C-to-A substitution. Exemplary nuclease / deaminase fusions are described by reference in Komor et al. Nature 533, 420-424 (19 May 2016) ("Komor"). Alternatively, genome editing systems may utilize cleavage-inactivated (i.e., "dead") nucleases such as dead Cas9 (dCas9), which form stable complexes on one or more target regions of cellular DNA, thereby functioning by disrupting, but not limited to, functions involving the target region, including mRNA transcription and chromatin remodeling.

[0173] In certain embodiments, the genome editing systems included in this disclosure will exhibit a specific minimum editing percentage in a standard assay. For example, but not limited to, certain genome editing systems included in this disclosure will exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% editing in a specific standard assay. One or more assays known in the art or assays described herein, such as those described in Example 1 below, may be used to evaluate CRISPR / Cpf1-mediated editing of a target nucleic acid sequence. For example, in Example 1 below, for example, CD34 +An evaluation comparing CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of its expression in specific cell types, such as HSCs, with CRISPR / Cas9-mediated editing is described. The CRISPR / Cpf1 genome editing system, i.e., a system comprising a Cpf1 RNA-induced nuclease and a gRNA complementary to at least a portion of the target nucleic acid containing the matching site target, is introduced into cells of the cell type of interest, for example, as an RNP or through the use of a vector encoding a component of the system. The editing of the target nucleic acid sequence and / or modification of its expression is detected as disclosed herein. The detected editing of the target nucleic acid sequence and / or modification of its expression can then be compared to the editing of the target nucleic acid sequence and / or modification of its expression detected using the CRISPR / Cas9 genome editing system for the same matching site target and the same cell type.

[0174] In certain embodiments, the genome editing system of the Disclosure may simultaneously knock out or knock down one or more, two or more, three or more, or four or more genes selected from the group consisting of B2M, TRAC, CIITA, and TRBC within a cell population. In certain embodiments, the genome editing system of the Disclosure may comprise one or more, two or more, three or more, or four or more gRNA molecules, each gRNA molecule comprising a targeting domain for a different gene, such as a gene selected from the B2M, TRAC, CIITA, and TRBC genes. For example, but not limited to, the multiple genome editing systems of the present disclosure may include: (i) a first RNP complex comprising a first guide RNA (gRNA) containing a first targeting domain complementary to the target sequence of a first gene and a first Cpf1 RNA-induced nuclease; (ii) a second RNP complex comprising a second gRNA molecule containing a second targeting domain complementary to the target sequence of a second gene and a second Cpf1 RNA-induced nuclease; (iii) a third RNP complex comprising a third gRNA molecule containing a third targeting domain complementary to the target sequence of a third gene and a fourth Cpf1 RNA-induced nuclease; and / or (iv) a fourth RNP complex comprising a fourth gRNA molecule containing a fourth targeting domain complementary to the target sequence of a fourth gene and a fourth Cpf1 RNA-induced nuclease. In certain embodiments, the first, second, third, and fourth genes are selected from the group consisting of B2M, TRAC, CIITA, and TRBC. In certain embodiments, the targeting domain of the gRNA molecule for targeting B2M includes the targeting domain sequences listed in Tables 6, 7, and 8. In certain embodiments, the targeting domain of the gRNA molecule for targeting TRAC includes the targeting domain sequences listed in Tables 2 and 3. In certain embodiments, the targeting domain of the gRNA molecule for targeting CIITA includes the targeting domain sequences listed in Table 9. In certain embodiments, the targeting domain of the gRNA molecule for targeting TRBC includes the targeting domain sequences listed in Tables 4 and 5. In certain embodiments, the editing efficiency may be >80%, >85%, >90%, >95%, >98%, or >99% for all target genes.In certain embodiments, the cell population may be a T cell population.

[0175] Guide RNA (gRNA) molecule The terms “guide RNA” and “gRNA” refer to any nucleic acid that facilitates the specific binding (or “targeting”) of RNA-induced nucleases, such as Cpf1, to target sequences within a cell, such as genomic or episomal sequences. gRNAs can be monomolecular (containing a single RNA molecule or also called a chimera) or modular (containing two or more, typically two separate RNA molecules, such as crRNA and tracrRNA, which usually bind to each other by duplication). gRNAs and their components are described in literature such as Briner et al. (Molecular Cell 56(2), 333-339, October 23, 2014 (Briner), cited by reference) and Cotta-Ramusino.

[0176] In bacteria and archaea, the type II CRISPR system generally includes an RNA-inducing nuclease protein such as Cas9; CRISPR RNA (crRNA) containing a 5' region complementary to the exogenous sequence; and a transactivating crRNA (tracrRNA) containing a 5' region complementary to the 3' region of the crRNA and forming a double helix with it. While not intended to be bound by any theory, this double helix is ​​thought to promote the formation of the Cas9 / gRNA complex and is necessary for its activity. While adapting the type II CRISPR system for use in gene editing, in one non-limiting example, it was discovered that crRNA and tracrRNA can be linked to a single monomolecule or chimeric guide RNA by a 4-nucleotide (e.g., GAAA) "tetraloop" or "linker" sequence that bridges the complementary regions of crRNA (its 3' end) and tracrRNA (its 5' end). (All of these are incorporated herein by reference: Mali et al. Science. 2013 Feb 15;339(6121):823-826 ("Mali"); Jiang et al. Nat Biotechnol. 2013 Mar;31(3):233-239 ("Jiang"); and Jinek et al., 2012 Science Aug. 17;337(6096):816-821 ("Jinek")).

[0177] Guide RNA, whether monolithic or modular, contains a “targeting domain” that is fully or partially complementary to the target domain in the target sequence, such as a DNA sequence in the genome of the cell to be edited. The targeting domain is referred to by various names in the literature, including, but is not limited to, “guide sequence” (Hsu et al., Nat Biotechnol. 2013 Sep;31(9):827-832, ("Hsu"), incorporated herein by reference), “complementary region” (Cotta-Ramusino et al.), “spacer” (Briner), and comprehensively “crRNA” (Jiang). Regardless of the names given to them, the targeting domain is typically 10–30 nucleotides long, and in certain embodiments 16–24 nucleotides long (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long), located at or near the 5' end in Cas9 gRNA, and at or near the 3' end in Cpf1 gRNA.

[0178] In addition to the targeting domain, gRNA typically (though not necessarily, as discussed below) contains multiple domains that can influence the formation or activity of the gRNA / Cas9 and gRNA / Cpf1 complexes. For example, as described above, the double-strand structure formed by the first and second complementary domains (repeats: also called anti-repeat double helixes) of gRNA can interact with the Cas9 recognition (REC) lobe to mediate the formation of the Cas9 / gRNA complex. (Nishimasu et al., Cell 156, 935-949, February 27, 2014 (Nishimasu 2014) and Nishimasu et al., Cell 162, 1113-1126, August 27, 2015 (Nishimasu 2015), both incorporated herein by reference). It should be noted that the first and / or second complementary domains may contain one or more poly(A) strands that can be recognized as termination signals by RNA polymerase. Therefore, the sequences of the first and second complementary domains may be selectively modified, for example, through the use of AG swaps or AU swaps as described by Briner, to remove these regions and facilitate complete in vitro transcription of the gRNA. These and other similar modifications to the first and second complementary domains are within the scope of this disclosure.

[0179] Along with the first and second complementary domains, Cas9g RNA typically contains two or more additional double-stranded regions that are involved in nuclease activity in vivo but not necessarily in vitro (Nishimasu 2015). The first stem-loop 1, located near the 3' portion of the second complementary domain, is referred to in various ways, such as the “proximal domain” (Cotta-Ramusino), “stem-loop 1” (Nishimasu 2014 and 2015), and “nexus” (Briner). One or more additional stem-loop structures are generally located near the 3' end of the gRNA, and their number varies by species. S. pyogenes gRNA typically contains two 3' stem-loop structures (a total of four stem-loop structures, including repeat / anti-repeat double helixes), while S. aureus and other species have only one (a total of three stem-loop structures). A species-by-species description of conserved stem-loop structures (and more generally, gRNA structures) is provided by Briner.

[0180] While the above explanation has focused on gRNAs for use with Cas9, it should be understood that other RNA-inducible nucleases have been discovered or invented (or may be discovered in the future) that utilize gRNAs that differ in some respects from those described so far. For example, Cpf1 (CRISPR derived from "Prevotella and Francicella 1") is a recently discovered RNA-inducible nuclease that does not require tracrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771 October 22, 2015 (Zetsche I), incorporated herein by reference). gRNAs for use in the Cpf1 genome editing system generally contain a targeting domain and a complementary domain (alternately referred to as "handles"). It should also be noted that in gRNAs intended for use with Cpf1, the targeting domain is usually located at or near the 3' end rather than the 5' end, as described above for Cas9 gRNA (the handle is at or near the 5' end of Cpf1 gRNA).

[0181] Those skilled in the art will understand that while structural differences may exist between gRNAs from different prokaryotic species or between Cpf1 and Cas9 gRNAs, the principle by which gRNAs function is generally consistent. Because of this consistency of function, gRNAs can be defined in a broad sense by their targeting domain sequences, and those skilled in the art will understand that a given targeting domain sequence can be incorporated into any suitable gRNA, including monomolecular or chimeric gRNAs, or gRNAs containing one or more chemical modifications and / or sequence modifications (substitutions, additional nucleotides, cleavage, etc.). Therefore, for the economy of presentation in this disclosure, gRNAs may be described solely in terms of their targeting domain sequences.

[0182] More generally, one of ordinary skill in the art will appreciate that some aspects of the present disclosure relate to systems, methods, and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise specified, the term gRNA should be understood to encompass not only gRNAs that are compatible with specific species of Cas9 or Cpf1, but also any suitable gRNA that can be used with any RNA-guided nuclease. By way of example, the term gRNA, in certain embodiments, can include gRNAs for use with class 2 CRISPR systems such as type II or type V, or any RNA-guided nuclease present in a CRISPR system, or an RNA-guided nuclease derived from or adapted from such a system.

[0183] The present disclosure provides gRNA molecules and compositions thereof that include any one of the sequences of gRNAs provided in Tables 2-9 and 19. The present disclosure further provides compositions and compositions thereof that include one or more gRNAs that include the sequences of gRNAs described in Tables 2-9 and 19. The present disclosure provides gRNAs and compositions thereof that target chromosomal regions (e.g., genomic coordinates) provided in Table 18.

[0184] The present disclosure provides, for example, gRNAs that effect more than about 10% editing at a target site in a cell population. By way of example and not limitation, the gRNAs of the present disclosure effect, for example, more than about 15% editing, more than about 20% editing, more than about 25% editing, more than about 30% editing, more than about 35% editing, more than about 40% editing, more than about 45% editing, more than about 50% editing, more than about 55% editing, more than about 60% editing, more than about 65% editing, more than about 70% editing, more than about 75% editing, more than about 80% editing, more than about 85% editing, more than about 90% editing, more than about 95% editing, more than about 96% editing, more than about 97% editing, more than about 98% editing, or more than about 99% editing at a target site in a cell population.

[0185] gRNA Design Methods for selecting and validating target sequences and for off-target analysis have been previously described, for example, in Mali;Hsu;Fu et al., 2014 Nat biotechnol 32(3):279-84, Heigwer et al., 2014 Nat methods 11(2):122-3, Bae et al. (2014) Bioinformatics 30(10):1473-5, and Xiao A et al. (2014) Bioinformatics 30(8):1180-1182. Each of these references is incorporated herein by reference. As a non-limiting example, gRNA design may involve, for example, the use of software tools to optimize the selection of potential target sequences corresponding to the user's target sequence in order to minimize total off-target activity across the genome. Although off-target activity is not limited to cleavage, the cleavage efficiency at each off-target sequence can be predicted, for example, using experimentally derived weighting schemes. These and other methods for selecting guides are described in detail by Maeder and Cotta-Ramusino et al.

[0186] gRNA modification The activity, stability, or other characteristics of gRNAs can be altered by incorporating specific modifications. For example, transiently expressed or delivered nucleic acids may be susceptible to degradation by cellular nucleases, for instance. Therefore, the gRNAs described herein may contain one or more modified nucleosides or nucleotides that introduce stability against nucleases. While we do not wish to be constrained by theory, it is also thought that certain modified gRNAs described herein may, upon introduction into cells, exhibit a reduction in innate immune responses. Those skilled in the art are aware of certain cellular responses commonly observed in cells, such as mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which may include the induction of cytokine expression and release and cell death, can be reduced or completely eliminated by the modifications presented herein.

[0187] The specific exemplary modifications discussed in this section may be located at any position in the gRNA sequence, such as the 5' end or its vicinity (e.g., within 1–10, 1–5, or 1–2 nucleotides of the 5' end) and / or the 3' end or its vicinity (e.g., within 1–10, 1–5, or 1–2 nucleotides of the 3' end). In some cases, the modifications are located within functional motifs such as repeat:antirepeat double helixes of Cas9 gRNA, stem-loop structures of Cas9 or Cpf1 gRNA, and / or targeting domains of gRNA.

[0188] As an example, the 5' end of a gRNA may contain a eukaryotic mRNA cap structure or G cap analogue (e.g., G(5')ppp(5')G cap analogue, m7G(5')ppp(5')G cap analogue, or 3'-O-Me-m7G(5')ppp(5')G anti-cap analogue (ARCA)) as shown below. TIFF2026048661000027.tif48161

[0189] Caps or cap analogues may be present during the chemical synthesis or in vitro transcription of gRNA.

[0190] Similarly, the 5' end of gRNA can lack a 5' triphosphate group. For example, the 5' triphosphate group can be removed by phosphatase treatment of in vitro transcribed gRNA (e.g., using calf intestinal alkaline phosphatase).

[0191] Another common modification involves adding a series of adenine (A) residues (e.g., 1–10, 10–20, or 25–200) to the 3' end of the gRNA, known as a poly-A tract. The poly-A sequence can be added to the gRNA by a polyadenylation sequence either during chemosynthesis following in vitro transcription using a polyadenosine polymerase (e.g., E. coli poly(A) polymerase) or in vivo, as described by Maeder.

[0192] It should be noted that the modifications described herein may be combined in any appropriate manner, for example, gRNA transcribed from a DNA vector in vivo or in vitro may contain either or both a 5' cap structure or a cap analogue, along with a 3' polyA sequence.

[0193] Guide RNA can be modified with 3' terminal U-ribose. For example, the two terminal hydroxyl groups of U-ribose can be oxidized to aldehyde groups, and simultaneous ring-opening of the ribose ring results in the modified nucleoside shown below. In formula TIFF2026048661000028.tif29161, "U" may be unmodified or modified uridine.

[0194] The 3'-terminal U-ribose may be modified with a 2'3'-cyclic phosphate ester, as shown below. In formula TIFF2026048661000029.tif36161, "U" may be unmodified or modified uridine.

[0195] The guide RNA may contain a 3' nucleotide that can be stabilized against degradation by incorporating, for example, one or more of the modified nucleotides described herein. In certain embodiments, uridine may be substituted with modified uridines such as, for example, 5-(2-amino)propyluridine and 5-bromouridine or any of the modified uridines described herein; adenosine and guanosine may be substituted with modified adenosine and guanosine, for example, 8-bromoguanosine, which is modified at position 8, or any of the modified adenosine or guanosines described herein.

[0196] In certain embodiments, a glycosylated ribonucleotide may be incorporated into the gRNA, for example, by substituting the 2'OH group with a group selected from H, -OR, -R (wherein R may be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), halo, -SH, -SR (wherein R may be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), amino (wherein amino may be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano(-CN). In certain embodiments, the phosphate backbone may be modified, for example, with a phosphorothioate (PhTx) group, as described herein. In certain embodiments, one or more nucleotides of the gRNA may independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications such as 2'-O-methyl, 2'-O-methoxyethyl, or 2'-fluoro modifications such as 2'-F or 2'-O-methyl, adenosine (A), 2'-F or 2'-O-methyl, cytidine (C), 2'-F or 2'-O-methyl, uridine (U), 2'-F or 2'-O-methyl, thymidine (T)1, 2'-F or 2'-O-methyl, guanosine (G), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ce0), and any combination thereof.

[0197] Guide gRNA may also include "locked" nucleic acids (LNAs), in which the 2'OH group may be linked, for example, by a C1-6 alkylene or C1-6 heteroalkylene crosslink to the 4' carbon of the same ribose sugar. To provide such crosslinks, but are not limited to methylene, propylene, ether or amino crosslinks; O-amino (wherein amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino) and aminoalkoxy or O(CH2) n Any suitable part may be used, including -amino (wherein amino may be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino).

[0198] In certain embodiments, gRNA may include polycyclic modified nucleotides (e.g., tricyclonucleotides) and "unlocked" forms such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, in which ribose is replaced by a glycol unit attached to a phosphate diester bond) or threose nucleic acids (TNAs, in which ribose is replaced by α-L-treophranosyl-(3'→2')).

[0199] Generally, gRNAs contain ribose, a five-membered ring sugar containing oxygen. Typical modified gRNAs include, but are not limited to, substitution of oxygen in ribose (e.g., by sulfur (S), selenium (Se), or alkylenes such as methylene or ethylene); addition of a double bond (e.g., substitution of ribose with cyclopentenyl or cyclohexenyl); ribose ring contraction (e.g., forming a four-membered ring of cyclobutane or oxetane); and ribose ring expansion (e.g., forming a six- or seven-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, althritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also has a phosphoramidate skeleton). Most sugar analog modifications are localized at the 2' position, but other sites, including the 4' position, are also receptive to modification. In certain embodiments, gRNAs include 4'-S, 4'-Se, or 4'-C-aminomethyl-2'-O-Me modifications.

[0200] In certain embodiments, deazanucleotides, such as 7-deaza-adenosine, may be incorporated into the gRNA. In certain embodiments, O- and N-alkylated nucleotides, such as N6-methyladenosine, may be incorporated into the gRNA. In certain embodiments, one, more, or all nucleotides in the gRNA are deoxyribonucleotides.

[0201] In certain embodiments, the gRNA may include one or more linkers and / or gRNA synthesis processes selected from those described in an international patent application having specification PCT / U.S. Patent Application Publication No. 17 / 69019, the entirety of which is incorporated herein by reference.

[0202] RNA-induced nuclease RNA-guided nucleases according to the present disclosure include, but are not limited to, naturally occurring class 2 CRISPR nucleases such as Cpf1, and other nucleases derived from or obtained from such, such as, for example, variants. RNA-guided nucleases can also be defined functionally. For example, an RNA-guided nuclease (a) interacts with (e.g., forms a complex with) a gRNA; and (b) together with the gRNA, binds to a target region of DNA that includes (i) a sequence complementary to the targeting domain of the gRNA, and optionally (ii) an additional sequence, referred to as a "protospacer adjacent motif" or "PAM", described in more detail below, and optionally cleaves or modifies it, as defined as a nuclease. As the following examples illustrate, RNA-guided nucleases can be defined, in a broad sense, by their PAM specificities and cleavage activities, even though there can be variation among individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Those skilled in the art will understand that some aspects of the present disclosure relate to systems, methods, and compositions that can be implemented using any suitable RNA-guided nuclease having a particular PAM specificity and / or cleavage activity. For this reason, unless otherwise specified, the term "RNA-guided nuclease" should be understood as a general term and is not limited to any particular type (e.g., Cpf1 as compared to Cas9), species (e.g., S. aureus as compared to S. pyogenes), or variation (e.g., truncated or split as compared to full length; engineered PAM specificity as compared to native PAM specificity, etc.) of an RNA-guided nuclease.

[0203] The PAM sequence derives its name from its sequence relationship to the "protospacer" sequence that is complementary to the gRNA targeting domain (or "spacer"). Together with the protospacer sequence, the PAM sequence defines the target region or sequence for a particular RNA-guided nuclease / gRNA combination.

[0204] Various RNA-induced nucleases may require different sequence relationships between the PAM and the protospacer. Generally, Cas9 recognizes the PAM sequence at the 3' position of the protospacer, while Cpf1 generally recognizes the PAM sequence at the 5' position of the protospacer.

[0205] In addition to recognizing the orientation of specific PAM and protospacer sequences, RNA-induced nucleases can also recognize specific PAM sequences. For example, S. aureus Cas9 recognizes the NNGRRT or NNGRRV PAM sequence, in which the N residue is adjacent to the 3' region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes the NGG PAM sequence. Also, F. nobicida Cpf1 recognizes the TTN PAM sequence. PAM sequences have been identified for various RNA-induced nucleases, and strategies for identifying novel PAM sequences are described by Shmakov et al., 2015, Molecular Cell 60, 385-397, November 5, 2015. It should also be noted that manipulated RNA-inducible nucleases may have PAM specificity different from that of the reference molecule (for example, in the case of a manipulated RNA-inducible nuclease, the reference molecule may be a naturally occurring mutant from which the RNA-inducible nuclease originates, or a naturally occurring mutant that has maximum amino acid sequence homology with the manipulated RNA-inducible nuclease).

[0206] In addition to their PAM specificity, RNA-induced nucleases can be characterized by their DNA cleavage activity. While native RNA-induced nucleases typically form DSBs in target nucleic acids, engineered mutants have been generated that produce only SSBs or do not cleave at all (as discussed above) Ran & Hsu, et al., Cell 154(6), 1380-1389, September 12, 2013 (Ran), incorporated herein by reference).

[0207] Cpf1 The crystal structure of Cpf1, a species of Acidaminococcus, when complexed with crRNA and double-stranded (ds)DNA targets such as the TTTN PAM sequence, has been analyzed by Yamano et al. (Cell. 2016 May 5;165(4):949-962 (Yamano), incorporated herein by reference). Similar to Cas9, Cpf1 has two lobes: a REC (recognition) lobe and a NUC (nuclease) lobe. The REC lobe contains REC1 and REC2 domains, which are not similar to any known protein structure. The NUC lobe, on the other hand, contains three RuvC domains (RuvC-I, -II, and -III) and one BH domain. However, in contrast to Cas9, the Cpf1 REC lobe lacks an HNH domain and is a different domain that lacks similarity to known protein structures, containing a structurally unique PI domain, three wedge (WED) domains (WED-I, -II, and -III), and a nuclease (Nuc) domain.

[0208] While Cas9 and Cpf1 share structural and functional similarities, it should be understood that certain Cpf1 activities are mediated by structural domains that are not similar to any Cas9 domain. For example, cleavage of the complementary strand of target DNA appears to be mediated by a Nuc domain that is sequencely and spatially distinct from the HNH domain of Cas9. Furthermore, the untargeting region (handle) of Cpf1 gRNA adopts a pseudo-knot structure rather than a stem-loop structure formed by repeat:anti-repeat double helix in Cas9 gRNA.

[0209] Modification of RNA-induced nucleases While the RNA-induced nucleases described above possess activities and properties that may be useful for a variety of applications, those skilled in the art will recognize that RNA-induced nucleases may also be modified to alter their cleavage activity, PAM specificity, or other structural or functional characteristics.

[0210] First, focusing on modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe are described above. Exemplary mutations that can be generated in the RuvC domain, Cas9 HNH domain, or Cpf1 Nuc domain are described by Ran and Yamano and Cotta-Ramusino. Generally, mutations that reduce or eliminate the activity of one of two nuclease domains result in an RNA-inducible nuclease with nickase activity, but it should be noted that the type of nickase activity changes depending on which domain is inactivated. For example, inactivation of the RuvC domain of Cas9 results in a nickase that cleaves the complementary or upper strand. On the other hand, inactivation of the Cas9 HNH domain results in a nickase that cleaves the lower or non-complementary strand.

[0211] Modifications of PAM specificity compared to naturally occurring Cas9 reference molecules have been described by Kleinstiver et al. for both S. pyogenes (Kleinstiver et al., Nature. 2015 Jul 23;523(7561):481-5 (Kleinstiver I)) and S. aureus (Kleinstiver et al., Nat Biotechnol. 2015 Dec;33(12):1293-1298 (Kleinstiver II)). Kleinstiver et al. also describe modifications that improve Cas9 targeting fidelity (Nature, 2016 January 28;529,490-495 (Kleinstiver III)). Modifications of PAM specificity compared to naturally occurring Cas9 reference molecules have been described by Kleinstiver et al. Both *S. pyogenes* (Kleinstiver et al., Nature. 2015 Jul 23;523(7561):481-5(Kleinstiver I)) are described. Each of these references is incorporated herein by reference.

[0212] Modifications of PAM specificity compared to naturally occurring Cpf1 reference molecules have been described by Gao et al. (Gao et al., Nat Biotechnol. 2017 Aug;35(8):789-792, as incorporated herein by reference). In certain embodiments, the RNA-induced nuclease may be a Cpf1 mutant, such as the AsCpf1 mutant. In certain embodiments, the Cpf1 mutant is the AsCpf1 mutant containing the S542R / K607R variation, which recognizes TYCV PAM. In certain embodiments, the Cpf1 mutant is the AsCpf1 mutant containing the S542R / K548V / N552R variation, which recognizes TATV PAM.

[0213] RNA-induced nucleases are divided into two or more parts, as described by Zetsche et al. (Nat Biotechnol. 2015 Feb; 33(2): 139-42 (Zetsche II), referenced) and Fine et al. (Sci Rep. 2015 Jul 1; 5: 10777 (Fine), referenced).

[0214] In certain embodiments, RNA-induced nucleases may be size-optimized or shortened through one or more deletions that reduce the size of the nuclease while still maintaining gRNA binding, target and PAM recognition, and cleavage activity. In certain embodiments, RNA-induced nucleases optionally bind covalently or noncovalently to another polypeptide, nucleotide, or other structure by a linker. Exemplary binding nucleases and linkers are described by Guilinger et al., Nature Biotechnology 32, 577-582 (2014), which are incorporated by reference for all purposes herein.

[0215] RNA-induced nucleases optionally include, but are not limited to, labels such as nuclear localization signals to facilitate the transfer of RNA-induced nuclease proteins into the nucleus. In certain embodiments, RNA-induced nucleases may incorporate nuclear localization signals at their C-terminus and / or N-terminus. Nuclear localization sequences are publicly known in the art and are described in Maeder and elsewhere.

[0216] The aforementioned list of modifications is intended to be illustrative in nature, and those skilled in the art will understand, in view of this disclosure, that other modifications may be possible or desirable in specific applications. Therefore, for the sake of brevity, the exemplary systems, methods, and compositions of this disclosure are presented with reference to specific RNA-inducing nucleases, but it should be understood that the RNA-inducing nucleases used may be modified in a manner that does not alter their operating principles. Such modifications are within the scope of this disclosure.

[0217] nucleic acids encoding RNA-induced nucleases For example, nucleic acids encoding RNA-inducible nucleases, such as Cpf1 or its functional fragments, are provided herein. Exemplary nucleic acids encoding RNA-inducible nucleases have been previously described (see, for example, Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

[0218] In some cases, the nucleic acid encoding the RNA-inducing nuclease may be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule may be chemically modified. In certain embodiments, the mRNA encoding the RNA-inducing nuclease may have one or more (e.g., all) of the following properties: it may be capped; it may be polyadenylated; and it may be substituted with 5-methylcytidine and / or pseudouridine.

[0219] Synthetic nucleic acid sequences can also be codon-optimized, for example, by replacing at least one uncommon or less common codon with a common codon. For example, synthetic nucleic acids can induce the synthesis of optimized messenger mRNA, optimized for expression in mammalian expression systems, as described herein. An example of a codon-optimized Cas9 coding sequence is presented in Cotta-Ramusino.

[0220] Furthermore, or alternatively, nucleic acids encoding RNA-induced nucleases may include nuclear localization sequences (NLSs). Nuclear localization sequences are known in the art.

[0221] Functional analysis of candidate molecules Candidate RNA-induced nucleases, gRNAs, and their complexes can be evaluated by standard methods known in the art. See, for example, Cotta-Ramusino et al. The stability of the RNP complex can be evaluated by differential scanning fluorescence quantification as described below.

[0222] Differential scanning fluorescence (DSF) The thermal stability of ribonucleoprotein (RNP) complexes containing gRNA and RNA-induced nucleases can be measured by DSF. DSF technology measures the thermal stability of proteins, which can be increased under favorable conditions, such as the addition of binding RNA molecules like gRNA.

[0223] DSF assays can be performed according to any suitable protocol, but are not limited to, and may be used in any suitable setting, including (a) testing different conditions (e.g., different stoichiometric ratios of gRNA:RNA-induced nuclease protein, different buffers, etc.) to identify optimal conditions for RNP formation; and (b) testing modifications of RNA-induced nucleases and / or gRNAs (e.g., chemical modifications, sequence alterations, etc.) to identify modifications that improve RNP formation or stability. One reading of a DSF assay is the change in melting temperature of the RNP complex; a relatively high change suggests that the RNP complex is more stable (and therefore may have greater activity or more favorable formation, degradation, or other functional properties) compared to a standard RNP complex characterized by a lower change. When a DSF assay is deployed as a screening tool, a threshold melting temperature change may be identified, thereby the result being one or more RNPs with a melting temperature change above the threshold. For example, the threshold could be 5-10°C (e.g., 5°C, 6°C, 7°C, 8°C, 9°C, 10°C) or greater, and the result could be one or more RNPs characterized by a change in melting temperature above the threshold.

[0224] Two non-limiting examples of DSF assay conditions are as follows (the conditions refer to the use of Cas9, but similar conditions may also be used for Cpf1):

[0225] To determine the best solution for RNP complex formation, Cas9+10×SYPRO Orange® (Life Technologies, catalog no. S-6650) at a fixed concentration (e.g., 2 μM) in water is dispensed into a 384-well plate. Next, equimolar amounts of gRNA, diluted in solutions of varying pH and salt, are added. After incubation at room temperature for 10 minutes and brief centrifugation to remove any bubbles, a gradient is run from 20°C to 90°C in 1°C increments every 10 seconds using a Bio-Rad CFX384® real-time system C1000 Touch® thermal cycler with Bio-Rad CFX Manager software.

[0226] The second assay consists of mixing various concentrations of gRNA with a fixed concentration (e.g., 2 μM) of Cas9i in the optimal buffer from assay 1 above, and incubating it in a 384-well plate (e.g., at room temperature for 10 minutes). Equivolutes of optimal buffer + 10 × SYPRO Orange® (Life Technologies, catalog no. S-6650) are added, and the plate is sealed with Microseal® B adhesive (MSB-1001). After briefly centrifugating to remove any bubbles, a gradient is run from 20°C to 90°C in increments of 1°C every 10 seconds using a Bio-Rad CFX384® real-time system C1000 Touch® thermal cycler with Bio-Rad CFX Manager software.

[0227] Genome editing strategies Using the genome editing systems described above, editing (i.e., modification) is performed in cells or within target regions of DNA obtained from cells, in various embodiments of this disclosure. Various strategies for performing specific edits are described herein, and these strategies are generally described by the desired repair outcome, the number and location of individual edits (e.g., SSBs or DSBs), and the target sites of such edits.

[0228] Genome editing strategies involving the formation of SSBs or DSBs are characterized by repair outcomes including (a) deletion of all or part of the target region; (b) insertion or substitution within all or part of the target region; or (c) interruption of all or part of the target region. This grouping is not intended to limit or be bound by any particular theory or model, but is provided solely for economics of presentation. Those skilled in the art will understand that the listed outcomes are not mutually exclusive and that some repairs may result in others. Descriptions of particular editing strategies or methods should not be understood as requiring a particular repair outcome unless otherwise specified.

[0229] Target region substitution generally involves the substitution of all or part of a sequence present within the target region by homologous sequences, through gene modification or gene conversion, which are two repair outcomes mediated by the HDR pathway. HDR is facilitated by the use of donor templates, which may be single-stranded or double-stranded, as described in more detail below. Single-stranded or double-stranded templates may be exogenous, in which case they facilitate gene modification, or they may be endogenous (e.g., homologous sequences in the cellular genome) and facilitate gene conversion. Exogenous templates may have asymmetric overhangs, as described, for example, by Richardson et al. (Nature Biotechnology 34, 339-344 (2016), (Richardson), reference cited) (i.e., the portion of the template complementary to the DSB site may be offset in the 3' or 5' direction rather than being centrally located within the template). If the template is a single strand, it can correspond to either the complementary (upper) or non-complementary (lower) strand of the target region.

[0230] As described by Ran and Cotta-Ramusino et al., gene transformation and gene modification are sometimes facilitated by forming one or more nicks within or around the target region. Sometimes, a double nicking strategy is used to form two offset SSBs, which are then formed into a single DSB with an overhang (e.g., a 5' overhang).

[0231] Interruption and / or deletion of all or part of a target sequence can be achieved by various repair outcomes. For example, as described by Maeder for the LCA10 mutation, a sequence can be deleted by simultaneously generating two or more double-strand breaks (DSBs) flanking the target region, which are then excised when the DSBs are repaired. Alternatively, a sequence can be interrupted by the formation of a double-strand break with a single-strand overhang, followed by deletion generated by the subsequent hydrolytic processing of the nucleotide terminals of the pre-repair overhang.

[0232] One particular subset of target sequence interruptions is mediated by the formation of indels in the target sequence, in which the repair outcome is typically mediated by the NHEJ pathway (including Alt-NHEJ). NHEJ is referred to as the “error-prone” repair pathway due to its association with indel mutations. However, in some cases, DSBs are repaired by NHEJ without alteration of the surrounding sequence (so-called “perfect” or “scarless” repair); this generally requires both ends of the DSB to be completely ligated. Indels, on the other hand, are thought to arise from the enzymatic processing of free DNA ends before they are ligated, which adds and / or removes nucleotides from one or both strands of one or both free ends.

[0233] Because the enzymatic processing of free DSB ends can be inherently probabilistic, indel mutations tend to be variable, occurring along distributions and influenced by various factors, including specific target sites, cell types used, and genome editing strategies employed. Nevertheless, it is possible to generalize to a limited extent about indel formation: deletions formed by single DSB repair are most commonly in the range of 1–50 bp, but can exceed 100–200 bp. Insertions formed by single DSB repair tend to be shorter and often contain short duplications of sequences directly surrounding the cleavage site. However, larger insertions are possible, and in these cases, the inserted sequence is often traced back to other regions of the genome or plasmid DNA present within the cell.

[0234] Indel mutations and genome editing systems configured to generate indels are useful for interrupting target sequences, for example, when the generation of a specific final sequence is not required and / or when frameshift mutations are tolerable. They may also be useful in situations where a specific sequence is preferred, as long as the desired specific sequence tends to preferentially arise from the repair of SSBs or DSBs at a given site. Indel mutations are also a useful tool for evaluating or screening the activity of specific genome editing systems and their components. In these and other settings, indels may be characterized by (a) their relative and absolute frequencies in the genome of the cell in contact with the genome editing system, and (b) a distribution of numerical differences, such as ±1, ±2, ±3, relative to the unedited sequence. As an example, in a read discovery setting, multiple gRNAs may be screened to identify the gRNA that most efficiently promotes cleavage at the target site based on indel readings under controlled conditions. Guides that generate indels above a threshold frequency or guides that generate a specific distribution of indels may be selected for further research and development. The frequency and distribution of indels can also be useful as a read for evaluating different genome editing system implementations or formulation and delivery methods, for example, by keeping the gRNA constant while varying other specific reaction conditions or delivery methods.

[0235] Multiple strategies While the exemplary strategies described above focus on repair outcomes mediated by a single DSB, the genome editing systems of this disclosure may be used to generate two or more DSBs at either the same locus or different loci. Editing strategies involving the formation of multiple DSBs or SSBs are described, for example, by Cotta-Ramusino et al. As described herein, the methods and compositions incorporated herein may be used to modify two or more T cell expression genes, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, B2M, TRAC, CIITA, and TRBC genes, thereby affecting T cell proliferation, survival, persistence, and / or function.

[0236] Donor mold design Donor template design is described in detail in literature such as that of Cotta-Ramusino. DNA oligomer donor templates (oligodeoxynucleotides or ODNs), which can be single-stranded (ssODN) or double-stranded (dsODN), can be used to facilitate HDR-based repair of DSBs, and are particularly useful for introducing modifications to the target DNA sequence, inserting a new sequence into the target sequence, or completely replacing the target sequence.

[0237] Whether single-stranded or double-stranded, the donor template generally includes regions homologous to the DNA region in or near the target sequence to be cleaved (e.g., lateral or adjacent). These homologous regions are referred to herein as “homology arms” and are schematically shown below. [5' homology arm]--[substitution sequence]--[3' homology arm]

[0238] Homology arms can have any appropriate length (including none if only one homology arm is used), and the 3' and 5' homology arms may have the same length or different lengths. The selection of appropriate homology arm lengths may be influenced by various factors, such as the desire to avoid homology or microhomology with specific sequences, such as Alu repeats or other very common elements. For example, the 5' homology arm may be shortened to avoid sequence repeats. In other embodiments, the 3' homology arm may be shortened to avoid sequence repeats. In certain embodiments, both the 5' and 3' homology arms may be shortened to avoid inclusion of specific sequence repeats. Furthermore, some homology arm designs may improve editing efficiency or increase the frequency of desired repair results. For example, Richardson et al., Nature Biotechnology 34,339-344 (2016) (Richardson), cited by reference, found that the relative asymmetry of the 3' and 5' homology arms of a single-stranded donor template affects the repair rate and / or outcome.

[0239] Substitution sequences in donor templates have been described elsewhere, including by Cotta-Ramusino et al. Substitution sequences can be of any appropriate length (including none if the desired repair outcome is a deletion) and typically involve one, two, three or more sequence modifications to the native intracellular sequence to be edited. One common sequence modification involves altering the native sequence to repair mutations associated with the disease or condition to be treated. Another common sequence modification involves altering one or more sequences that are complementary to or encode the PAM sequence of an RNA-induced nuclease or the targeting domain of a gRNA used to generate SSBs or DSBs, thereby reducing or eliminating repeat breaks at the target site after the substitution sequence has been incorporated into the target site.

[0240] When a linear ssODN is used, it may be configured to (i) anneal to the nicked strand of the target nucleic acid, (ii) anneal to the intact target nucleic acid strand, (iii) anneal to the positive strand of the target nucleic acid, and / or (iv) anneal to the negative strand of the target nucleic acid. The ssODN may have any suitable length, such as approximately or at least 150-200 nucleotides or less (e.g., 150, 160, 170, 180, 190, or 200 nucleotides).

[0241] It should be noted that the template nucleic acid may be a nucleic acid vector such as a viral genome or a circular double-stranded DNA such as a plasmid. Nucleic acid vectors containing a donor template may contain other coding or non-coding elements. For example, the template nucleic acid may contain specific genomic backbone elements (e.g., reverse terminal repeats in the case of an AAV genome) and may be delivered as part of a viral genome (e.g., in an AAV or lentiviral genome) that optionally contains additional sequences encoding gRNA and / or RNA-induced nucleases. In certain embodiments, the donor template may be adjacent to or sandwiched between target sites recognized by one or more gRNAs, facilitating the formation of free DSBs at one or both ends of the donor template, which may be involved in the repair of corresponding SSBs or DSBs formed in cellular DNA using the same gRNA. Exemplary nucleic acid vectors suitable for use as donor templates are described by Cotta-Ramusino et al.

[0242] Regardless of the form used, the template nucleic acid may be designed to avoid undesirable sequences. In certain embodiments, one or both homology arms may be shortened to avoid overlap with certain sequence repeat elements, such as Alu repeats and LINE elements.

[0243] Targeted embedding This disclosure further provides a genome editing system including a donor template specifically designed to enable quantitative evaluation of gene editing events occurring during cleavage and separation events at cleavage sites of target nucleic acids within cells. The donor template of the genome editing system described herein is a DNA oligodeoxynucleotide (ODN), which may be single-stranded (ssODN) or double-stranded (dsODN) and may be used to facilitate HDR-based repair of double-strand breaks. The donor template is particularly useful for introducing modifications to a target DNA sequence, inserting a novel sequence into a target sequence, or replacing an entire target sequence. This disclosure provides a donor template including a cargo, one or two homology arms, and one or more priming sites. The priming sites of the donor template are spatially arranged in such a manner that the frequency of integration of parts of the donor template into the target nucleic acid can be easily evaluated and quantified.

[0244] Figures 44A, 44B, and 44C are schematic diagrams illustrating typical donor templates and the potential targeted integration results obtained from the use of these donor templates. The use of the exemplary donor templates described herein results in targeted integration of at least one priming site in the targeted nucleic acid, which can be used to generate an amplicon that can be sequenced to determine the frequency of targeted integration of cargo (e.g., transgene) into the targeted nucleic acid within the target cell.

[0245] For example, Figure 44A illustrates an exemplary donor template including a first homology arm (A1), a first stuffer sequence (S1), a second priming site (P2'), a cargo, a first priming site, a second stuffer sequence, and a second homology arm, oriented from 5' to 3'. The first homology arm (A1) of the donor template is substantially identical to the first homology arm of the target nucleic acid, while the second homology arm (A2) of the donor template is substantially identical to the second homology arm of the target nucleic acid. The donor template is designed such that the second priming site (P2') is substantially identical to the first priming site (P1) of the target nucleic acid, and the first priming site (P1') is substantially identical to the second priming site (P2) of the target nucleic acid. During cleavage and separation events of a target nucleic acid using the nucleases described herein, a single primer pair set may be used to amplify the nucleic acid sequences surrounding the cleavage sites of the target nucleic acid (i.e., the nucleic acids present between P1 and P2, between P1 and P2', and between P1' and P2). Advantageously, the sizes of the amplicons (indicated as amplicons X, Y, and Z) obtained from cleavage and separation events with or without targeted incorporation are approximately the same. The amplicons can then be evaluated, for example, by sequencing or hybridization to a probe sequence, to determine the frequency of targeted incorporation.

[0246] Instead, Figures 44B and 44C depict exemplary donor templates containing a single priming site located either 3' (Figure 44B) or 5' (Figure 44C) from the cargo nucleic acid sequence. In this case as well, during cleavage and separation events of the target nucleic acid using the nucleases described herein, these exemplary donor templates are designed to amplify the nucleic acid sequence around the cleavage site of the target nucleic acid using a single primer pair, yielding two amplicons of approximately the same size. If the priming site of the donor template is located 3' from the cargo nucleic acid, an amplicon corresponding to a non-targeted integration event or an amplicon corresponding to the 5' junction of a targeted integration site may be amplified. If the priming site of the donor template is located 5' from the cargo nucleic acid, an amplicon corresponding to a non-targeted integration event or an amplicon corresponding to the 3' junction of a targeted integration site may be amplified. These amplicons can be sequenced to determine the frequency of targeted integration.

[0247] The donor templates provided in this disclosure can be implemented in any suitable form, including, but not limited to, linear or circular, bare single-stranded or double-stranded DNA, or can be contained within a vector and / or can be covalently or acovalently bound to guide RNA (e.g., by direct hybridization or sprint hybridization). In certain embodiments, the donor template is an ssODN. When a linear ssODN is used, it may be configured to (i) anneal to the nicked strand of the target nucleic acid, (ii) anneal to the intact target nucleic acid strand, (iii) anneal to the positive strand of the target nucleic acid, and / or (iv) anneal to the negative strand of the target nucleic acid. The ssODN may have any suitable length, such as about 150–200 nucleotides or less (e.g., 150, 160, 170, 180, 190, or 200 nucleotides). In other embodiments, the donor template is a dsODN. In certain embodiments, the donor template includes a first chain. In other embodiments, the donor template includes a first chain and a second chain. In certain embodiments, the donor template is an exogenous oligonucleotide, such as an oligonucleotide that is not naturally present in cells.

[0248] It should be noted that donor templates may also be contained within nucleic acid vectors such as viral genomes or circular double-stranded DNA, such as plasmids. In certain embodiments, the donor template may be bone-shaped DNA for dogs (see, for example, U.S. Patent No. 9,499,847). Nucleic acid vectors containing donor templates may contain other coding or non-coding elements. For example, a donor template nucleic acid may contain specific genomic backbone elements (e.g., reverse-ended repeats in the case of an AAV genome) and may be delivered as part of a viral genome (e.g., in an AAV or lentiviral genome) that includes additional sequences encoding gRNA and / or RNA-inducible nucleases. In certain embodiments, the donor template may be adjacent to or sandwiched between target sites recognized by one or more gRNAs, facilitating the formation of free DSBs at one or both ends of the donor template, which may be involved in the repair of corresponding SSBs or DSBs formed in cellular DNA using the same gRNA. Exemplary nucleic acid vectors suitable for use as donor templates are described by Cotta-Ramusino et al.

[0249] homology arm Whether single-stranded or double-stranded, a donor template generally includes one or more regions homologous to a region of DNA, such as a target nucleic acid, within or near (e.g., flanking or adjacent to) the target sequence to be cleaved, such as a cleavage site. These homologous regions are referred to herein as “homology arms” and are schematically shown below. [5' homology arm]-[substitution sequence]-[3' homology arm]

[0250] The homology arms of the donor template described herein may be of any suitable length, as long as they are long enough to allow for the efficient separation of the cleavage site of the target nucleic acid by the DNA repair process requiring the donor template. For example, in a particular embodiment where homology arm amplification by PCR is desired, the homology arms are of a length that allows amplification to be performed. In a particular embodiment where sequencing of the homology arms is desired, the homology arms are of a length that allows sequencing to be performed. In a particular embodiment where quantitative evaluation of the amplicons is desired, the homology arms are of a length that allows for similar amplification cycles of each amplicon, for example, by having similar G / C content, amplification temperature, etc. In a particular embodiment, the homology arms are double-stranded. In a particular embodiment, the double-stranded homology arms are single-stranded.

[0251] In certain embodiments, the 5' homology arm has a length of 50 to 250 nucleotides. In certain embodiments, the 5' homology arm has a length of 50 to 2000 nucleotides. In certain embodiments, the 5' homology arm has a length of 50 to 1500 nucleotides. In certain embodiments, the 5' homology arm has a length of 50 to 1000 nucleotides. In certain embodiments, the 5' homology arm has a length of 50 to 500 nucleotides. In certain embodiments, the 5' homology arm has a length of 150 to 250 nucleotides. In certain embodiments, the 5' homology arm has a length of 2000 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 1500 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 1000 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 700 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 650 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 600 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 550 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 500 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 400 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 300 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 250 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 200 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 150 nucleotides or less. In certain embodiments, the 5' homology arm has a length of less than 100 nucleotides. In certain embodiments, the 5' homology arm has a length of 50 nucleotides or less. In certain embodiments, the 5' homology arm has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides.In certain embodiments, the 5' homology arm is at least 20 nucleotides long. In certain embodiments, the 5' homology arm is at least 40 nucleotides long. In certain embodiments, the 5' homology arm is at least 50 nucleotides long. In certain embodiments, the 5' homology arm is at least 70 nucleotides long. In certain embodiments, the 5' homology arm is at least 100 nucleotides long. In certain embodiments, the 5' homology arm is at least 200 nucleotides long. In certain embodiments, the 5' homology arm is at least 300 nucleotides long. In certain embodiments, the 5' homology arm is at least 400 nucleotides long. In certain embodiments, the 5' homology arm is at least 500 nucleotides long. In certain embodiments, the 5' homology arm is at least 600 nucleotides long. In certain embodiments, the 5' homology arm is at least 700 nucleotides long. In certain embodiments, the 5' homology arm is at least 1000 nucleotides long. In certain embodiments, the 5' homology arm is at least 1500 nucleotides long. In certain embodiments, the 5' homology arm is at least 2000 nucleotides long. In certain embodiments, the 5' homology arm is about 20 nucleotides long. In certain embodiments, the 5' homology arm is about 40 nucleotides long. In certain embodiments, the 5' homology arm is 250 nucleotides or less long. In certain embodiments, the 5' homology arm is about 100 nucleotides long. In certain embodiments, the 5' homology arm is about 200 nucleotides long.

[0252] In certain embodiments, the 3' homology arm has a length of 50 to 250 nucleotides. In certain embodiments, the 3' homology arm has a length of 50 to 2000 nucleotides. In certain embodiments, the 3' homology arm has a length of 50 to 1500 nucleotides. In certain embodiments, the 3' homology arm has a length of 50 to 1000 nucleotides. In certain embodiments, the 3' homology arm has a length of 50 to 500 nucleotides. In certain embodiments, the 3' homology arm has a length of 150 to 250 nucleotides. In certain embodiments, the 3' homology arm has a length of 2000 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 1500 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 1000 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 700 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 650 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 600 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 550 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 500 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 400 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 300 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 200 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 150 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 100 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 50 nucleotides or less. In certain embodiments, the 3' homology arm has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides. In certain embodiments, the 3' homology arm has a length of at least 20 nucleotides.In certain embodiments, the 3' homology arm is at least 40 nucleotides long. In certain embodiments, the 3' homology arm is at least 50 nucleotides long. In certain embodiments, the 3' homology arm is at least 70 nucleotides long. In certain embodiments, the 3' homology arm is at least 100 nucleotides long. In certain embodiments, the 3' homology arm is at least 200 nucleotides long. In certain embodiments, the 3' homology arm is at least 300 nucleotides long. In certain embodiments, the 3' homology arm is at least 400 nucleotides long. In certain embodiments, the 3' homology arm is at least 500 nucleotides long. In certain embodiments, the 3' homology arm is at least 600 nucleotides long. In certain embodiments, the 3' homology arm is at least 700 nucleotides long. In certain embodiments, the 3' homology arm is at least 1000 nucleotides long. In certain embodiments, the 3' homology arm is at least 1500 nucleotides long. In certain embodiments, the 3' homology arm is at least 2000 nucleotides long. In certain embodiments, the 3' homology arm is about 20 nucleotides long. In certain embodiments, the 3' homology arm is about 40 nucleotides long. In certain embodiments, the 3' homology arm is 250 nucleotides or less long. In certain embodiments, the 3' homology arm is about 100 nucleotides long. In certain embodiments, the 3' homology arm is about 200 nucleotides long.

[0253] In certain embodiments, the 5' homology arm has a length of 50 to 250 base pairs. In certain embodiments, the 5' homology arm has a length of 50 to 2000 base pairs. In certain embodiments, the 5' homology arm has a length of 50 to 1500 base pairs. In certain embodiments, the 5' homology arm has a length of 50 to 1000 base pairs. In certain embodiments, the 5' homology arm has a length of 50 to 500 base pairs. In certain embodiments, the 5' homology arm has a length of 150 to 250 base pairs. In certain embodiments, the 5' homology arm has a length of 2000 base pairs or less. In certain embodiments, the 5' homology arm has a length of 1500 base pairs or less. In certain embodiments, the 5' homology arm has a length of 1000 base pairs or less. In certain embodiments, the 5' homology arm has a length of 700 base pairs or less. In certain embodiments, the 5' homology arm is 650 base pairs or less in length. In certain embodiments, the 5' homology arm is 600 base pairs or less in length. In certain embodiments, the 5' homology arm is 550 base pairs or less in length. In certain embodiments, the 5' homology arm is 500 base pairs or less in length. In certain embodiments, the 5' homology arm is 400 base pairs or less in length. In certain embodiments, the 5' homology arm is 300 base pairs or less in length. In certain embodiments, the 5' homology arm is 250 base pairs or less in length. In certain embodiments, the 5' homology arm is 200 base pairs or less in length. In certain embodiments, the 5' homology arm is 150 base pairs or less in length. In certain embodiments, the 5' homology arm is less than 100 base pairs in length. In certain embodiments, the 5' homology arm is 50 base pairs or less in length. In certain embodiments, the 5' homology arm has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs. In certain embodiments, the 5' homology arm has a length of at least 20 base pairs.In certain embodiments, the 5' homology arm is at least 40 base pairs long. In certain embodiments, the 5' homology arm is at least 50 base pairs long. In certain embodiments, the 5' homology arm is at least 70 base pairs long. In certain embodiments, the 5' homology arm is at least 100 base pairs long. In certain embodiments, the 5' homology arm is at least 200 base pairs long. In certain embodiments, the 5' homology arm is at least 300 base pairs long. In certain embodiments, the 5' homology arm is at least 400 base pairs long. In certain embodiments, the 5' homology arm is at least 500 base pairs long. In certain embodiments, the 5' homology arm is at least 600 base pairs long. In certain embodiments, the 5' homology arm is at least 700 base pairs long. In certain embodiments, the 5' homology arm is at least 1000 base pairs long. In certain embodiments, the 5' homology arm is at least 1500 base pairs long. In certain embodiments, the 5' homology arm is at least 2000 base pairs long. In certain embodiments, the 5' homology arm is about 20 base pairs long. In certain embodiments, the 5' homology arm is about 40 base pairs long. In certain embodiments, the 5' homology arm is 250 base pairs or less long. In certain embodiments, the 5' homology arm is about 100 base pairs long. In certain embodiments, the 5' homology arm is about 200 base pairs long.

[0254] In certain embodiments, the 3' homology arm has a length of 50 to 250 base pairs. In certain embodiments, the 3' homology arm has a length of 50 to 2000 base pairs. In certain embodiments, the 3' homology arm has a length of 50 to 1500 base pairs. In certain embodiments, the 3' homology arm has a length of 50 to 1000 base pairs. In certain embodiments, the 3' homology arm has a length of 50 to 500 base pairs. In certain embodiments, the 3' homology arm has a length of 150 to 250 base pairs. In certain embodiments, the 3' homology arm has a length of 2000 base pairs or less. In certain embodiments, the 3' homology arm has a length of 1500 base pairs or less. In certain embodiments, the 3' homology arm has a length of 1000 base pairs or less. In certain embodiments, the 3' homology arm has a length of 700 base pairs or less. In certain embodiments, the 3' homology arm is 650 base pairs or less in length. In certain embodiments, the 3' homology arm is 600 base pairs or less in length. In certain embodiments, the 3' homology arm is 550 base pairs or less in length. In certain embodiments, the 3' homology arm is 500 base pairs or less in length. In certain embodiments, the 3' homology arm is 400 base pairs or less in length. In certain embodiments, the 3' homology arm is 300 base pairs or less in length. In certain embodiments, the 3' homology arm is 250 base pairs or less in length. In certain embodiments, the 3' homology arm is 200 base pairs or less in length. In certain embodiments, the 3' homology arm is 150 base pairs or less in length. In certain embodiments, the 3' homology arm is less than 100 base pairs in length. In certain embodiments, the 3' homology arm is 50 base pairs or less in length. In certain embodiments, the 3' homology arm has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs. In certain embodiments, the 3' homology arm has a length of at least 20 base pairs.In certain embodiments, the 3' homology arm is at least 40 base pairs long. In certain embodiments, the 3' homology arm is at least 50 base pairs long. In certain embodiments, the 3' homology arm is at least 70 base pairs long. In certain embodiments, the 3' homology arm is at least 100 base pairs long. In certain embodiments, the 3' homology arm is at least 200 base pairs long. In certain embodiments, the 3' homology arm is at least 300 base pairs long. In certain embodiments, the 3' homology arm is at least 400 base pairs long. In certain embodiments, the 3' homology arm is at least 500 base pairs long. In certain embodiments, the 3' homology arm is at least 600 base pairs long. In certain embodiments, the 3' homology arm is at least 700 base pairs long. In certain embodiments, the 3' homology arm is at least 1000 base pairs long. In certain embodiments, the 3' homology arm is at least 1500 base pairs long. In certain embodiments, the 3' homology arm is at least 2000 base pairs long. In certain embodiments, the 3' homology arm is about 20 base pairs long. In certain embodiments, the 3' homology arm is about 40 base pairs long. In certain embodiments, the 3' homology arm is 250 base pairs or less long. In certain embodiments, the 3' homology arm is about 100 base pairs long. In certain embodiments, the 3' homology arm is about 200 base pairs long. In certain embodiments, the 3' homology arm is 250 base pairs or less long. In certain embodiments, the 3' homology arm is 200 base pairs or less long. In certain embodiments, the 3' homology arm is 150 base pairs or less long. In certain embodiments, the 3' homology arm is 100 base pairs or less long. In certain embodiments, the 3' homology arm is 50 base pairs or less in length.In certain embodiments, the 3' homology arm has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs. In certain embodiments, the 3' homology arm has a length of 40 base pairs.

[0255] The 5' and 3' homology arms may be the same length or may differ in length. In certain embodiments, the 5' and 3' homology arms are amplified to enable quantitative evaluation of gene editing events, such as targeted integration in the target nucleic acid. In certain embodiments, quantitative evaluation of gene editing events may depend on amplification of both the 5' and 3' junctions at the targeted integration site by amplifying all or part of the homology arms using a pair of PCR primers in a single amplification reaction. Therefore, although the lengths of the 5' and 3' homology arms may differ, the length of each homology arm must be amplified as needed (e.g., using PCR). Furthermore, if both amplification of the 5' homology arm and the difference in length between the 5' and 3' homology arms are desired in a single PCR reaction, the difference in length between the 5' and 3' homology arms must be PCR amplified using a pair of PCR primers.

[0256] In certain embodiments, the lengths of the 5' and 3' homology arms do not differ by more than 75 nucleotides. Therefore, in certain embodiments, when the lengths of the 5' and 3' homology arms differ, the difference in length between the homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide or base pair. In certain embodiments, the 5' and 3' homology arms differ in length by at least 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, 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 nucleotides. In certain embodiments, the difference in length between the 5' and 3' homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 base pairs. In certain embodiments, the 5' and 3' homology arms differ in length by at least 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, 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 base pairs.

[0257] The donor template of this disclosure is designed to promote homologous recombination with a target nucleic acid having a cleavage site, wherein the target nucleic acid contains P1--H1--X--H2--P2 in the 5' to 3' direction. Here, P1 is the first priming area; H1 is the first homology arm; X is the cutting area; H2 is the second homology arm; P2 is the second priming area; and the donor mold includes A1--P2'--N--A2 or A1--N--P1'--A2 in the 5' to 3' direction. Here, A1 is a homology arm substantially identical to H1; P2' is a priming site substantially identical to P2; N is a cargo; P1' is a priming site substantially identical to P1; and A2 is a homology arm substantially identical to H2. In certain embodiments, the target nucleic acid is double-stranded. In certain embodiments, the target nucleic acid comprises a first strand and a second strand. In other embodiments, the target nucleic acid is single-stranded. In certain embodiments, the target nucleic acid comprises a first strand.

[0258] In a particular embodiment, the donor mold includes A1--P2'--N--A2 in the 5' to 3' direction.

[0259] In a particular embodiment, the donor mold includes A1--P2'--N--P1'--A2 in the 5' to 3' direction.

[0260] In certain embodiments, the target nucleic acid comprises P1--H1--X--H2--P2 in the 5' to 3' direction, Here, P1 is the first priming site; H1 is the first homology arm; X is the cutting site; H2 is the second homology arm; P2 is the second priming site; and the first chain of the donor mold includes A1--P2'--N--A2 or A1--N--P1'--A2 in the 5' to 3' direction. Here, A1 is a homologous arm substantially identical to H1; P2' is a priming area substantially identical to P2; N is the cargo; P1' is a priming area substantially identical to P1; and A2 is a homologous arm substantially identical to H2.

[0261] In a particular embodiment, the first chain of the donor mold includes A1--P2'--N--P1'--A2 in the 5' to 3' direction.

[0262] In a particular embodiment, the first chain of the donor mold includes A1--N--P1'--A2 in the 5' to 3' direction.

[0263] In certain embodiments, A1 has a length of 700 base pairs or less. In certain embodiments, A1 has a length of 650 base pairs or less. In certain embodiments, A1 has a length of 600 base pairs or less. In certain embodiments, A1 has a length of 550 base pairs or less. In certain embodiments, A1 has a length of 500 base pairs or less. In certain embodiments, A1 has a length of 400 base pairs or less. In certain embodiments, A1 has a length of 300 base pairs or less. In certain embodiments, A1 has a length of less than 250 base pairs. In certain embodiments, A1 has a length of less than 200 base pairs. In certain embodiments, A1 has a length of less than 150 base pairs. In certain embodiments, A1 has a length of less than 100 base pairs. In certain embodiments, A1 has a length of less than 50 base pairs. In certain embodiments, A1 has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs. In certain embodiments, A1 has a length of 40 base pairs. In certain embodiments, A1 has a length of 30 base pairs. In certain embodiments, A1 has a length of 20 base pairs.

[0264] In certain embodiments, A2 has a length of 700 base pairs or less. In certain embodiments, A2 has a length of 650 base pairs or less. In certain embodiments, A2 has a length of 600 base pairs or less. In certain embodiments, A2 has a length of 550 base pairs or less. In certain embodiments, A2 has a length of 500 base pairs or less. In certain embodiments, A2 has a length of 400 base pairs or less. In certain embodiments, A2 has a length of 300 base pairs or less. In certain embodiments, A2 has a length of less than 250 base pairs. In certain embodiments, A2 has a length of less than 200 base pairs. In certain embodiments, A2 has a length of less than 150 base pairs. In certain embodiments, A2 has a length of less than 100 base pairs. In certain embodiments, A2 has a length of less than 50 base pairs. In certain embodiments, A2 has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs. In certain embodiments, A2 has a length of 40 base pairs. In certain embodiments, A2 has a length of 30 base pairs. In certain embodiments, A2 has a length of 20 base pairs.

[0265] In certain embodiments, A1 has a length of 700 nucleotides or less. In certain embodiments, A1 has a length of 650 nucleotides or less. In certain embodiments, A1 has a length of 600 nucleotides or less. In certain embodiments, A1 has a length of 550 nucleotides or less. In certain embodiments, A1 has a length of 500 nucleotides or less. In certain embodiments, A1 has a length of 400 nucleotides or less. In certain embodiments, A1 has a length of 300 nucleotides or less. In certain embodiments, A1 has a length of less than 250 nucleotides. In certain embodiments, A1 has a length of less than 200 nucleotides. In certain embodiments, A1 has a length of less than 150 nucleotides. In certain embodiments, A1 has a length of less than 100 nucleotides. In certain embodiments, A1 has a length of less than 50 nucleotides. In certain embodiments, A1 has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides. In certain embodiments, A1 has a length of at least 40 nucleotides. In certain embodiments, A1 has a length of at least 30 nucleotides. In certain embodiments, A1 has a length of at least 20 nucleotides.

[0266] In certain embodiments, A2 has a length of 700 nucleotides or less. In certain embodiments, A2 has a length of 650 base pairs or less. In certain embodiments, A2 has a length of 600 nucleotides or less. In certain embodiments, A2 has a length of 550 nucleotides or less. In certain embodiments, A2 has a length of 500 nucleotides or less. In certain embodiments, A2 has a length of 400 nucleotides or less. In certain embodiments, A2 has a length of 300 nucleotides or less. In certain embodiments, A2 has a length of less than 250 nucleotides. In certain embodiments, A2 has a length of less than 200 nucleotides. In certain embodiments, A2 has a length of less than 150 nucleotides. In certain embodiments, A2 has a length of less than 100 nucleotides. In certain embodiments, A2 has a length of less than 50 nucleotides. In certain embodiments, A2 has a length of 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides. In certain embodiments, A2 has a length of at least 40 nucleotides. In certain embodiments, A2 has a length of at least 30 nucleotides. In certain embodiments, A2 has a length of at least 20 nucleotides.

[0267] In certain embodiments, the nucleic acid sequence of A1 is substantially identical to that of H1. In certain embodiments, A1 is identical to H1, or has a sequence that differs by only 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides or less. In certain embodiments, A1 is identical to H1, or has a sequence that differs by only 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 base pairs or less.

[0268] In certain embodiments, the nucleic acid sequence of A2 is substantially identical to that of H2. In certain embodiments, A2 is identical to that of H2, or has a sequence that differs by only 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides or less. In certain embodiments, A2 is identical to H2, or has a sequence that differs by only 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 base pairs or less.

[0269] In any form, the donor template may be designed to avoid undesirable arrangements. In certain embodiments, one or both homology arms may be shortened to avoid overlap with certain arrangement repeat elements, such as Alu repeats or LINE elements.

[0270] Priming area The donor templates described herein include at least one priming site having a sequence that is substantially similar to or identical to the sequence of a priming site in the target nucleic acid, but which is in a different spatial order or orientation with respect to homologous sequences / homology arms in the donor template. When the donor template is homologously recombined with the target nucleic acid, the priming site is advantageously incorporated into the target nucleic acid, thereby enabling amplification of a portion of the modified nucleic acid sequence resulting from the recombination event. In certain embodiments, the donor template includes at least one priming site. In certain embodiments, the donor template includes first and second priming sites. In certain embodiments, the donor template includes three or more priming sites.

[0271] In certain embodiments, the donor template includes a priming site P1' in the target nucleic acid that is substantially similar to or identical to priming site P, and when the donor template is incorporated into the target nucleic acid, P1' is incorporated downstream of P1. In certain embodiments, the donor template includes a first priming site P1' and a second priming site P2'; P1' is substantially similar to or identical to the first priming site P1 in the target nucleic acid; P2' is substantially similar to or identical to the second priming site P2 in the target nucleic acid; and P1 and P2 are substantially different or not identical. In a particular embodiment, the donor template includes a first priming site P1' and a second priming site P2'; P1' is substantially similar to or identical to the first priming site P1 in the target nucleic acid; P2' is substantially similar to or identical to the second priming site P2 in the target nucleic acid; P2 is located downstream of P1 on the target nucleic acid; P1 and P2 are substantially dissimilar or dissimilar; when the donor template is incorporated into the target nucleic acid, P1' is incorporated downstream of P1; P2' is incorporated upstream of P2; and P2' is incorporated upstream of P1.

[0272] In certain embodiments, the target nucleic acid includes a first priming site (P1) and a second priming site (P2). The first priming site of the target nucleic acid may be located within a first homology arm. Alternatively, the first priming site of the target nucleic acid may be located at 5' and adjacent to the first homology arm. The second priming site of the target nucleic acid may be located within a second homology arm. Alternatively, the second priming site of the target nucleic acid may be located at 3' and adjacent to the second homology arm.

[0273] The donor template may include a cargo sequence, a first priming site (P1'), and a second priming site (P2'), where P2' is located at 5' of the cargo sequence and P1' is located at 3' of the cargo sequence (i.e., A1--P2'--N--P1'--A2), where P1' is substantially identical to P1 and P2' is substantially identical to P2. In this scenario, the targeted locus may be amplified using a primer pair containing oligonucleotides targeting P1' and P1, as well as oligonucleotides containing P2' and P2, thereby generating three amplicons of similar size, which can be sequenced to determine whether targeted incorporation has occurred. The first amplicon, amplicon X, arises from the amplification of the nucleic acid sequence between P1 and P2 as a result of untargeted incorporation in the target nucleic acid. A second amplicon, amplicon Y, arises from the amplification of the nucleic acid sequence between P1 and P2' after the target nucleic acid targeting integration event, thereby amplifying the 5' junction. A third amplicon, amplicon Z, arises from the amplification of the nucleic acid sequence between P1' and P2 after the target nucleic acid targeting integration event, thereby amplifying the 3' junction. In other embodiments, P1' may be identical to P1. Furthermore, P2' may be identical to P2.

[0274] In certain embodiments, the donor template includes a cargo and a priming site (P1'), where P1' is located at 3' of the cargo nucleic acid sequence (rnpA1--N--P1'--A2), and P1' is substantially identical to P1. In this scenario, the targeted locus may be amplified using a primer pair containing oligonucleotides targeting P1' and P1, and an oligonucleotide targeting P2, thereby generating two amplicons of similar size, which can be sequenced to determine whether targeted integration has occurred. The first amplicon, amplicon X, arises from amplification of the nucleic acid sequence between P1 and P2 as a result of untargeted integration in the target nucleic acid. The second amplicon, amplicon Z, arises from amplification of the nucleic acid sequence between P1' and P2 after the targeted integration event in the target nucleic acid, thereby amplifying the 3' junction. In other embodiments, P1' may be identical to P1. Furthermore, P2' may be identical to P2.

[0275] In certain embodiments, the target nucleic acid comprises a first priming site (P1) and a second priming site (P2), and the donor template comprises a priming site P2', where P2' is located at 5' of the cargo nucleic acid sequence (i.e., A1--P2'--N--A2), and P2' is substantially identical to P2. In this scenario, the targeted locus may be amplified using a primer pair containing oligonucleotides targeting P2' and P2, and an oligonucleotide targeting P1, thereby generating two amplicons of similar size, which can be sequenced to determine whether targeted integration has occurred. The first amplicon, amplicon X, arises from amplification of the nucleic acid sequence between P1 and P2 as a result of untargeted integration in the target nucleic acid. The second amplicon, amplicon Y, arises from amplification of the nucleic acid sequence between P1 and P2' after the targeted integration event in the target nucleic acid, thereby amplifying the 5' junction. In other embodiments, P1' may be identical to P1. Furthermore, P2' may be identical to P2.

[0276] The priming site of the donor template can be of any length that allows for quantitative evaluation of the gene editing event in the target nucleic acid by amplification and / or sequencing of a portion of the target nucleic acid. For example, in certain embodiments, the target nucleic acid includes a first priming site (P1), and the donor template includes a priming site (P1'). In these embodiments, the lengths of the P1' priming site and the P1 primer site are such that a single primer can specifically anneal to both priming sites (for example, in certain embodiments, the lengths of the P1' priming site and the P1 priming site are such that they both have the same or very similar GC content).

[0277] In certain embodiments, the priming site of the donor template is 60 nucleotides long. In certain embodiments, the priming site of the donor template is less than 60 nucleotides long. In certain embodiments, the priming site of the donor template is less than 50 nucleotides long. In certain embodiments, the priming site of the donor template is less than 40 nucleotides long. In certain embodiments, the priming site of the donor template is less than 30 nucleotides long. In certain embodiments, the priming site of the donor template is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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, or 60 nucleotides long. In certain embodiments, the priming region of the donor template is 60 base pairs long. In certain embodiments, the priming region of the donor template is less than 60 base pairs long. In certain embodiments, the priming region of the donor template is less than 50 base pairs long. In certain embodiments, the priming region of the donor template is less than 40 base pairs long. In certain embodiments, the priming region of the donor template is less than 30 base pairs long. In certain embodiments, the priming region of the donor template has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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, or 60 base pairs.

[0278] In certain embodiments, during cleavage separation events at the cleavage site of the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the first priming site (P1) of the target nucleic acid and the currently incorporated P2' priming site is 600 base pairs or less. In certain embodiments, during cleavage separation events at the cleavage site of the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the first priming site (P1) of the target nucleic acid and the currently incorporated P2' priming site is 550, 500, 450, 400, 350, 300, 250, 200, and 150 base pairs or less. In certain embodiments, during cleavage separation events in the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the first priming site (P1) of the target nucleic acid and the currently incorporated P2' priming site is 600 nucleotides or less. In certain embodiments, during cleavage and separation events in the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the first priming site (P1) of the target nucleic acid and the currently incorporated P2' priming site is 550, 500, 450, 400, 350, 300, 250, 200, and 150 nucleotides or less.

[0279] In certain embodiments, the target nucleic acid includes a second priming site (P2), and the donor template includes a priming site (P2') substantially identical to P2. In certain embodiments, during cleavage separation events in the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the second priming site (P2) of the target nucleic acid and the currently incorporated P1' priming site is 600 base pairs or less. In certain embodiments, during cleavage separation events in the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the second priming site (P2) of the target nucleic acid and the currently incorporated P1' priming site is 550, 500, 450, 400, 350, 300, 250, 200, and 150 base pairs or less. In certain embodiments, during cleavage separation events in the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the second priming site (P2) of the target nucleic acid and the currently incorporated P1' priming site is 600 nucleotides or less. In certain embodiments, during cleavage and separation events in the target nucleic acid and homologous recombination of the donor template by the target nucleic acid, the distance between the second priming site (P2) of the target nucleic acid and the currently incorporated P1' priming site is 550, 500, 450, 400, 350, 300, 250, 200, and 150 nucleotides or less.

[0280] In certain embodiments, the nucleic acid sequence of P2' is contained within the nucleic acid sequence of A1. In certain embodiments, the nucleic acid sequence of P2' is directly adjacent to the nucleic acid sequence of A1. In certain embodiments, the nucleic acid sequence of P2' is directly adjacent to the nucleic acid sequence of N. In certain embodiments, the nucleic acid sequence of P2' is contained within the nucleic acid sequence of N.

[0281] In certain embodiments, the nucleic acid sequence of P1' is contained within the nucleic acid sequence of A2. In certain embodiments, the nucleic acid sequence of P1' is directly adjacent to the nucleic acid sequence of A2. In certain embodiments, the nucleic acid sequence of P1' is directly adjacent to the nucleic acid sequence of N. In certain embodiments, the nucleic acid sequence of P1' is contained within the nucleic acid sequence of N.

[0282] In certain embodiments, the nucleic acid sequence P2' is contained within the nucleic acid sequence S1. In certain embodiments, the nucleic acid sequence P2' is directly adjacent to the nucleic acid sequence S1. In certain embodiments, the nucleic acid sequence P1' is contained within the nucleic acid sequence S2. In certain embodiments, the nucleic acid sequence P1' is directly adjacent to the nucleic acid sequence S2.

[0283] cargo The donor template for the gene editing system described herein includes a cargo (N). The cargo may be of any length necessary to achieve the desired result. For example, the cargo sequence may be less than 2500 base pairs or less than 2500 nucleotides in length. In other embodiments, the cargo sequence may be 12 kb or less. In other embodiments, the cargo sequence may be 10 kb or less. In other embodiments, the cargo sequence may be 7 kb or less. In other embodiments, the cargo sequence may be 5 kb or less. In other embodiments, the cargo sequence may be 4 kb or less. In other embodiments, the cargo sequence may be 3 kb or less. In other embodiments, the cargo sequence may be 2 kb or less. In other embodiments, the cargo sequence may be 1 kb or less. In a particular embodiment, the cargo may be about 5 to 10 kb in length. In another embodiment, the cargo may be about 1 to 5 kb in length. In another embodiment, the cargo may be about 0 to 1 kb in length. For example, in exemplary embodiments, the cargo may have a length of approximately 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs or nucleotides. In other exemplary embodiments, the cargo may have a length of approximately 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 base pairs or nucleotides. Those skilled in the art will readily see that when delivering donor templates using a size-limited delivery vehicle (e.g., viral delivery vehicles such as adeno-associated virus (AAV), adenovirus, lentivirus, embedded-deficient lentivirus (IDLV), or herpes simplex virus (HSV) delivery vehicles), the size of the donor template, including the cargo, must not exceed the size limits of the delivery system.

[0284] In certain embodiments, the cargo includes a substitution sequence. In certain embodiments, the cargo includes an exon of a gene sequence. In certain embodiments, the cargo includes an intron of a gene sequence. In certain embodiments, the cargo includes a cDNA sequence. In certain embodiments, the cargo includes a transcriptional regulatory element. In certain embodiments, the cargo includes the reverse complement of a substitution sequence, an exon of a gene sequence, an intron of a gene sequence, a cDNA sequence, or a transcriptional regulatory element. In certain embodiments, the cargo includes a portion of a substitution sequence, an exon of a gene sequence, an intron of a gene sequence, a cDNA sequence, or a transcriptional regulatory element. In certain embodiments, the cargo is a transgene sequence. In certain embodiments, the cargo introduces a deletion into a target nucleic acid. In certain embodiments, the cargo includes an exogenous sequence. In other embodiments, the cargo includes an endogenous sequence.

[0285] Substitution sequences in donor templates are described elsewhere, including by Cotta-Ramusino et al. Substitution sequences can be of any appropriate length (including none if the desired repair outcome is a deletion) and typically involve one, two, three, or more sequence modifications to the native intracellular sequence to which editing is desired. One common sequence modification involves altering the native sequence to repair mutations associated with the disease or condition to which treatment is desired. Another common sequence modification involves altering one or more sequences that are complementary to or encode the PAM sequence of an RNA-induced nuclease or the targeting domain of a gRNA used to generate SSBs or DSBs, thereby reducing or eliminating repeat breaks at the target site after the substitution sequence has been incorporated into the target site.

[0286] Based on the cell type to be edited, the target nucleic acid, and the effect to be achieved, a specific cargo may be selected for a given application.

[0287] For example, in certain embodiments, it may be desirable to "knock in" a desired gene sequence at a selected chromosomal locus within a target cell. In such cases, the cargo may include the desired gene sequence. In certain embodiments, the gene sequence encodes a desired protein, such as an exogenous protein, an orthologous protein, or an endogenous protein, or a combination thereof.

[0288] In certain embodiments, the cargo may contain a wild-type sequence or a sequence with one or more modifications to the wild-type sequence. For example, in some embodiments where it is desirable to correct mutations in a target gene within a cell, the cargo may be designed to restore the wild-type sequence to the target protein.

[0289] In other embodiments, it may be desirable to “knock out” a gene sequence at a selected chromosomal locus within the target cell. In such cases, the cargo may be designed to be incorporated into a site that interferes with the expression of the target gene sequence, such as the coding region of the target gene sequence or the expression regulatory region of the target gene sequence, such as the promoter or enhancer of the target gene sequence. In other embodiments, the cargo may be designed to disrupt the target gene sequence. For example, in certain embodiments, the cargo may introduce a deletion, insertion, stop codon, or frameshift mutation into the target nucleic acid.

[0290] In certain embodiments, the donor is designed to delete all or part of the target nucleic acid sequence. In certain embodiments, the homology arms of the donor may be designed to flank the desired deletion site. In certain embodiments, the donor does not contain a cargo sequence between the homology arms, and following targeted integration of the donor, results in a deletion of a portion of the target nucleic acid located between the homology arms. In other embodiments, the donor contains a cargo sequence homologous to the target nucleic acid, and one or more nucleotides of the target nucleic acid sequence are absent from the cargo. Following targeted integration of the donor, the target nucleic acid will contain a deletion at a residue absent from the cargo sequence. The size of the deletion may be selected based on the size of the target nucleic acid and the desired effect. In certain embodiments, the donor is designed to introduce a deletion of 1 to 2000 nucleotides into the target nucleic acid following targeted integration. In other embodiments, the donor is designed to introduce a deletion of 1 to 1000 nucleotides into the target nucleic acid following targeted integration. In other embodiments, the donor is designed to introduce a deletion of 1 to 500 nucleotides into the target nucleic acid following target integration. In other embodiments, the donor is designed to introduce a deletion of 1 to 100 nucleotides into the target nucleic acid following target integration. In exemplary embodiments, the donor is designed to introduce a deletion of approximately 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide into the target nucleic acid following target integration. In other embodiments, the donor is designed to introduce deletions exceeding 2,000 nucleotides from the target nucleic acid, such as deletions of approximately 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 nucleotides or more, following target integration.

[0291] In certain embodiments, the cargo may include a promoter sequence. In other embodiments, the cargo is designed to be incorporated into a site under the control of an endogenous promoter in a target cell.

[0292] In certain embodiments, a cargo encoding an exogenous or orthologous protein or polypeptide may be incorporated into the protein-coding chromosome sequence, such that the chromosome sequence is inactivated but the exogenous sequence is expressed. In other embodiments, the cargo sequence may be incorporated into the chromosome sequence without altering the expression of the chromosome sequence. This can be achieved by incorporating the cargo into a “safe harbor” locus, such as the Rosa26 locus, the HPRT locus, or the AAV locus.

[0293] In certain embodiments, the cargo encodes a protein associated with a disease or disorder. In certain embodiments, the cargo may encode the wild-type form of the protein, or is designed to restore the expression of the wild-type form of the protein if the protein is deficient in a subject suffering from a disease or disorder. In other embodiments, the cargo encodes a protein associated with a disease or disorder, and the protein encoded by the cargo includes at least one modification such that the modified version of the protein protects against the onset of the disease or disorder. In other embodiments, the cargo encodes a protein that includes at least one modification such that the modified version of the protein causes or enhances the disease or disorder.

[0294] In certain embodiments, cargo can be used to insert genes from one species into the genome of a different species. For example, “humanized” animal models and / or “humanized” animal cells can be generated through targeted incorporation of human genes into the genome of a non-human animal species, such as a mouse, rat, or non-human primate species. In certain embodiments, such humanized animal models and animal cells contain incorporated sequences encoding one or more human proteins.

[0295] In another embodiment, Cargo encodes proteins that benefit plant species, including crops such as grains, fruits, or vegetables. For example, Cargo may encode proteins that enable plants to be grown at higher temperatures, extending their shelf life after harvest or conferring disease resistance. In certain embodiments, Cargo may encode proteins that confer resistance to diseases and pests (see, for example, Jones et al. (1994) Science 266:789 (Cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089 (RSP2 gene for resistance to Pseudomonas syringae); see International Publication No. 96 / 30517 (Resistance to soybean cyst nematode)). In other embodiments, the cargo may encode a protein encoding resistance to herbicides, as described in its entirety in U.S. Patent Application Publication No. 2013 / 0326645A1, which is incorporated herein by reference.In another embodiment, the cargo encodes a protein that imparts value-added traits to plant cells, such as altered fatty acid metabolism, reduced phytate content, and changes in carbohydrate composition, which are brought about by transforming the plant with a gene encoding an enzyme that alters the branching pattern of starch, for example (e.g., Shiroza et al. (1988) J. Bacteol. 170:810 (nucleotide sequence of the fructosyltransferase gene of a Streptococcus mutant); Steinmetz et al. (1985) Mol. Gen. Genet. 20:220 (levansulacase gene); Pen et al. (1992) Bio / Technology 10:292 (α-amylase); Elliot et al. (1993) Plant Molec. Biol. 21:515 (nucleotide sequence of the tomato invertase gene); Sogaard et al. See al. (1993) J. Biol. Chem. 268:22480 (Barley α-amylase gene); and Fisher et al. (1993) Plant Physiol. 102:1045 (Maize endosperm starch branching enzyme II). Other exemplary cargo useful for targeted incorporation in plant cells are described in their entirety in U.S. Patent Application Publication No. 2013 / 0326645A1, which is incorporated herein by reference.

[0296] Additional cargo can be selected by those skilled in the art for a given application, based on the cell type to be edited, the target nucleic acid, and the effect to be achieved.

[0297] Staffer In certain embodiments, the donor template may optionally include one or more stuffer sequences. Generally, the stuffer sequences are heterogeneous or random nucleic acid sequences that (a) promote (or do not inhibit) targeted incorporation of the donor template to a target site and subsequent amplification of the amplicon containing the stuffer sequence by a particular method of the disclosure, but (b) avoid promoting incorporation to another site of the donor template. The stuffer sequences may be positioned, for example, between homology arm A1 and primer site P2' to regulate the size of the amplicon produced when the donor template sequence is incorporated to a target site. Using such size regulation, for example, equilibrium is maintained in the size of the amplicons produced by the incorporated target and the unincorporated target sites, and as a result equilibrium in the efficiency of each amplicon being produced in a single PCR reaction; this may then facilitate quantitative evaluation of targeted incorporation based on the relative abundance of the two amplicons in the reaction mixture.

[0298] To facilitate targeted integration and amplification, stuffer sequences may be selected to minimize the formation of secondary structures that could prevent separation of the cleavage site or amplification by DNA repair mechanisms (e.g., via homologous recombination). In certain embodiments, the donor template includes A1--S1--P2'--N--A2 or A1--N--P1'--S2--A2 in the 5' to 3' direction; Here, S1 is the first stuffer sequence, and S2 is the second stuffer sequence.

[0299] In a particular embodiment, the donor mold includes A1--S1--P2'--N--P1'--S2--A2 in the 5' to 3' direction, Here, S1 is the first stuffer sequence, and S2 is the second stuffer sequence.

[0300] In certain embodiments, the stuffer sequence contains approximately the same guanine-cytosine content ("GC content") as the entire cell genome. In certain embodiments, the stuffer sequence contains approximately the same GC content as the targeted locus. For example, if the target cell is a human cell, the stuffer sequence contains approximately 40% GC content. In certain embodiments, the stuffer sequence may be designed by creating a random nucleic acid sequence containing a desired GC content. For example, to produce a stuffer sequence containing 40% GC content, a nucleic acid sequence having the following nucleotide distribution may be designed: A=30%, T=30%, G=20%, C=20%. Methods for determining the GC content of a genome or a target locus are known to those skilled in the art. Therefore, in certain embodiments, the stuffer sequence may contain GC content of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. Exemplary 2.0 kilobase stuffer sequences having a GC content of 40 ± 5% are provided herein as SEQ ID NOs. 23 to 123.

[0301] Specifically, the first stuffer contains at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, and at least 15 of the sequences described in sequence numbers 23 to 123. The sequence has at least 0, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, or at least 500 polypeptides.In another embodiment, the second stuffer is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, and at least 15 of the sequences listed in sequence numbers 23-123. The sequence has at least 0, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, or at least 500 polypeptides.

[0302] The stuffer sequence preferably does not interfere with the separation of the cleavage site in the target nucleic acid. Therefore, the stuffer sequence should have the minimum sequence identity with the nucleic acid sequence at the cleavage site of the target nucleic acid. In certain embodiments, the stuffer sequence is identical to any nucleic acid sequence within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 nucleotides from the cleavage site of the target nucleic acid by 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10%. In certain embodiments, the stuffer sequence is identical to any nucleic acid sequence within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 base pairs from the cleavage site of the target nucleic acid by 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10%.

[0303] To avoid off-target molecular recombination events, the stuffer sequence preferably has minimal homology to nucleic acid sequences in the target cell genome. In certain embodiments, the stuffer sequence has minimal sequence identity to nucleic acids in the target cell genome. In certain embodiments, the stuffer sequence is 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10% identical to any nucleic acid sequence of the same length (measured in base pairs or nucleotides) in the target cell genome. In certain embodiments, a 20-base-pair stretch of the stuffer sequence is 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10% identical to any stretch of at least 20 base pairs of nucleic acids in the target cell genome. In certain embodiments, a 20-nucleotide stretch of the stuffer sequence is identical to any stretch of at least 20 nucleotides of the nucleic acid of the target cell genome by 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10%.

[0304] In certain embodiments, the stuffer sequence has minimal sequence identity with the nucleic acid sequence in the donor template (e.g., the nucleic acid sequence of the cargo or the nucleic acid sequence of the priming site present in the donor template). In certain embodiments, the stuffer sequence is identical to any nucleic acid sequence of the same length (measured by base pairs or nucleotides) in the donor template by 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10%. In certain embodiments, a 20-base-pair stretch of the stuffer sequence is identical to any 20-base-pair stretch of the nucleic acid in the donor template by 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10%. In certain embodiments, a 20-nucleotide stretch of the stuffer sequence is identical to any 20-nucleotide stretch of the donor template nucleic acid by 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or less than 10%.

[0305] In certain embodiments, the length of the first homology arm and its adjacent stuffer sequence (i.e., A1+S1) is approximately equal to the length of the second homology arm and its adjacent stuffer sequence (i.e., A2+S2). For example, in certain embodiments, the length of A1+S1 is the same as the length of A2+S2 (determined by base pairs or nucleotides). In certain embodiments, the length of A1+S1 differs from the length of A2+S2 by only 25 nucleotides or less. In certain embodiments, the length of A1+S1 differs from the length of A2+S2 by only 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides or less. In certain embodiments, the length of A1+S1 differs from the length of A2+S2 by only 25 base pairs or less. In certain embodiments, the length of A1+S1 differs from the length of A2+S2 by only 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2 base pairs.

[0306] In certain embodiments, the length of A1+H1 is 250 base pairs or less. In certain embodiments, the length of A1+H1 is 200 base pairs or less. In certain embodiments, the length of A1+H1 is 150 base pairs or less. In certain embodiments, the length of A1+H1 is 100 base pairs or less. In certain embodiments, the length of A1+H1 is 50 base pairs or less. In certain embodiments, the length of A1+H1 is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs. In certain embodiments, the length of A1+H1 is 40 base pairs. In certain embodiments, the length of A2+H2 is 250 base pairs or less. In certain embodiments, the length of A2+H2 is 200 base pairs or less. In certain embodiments, the length of A2+H2 is 150 base pairs or less. In certain embodiments, the length of A2+H2 is 100 base pairs or less. In certain embodiments, the length of A2+H2 is 50 base pairs or less. In certain embodiments, the length of A2+H2 is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs. In certain embodiments, the length of A2+H2 is 40 base pairs.

[0307] In certain embodiments, the length of A1+S1 is the same as the length of H1+X+H2 (determined by nucleotides or base pairs). In certain embodiments, the length of A1+S1 differs from the length of H1+X+H2 by less than 25 nucleotides. In certain embodiments, the length of A1+S1 differs from the length of H1+X+H2 by only 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. In certain embodiments, the length of A1+S1 differs from the length of H1+X+H2 by less than 25 base pairs. In certain embodiments, the length of A1+S1 differs from the length of H1+X+H2 by only 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 base pairs.

[0308] In certain embodiments, the length of A2+S2 is the same as the length of H1+X+H2 (determined by nucleotides or base pairs). In certain embodiments, the length of A2+S2 differs from the length of H1+X+H2 by less than 25 nucleotides. In certain embodiments, the length of A2+S2 differs from the length of H1+X+H2 by only 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. In certain embodiments, the length of A2+S2 differs from the length of H1+X+H2 by less than 25 base pairs. In certain embodiments, the length of A2+S2 differs from the length of H1+X+H2 by only 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 base pairs.

[0309] target cell Using the genome editing systems described herein, cells may be manipulated or modified, for example, by editing or modifying a target nucleic acid. The manipulation may be performed in vivo or ex vivo in various embodiments.

[0310] According to embodiments of the present disclosure, various cell types can be manipulated or modified. In the case of in vivo applications, for example, by delivering a genome editing system according to the present disclosure to a plurality of cell types, the plurality of cell types can be modified or manipulated. However, in other cases, it may be desirable to limit the manipulation or modification to a specific cell type. For example, in some instances, it may be desirable to edit cells with limited differentiation potential or terminally differentiated cells, such as photoreceptor cells in the Maeder example, where modification of the genotype is expected to result in a change in the cell phenotype. However, in other cases, it may be desirable to edit undifferentiated, multipotent or pluripotent, stem or progenitor cells. By way of example, the cells can be embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs) or other stem or progenitor cell types that differentiate into cell types relevant to a given application or indication.

[0311] In certain embodiments, the cells being manipulated are eukaryotic cells. For example, without limitation, the cells can be vertebrate, mammalian, rodent, goat, pig, avian, chicken, quail, bovine, equine, sheep, fish, primate or human cells. In certain embodiments, the cells being manipulated are somatic cells, germ cells or prenatal cells. In certain embodiments, the cells being manipulated are zygotic somatic cells, blastocyst cells, embryonic germ cells, stem cells, mitotically competent cells or meiotically competent cells. In certain embodiments, the cells being manipulated are not part of a human embryo. In certain embodiments, the cells being manipulated are T cells, CD8 + T cells, CD8 + naïve T cells, CD4 + central memory T cells, CD8 + central memory T cells, CD4 + effector memory T cells, CD4 + effector memory T cells, CD4 + T cells, CD4 + stem cell memory T cells, CD8 + stem cell memory T cells, CD4 + helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, CD4+ naïve T cells, TH17 CD4 +T cells, TH1 CD4 + T cells, TH2 CD4 + T cells, TH9 CD4 + T cells, CD4 + Foxp3 + T cells, CD4 + CD25 + CD127 - T cells, CD4 + CD25 + CD127 - Foxp3 + It is a T cell. In certain embodiments, the cells to be engineered are long-term hematopoietic stem cells, short-term hematopoietic stem cells, multipotent progenitor cells, lineage-restricted progenitor cells, lymphoid progenitor cells, myeloid progenitor cells, common myeloid progenitor cells, erythroid progenitor cells, megakaryocyte-erythroid progenitor cells, retinal cells, photoreceptor cells, rod cells, cone cells, retinal pigment epithelial cells, bipolar cells, cochlear hair cells, outer hair cells, inner hair cells, lung epithelial cells, bronchial epithelial cells, alveolar epithelial cells, lung epithelial progenitor cells, skeletal muscle cells, cardiomyocytes, muscle satellite cells, neurons, neural stem cells, mesenchymal stem cells, induced pluripotent stem (iPS) cells, embryonic stem cells, monocytes, megakaryocytes, neutrophils, eosinophils, basophils, mast cells, reticulocytes, B cells (e.g., progenitor B cells, pre-B cells, pro-B cells, memory B cells, plasma B cells, etc.), gastrointestinal epithelial cells, bile duct epithelial cells, pancreatic duct epithelial cells, intestinal stem cells, hepatocytes, hepatic stellate cells, Kupffer cells, osteoblasts, osteoclasts, adipocytes, preadipocytes, pancreatic islet cells (e.g., beta cells, alpha cells, delta cells), pancreatic exocrine cells, Schwann cells or oligodendrocytes. In certain embodiments, the cells to be engineered are plant cells, such as, for example, monocotyledonous or dicotyledonous plant cells.

[0312] In certain embodiments, the target cells are circulating blood cells such as reticulocytes, megakaryocyte progenitor cells (MEP) cells, myeloid progenitor cells (CMP / GMP), lymphoid progenitor cells (LP) cells, hematopoietic stem / progenitor cells (HSC) or endothelial cells (EC). In certain embodiments, the target cells are myeloid cells (e.g., reticulocytes, erythroid cells (e.g., erythroblasts), MEP cells, myeloid progenitor cells (CMP / GMP), LP cells, erythrocyte progenitor (EP) cells, HSCs, pluripotent progenitor (MPP) cells, endothelial cells (EC), hematopoietic endothelial (HE) cells or mesenchymal stem cells). In certain embodiments, the target cells are myeloid progenitor cells (e.g., common myeloid progenitor (CMP) cells or granulocyte-macrophage progenitor (GMP) cells). In certain embodiments, the target cells are lymphoid progenitor cells, such as common lymphoid progenitor (CLP) cells. In certain embodiments, the target cells are erythrocyte progenitor cells (e.g., MEP cells). In certain embodiments, the target cells are hematopoietic stem / progenitor cells (e.g., long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), MPP cells, or lineage-limiting progenitor (LRP) cells). In certain embodiments, the target cells are CD34 + cells, CD34 + CD90 + cells, CD34 + CD38 - cells, CD34 + CD90 + CD49f + CD38 - CD45RA - cells, CD105 + cells, CD31 + Alternatively, CD133 + Cells or CD34 + CD90 + CD133 + These are cells. In certain embodiments, the target cells are umbilical cord blood CD34 + HSPC, umbilical vein endothelial cells, umbilical artery endothelial cells, amniotic fluid CD34 + Cells, amniotic endothelial cells, placental endothelial cells, or placental hematopoietic CD34 + These are cells. In certain embodiments, the target cells are mobilized peripheral blood hematopoietic CD34 cells. +These are cells (after the patient has been treated with a mobilizing agent such as G-CSF or plerixafor). In certain embodiments, the target cells are peripheral blood endothelial cells.

[0313] Naturally, the cells being modified or manipulated can be dividing or non-dividing cells, depending on the target cell type and / or the desired editing result.

[0314] When cells are manipulated or modified ex vivo, they can be used immediately (e.g., administered to a control) or they can be maintained or stored for later use. Those skilled in the art will understand that cells can be maintained or stored in a culture (e.g., frozen in liquid nitrogen) using any suitable method known in the art.

[0315] Implementation of genome editing systems: delivery, formulation, and administration routes. As discussed above, the genome editing systems of this disclosure may be implemented in any suitable manner, meaning that the components of the system, including, but not limited to, RNA-induced nucleases, gRNAs, and any donor template nucleic acids, may be delivered, formulated, or administered in any suitable form or combination of forms that results in the transduction, expression, or introduction of the genome editing system and / or cause the desired repair outcome in a cell, tissue, or subject. Several non-limiting examples of genome editing system implementations are given in Tables 10 and 11. However, those skilled in the art will understand that these lists are not exhaustive and that other implementations are possible. Referring in particular to Table 10, this table lists several exemplary implementations of genome editing systems that include a single gRNA and any donor template. However, the genome editing systems of this disclosure may incorporate other components such as multiple gRNAs, multiple RNA-induced nucleases, and proteins, and those skilled in the art will see various implementations based on the principles shown in the table. In the table, [N / A] indicates that the genome editing system does not include the components shown.

[0316] TIFF2026048661000030.tif237170

[0317] Table 11 summarizes various delivery methods for components of genome editing systems as described herein. Again, the list is intended to be illustrative rather than restrictive.

[0318] TIFF2026048661000031.tif242170

[0319] Nucleic acid-based delivery of genome editing systems Nucleic acids encoding various elements of the genome editing systems described herein may be administered to a subject or delivered intracellularly by methods known in the art or as described herein. For example, DNA encoding RNA-induced nucleases and / or gRNAs, as well as donor template nucleic acids, may be delivered, for example, by vectors (e.g., viral vectors or non-viral vectors), non-vector-based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.

[0320] Nucleic acids or their components that encode genome editing systems can be delivered directly to cells as naked DNA or RNA, for example, by translocation or electroporation, or they can be conjugated to molecules (e.g., N-acetylgalactosamine) that promote uptake by target cells (e.g., red blood cells, HSCs). Nucleic acid vectors, such as those summarized in Table 11, may also be used.

[0321] A nucleic acid vector may contain one or more sequences encoding components of a genome editing system, such as RNA-induced nucleases, gRNAs, and / or donor templates. The vector may also contain sequences encoding signal peptides (e.g., for nuclear, nucleolar, or mitochondrial localization) that bind to (e.g., are inserted into or fused to) protein-coding sequences. As an example, a nucleic acid vector may contain a Cpf1 coding sequence containing one or more nuclear localization sequences (e.g., nuclear localization sequences derived from SV40).

[0322] Nucleic acid vectors may also include any number of appropriate regulatory elements, such as promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRESs). These elements are well known in the art and are described by Cotta-Ramusino et al.

[0323] The nucleic acid vectors described herein include recombinant viral vectors. Exemplary viral vectors are listed in Table 11, and additional suitable viral vectors and their uses and manufactures are described by Cotta-Ramusino et al. Other viral vectors known in the art may also be used. Furthermore, viral particles may be used to deliver components of genome editing systems in the form of nucleic acids and / or peptides. For example, “empty” viral particles may be assembled to contain any suitable cargo. Viral vectors and viral particles may also be manipulated to incorporate targeted ligands to modify their target tissue specificity.

[0324] In addition to viral vectors, non-viral vectors may be used to deliver nucleic acids encoding genome editing systems according to this disclosure. One important category of non-viral nucleic acid vectors is nanoparticles, which may be organic or inorganic. Nanoparticles are well known in the art and summarized by Cotta-Ramusino et al. Using any suitable nanoparticle design, genome editing system components or nucleic acids encoding such components can be delivered. For example, organic (e.g., lipids and / or polymers) nanoparticles may be suitable for use as delivery vehicles in certain implementations of this disclosure. Exemplary lipids for use in nanoparticle formulations and / or gene transfer are shown in Table 12, and exemplary polymers for use in gene transfer and / or nanoparticle formulations are listed in Table 13.

[0325] TIFF2026048661000032.tif138170

[0326] TIFF2026048661000033.tif214170

[0327] TIFF2026048661000034.tif250170

[0328] Nonviral vectors optionally contain targeted modifications to improve uptake and / or selectively target specific cell types. These targeted modifications include, for example, cell-specific antigens, monoclonal antibodies, single-chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell-permeable peptides. Such vectors also optionally utilize fusion-inducible and endosomal-destabilizing peptides / polymers, incorporating polymers that undergo acid-induced conformational changes (e.g., to facilitate endosomal leakage of cargo) and / or polymers that are stimulably cleavable for release into cellular compartments. For example, disulfide-based cationic polymers cleaved in a reducing cellular environment may be used.

[0329] In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of the genome editing system, such as the RNA-induced nuclease components and / or gRNA components described herein, are delivered. In certain embodiments, the nucleic acid molecules are delivered simultaneously with one or more components of the genome editing system. In certain embodiments, the nucleic acid molecules are delivered before or after one or more components of the genome editing system are delivered (e.g., about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or less than 4 weeks). In certain embodiments, the nucleic acid molecules are delivered by means different from those used to deliver one or more components of the genome editing system, such as the RNA-induced nuclease components and / or gRNA components. The nucleic acid molecules may be delivered by any of the delivery methods described herein. For example, nucleic acid molecules may be delivered by viral vectors, such as embedded deficient lentiviruses, so that the toxicity caused by nucleic acids (e.g., DNA) can be reduced, and RNA-induced nuclease molecular components and / or gRNA components may be delivered by electroporation. In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, such as the proteins described herein. In certain embodiments, the nucleic acid molecule encodes an RNA molecule, such as the RNA molecules described herein.

[0330] Delivery of genome editing system components encoding RNP and / or RNA RNAs encoding RNPs (complexes of gRNA and RNA-inducing nucleases) and / or RNA-inducing nucleases and / or gRNAs may be delivered to cells or administered to subjects by methods known in the art, some of which are described by Cotta-Ramusino et al. In vitro, RNAs encoding RNA-inducing nucleases and / or gRNAs may be delivered, for example, by microinjection, electroporation, transient cell compression, or squeezing (see, e.g., Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc or other conjugate-mediated delivery, and combinations thereof may also be used for in vitro and in vivo delivery.

[0331] In vitro, delivery by electroporation comprises the steps of mixing cells with RNA encoding an RNA-inducing nuclease and / or gRNA in the presence or absence of a donor template nucleic acid molecule within a cartridge, chamber, or cuvette, and applying one or more electrical impulses of a specified length and amplitude. Electroporation systems and protocols are known in the art, and any suitable electroporation tool and / or protocol may be used in relation to various embodiments of this disclosure. Exemplary systems include, but are not limited to, Nucleofector® technologies (Lonza), Gene Pulser Xcell® (BioRad), Flow Electroporation® transfusion system (MaxCyte), and Neon® transfusion system (ThermoFisher).

[0332] Route of administration Genome editing systems or cells modified or manipulated using such systems may be administered to a target by any appropriate mode or route, whether local or systemic. Systemic administration methods include oral and parenteral routes. Parenteral routes include, for example, intravenous, intramedullary, intraarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. Components administered systemically may be modified or formulated to target, for example, HSCs, hematopoietic stem / progenitor cells, or erythrocyte precursors or progenitor cells.

[0333] Local administration methods include, for example, intramedullary injection into the trabeculae or intrafemoral injection into the bone marrow cavity, and infusion into the portal vein. In certain embodiments, significantly smaller amounts of components may be effective when administered locally (e.g., directly into the bone marrow) compared to systemic administration (e.g., intravenously). Local administration methods can reduce or eliminate the occurrence of potentially toxic side effects that may occur when a therapeutically effective dose of the components is administered systemically.

[0334] The drug may be administered as a periodic bolus (e.g., intravenously) or as a continuous infusion from an internal or external reservoir (e.g., from an intravenous bag or implantable pump). The components may be administered locally, for example, by continuous release from a sustained-release drug delivery device.

[0335] In addition, the components may be formulated to be released over a long period of time. The release system may include a matrix of biodegradable materials or materials that release components incorporated by diffusion. The components may be distributed uniformly or non-uniformly within the release system. A variety of release systems may be useful, but the selection of an appropriate system depends on the release rate required by the specific application. Both non-degradable and degradable release systems may be used. Suitable release systems include, but are not limited to, polymers and polymer matrices, non-polymer matrices, or calcium carbonate and sugars (e.g., trehalose), as well as inorganic and organic excipients and diluents. The release system may be natural or synthetic. However, synthetic release systems are generally preferred because they are usually more reliable, more reproducible, and produce a more defined release profile. The release system material may be selected so that components with different molecular weights are released by diffusion, either through the material or by the decomposition of the material.

[0336] Typical synthetic biodegradable polymers include, for example, polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic acid-coglycolic acid) and poly(caprolactone); poly(anhydride); polyorthoesters; polycarbonates; and their chemical derivatives (e.g., substitution and addition of chemical groups such as alkyl and alkylene, hydroxylation, oxidation, and other modifications routinely performed by those skilled in the art), copolymers, and mixtures thereof. Typical synthetic non-degradable polymers include, for example, polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acid, and poly(vinyl alcohol), poly(vinylpyrrolidone), and poly(vinyl acetate); poly(urethane); cellulose and its derivatives such as alkyl, hydroxyalkyl, ether, ester, nitrocellulose, and various cellulose acetates; polysiloxanes; any chemical derivatives thereof (e.g., substitution, addition, hydroxylation, oxidation, and other modifications customarily made by those skilled in the art, such as alkyl and alkylene), copolymers, and mixtures thereof.

[0337] Poly(lactide-co-glycolide) microspheres may also be used. Typically, the microspheres consist of lactic acid and glycolic acid polymers structured to form hollow spheres. The spheres may have a diameter of approximately 15 to 30 microns and may be loaded with the components described herein.

[0338] Multimodality or differential delivery of components Those skilled in the art will understand, in view of this disclosure, that the different components of the genome editing systems disclosed herein may be delivered together or separately, and simultaneously or asynchronously. Separate and / or asynchronous delivery of genome editing system components may be particularly desirable to provide temporal or spatial control over the function of the genome editing system and to limit the specific effects caused by their activity.

[0339] Different or differential modes, as used herein, refer to delivery modes that impart different pharmacodynamic or pharmacokinetic properties to a target component molecule, such as an RNA-induced nuclease molecule, gRNA, template nucleic acid, or payload. For example, a delivery mode may result in different tissue distribution, different half-lives, or different temporal distribution in a selected compartment, tissue, or organ.

[0340] For example, some delivery methods, such as delivery by nucleic acid vectors that remain within cells or cell progeny through autonomous replication or insertion into cellular nucleic acids, result in more persistent expression and presence of components. Examples include viral delivery, such as AAV or lentiviral delivery.

[0341] For example, components of genome editing systems, such as RNA-induced nucleases and gRNAs, may be delivered in different ways with respect to the resulting half-life or persistence of the components delivered to the body, a specific compartment, tissue, or organ. In certain embodiments, gRNAs may be delivered in such a manner. RNA-induced nuclease molecular components may be delivered in a manner that results in lower persistence or less exposure to the body, a specific compartment, tissue, or organ.

[0342] More generally, in certain embodiments, a first delivery mode is used to deliver a first component, and a second delivery mode is used to deliver a second component. The first delivery mode imparts a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property may be, for example, the distribution, persistence, or exposure of the component or the nucleic acid encoding the component within the body, compartment, tissue, or organ. The second delivery mode imparts a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property may be, for example, the distribution, persistence, or exposure of the component or the nucleic acid encoding the component within the body, compartment, tissue, or organ.

[0343] In certain embodiments, the first pharmacodynamic or pharmacokinetic properties, such as distribution, persistence, or exposure, are more limited than the second pharmacodynamic or pharmacokinetic properties.

[0344] In certain embodiments, the first delivery mode is selected to optimize, for example, minimize pharmacodynamic or pharmacokinetic properties such as distribution, duration, or exposure.

[0345] In certain embodiments, the second delivery mode is selected to optimize, for example, maximize pharmacodynamic or pharmacokinetic properties such as distribution, duration, or exposure.

[0346] In certain embodiments, the first delivery mode involves the use of relatively persistent elements, such as nucleic acids, plasmids, or viral vectors, such as AAV or lentiviruses. Because such vectors are relatively persistent, the products transcribed from them are also relatively persistent.

[0347] In certain embodiments, the second delivery mode includes, for example, a relatively transient element such as RNA or a protein.

[0348] In certain embodiments, the first component comprises gRNA, and the delivery mode is relatively persistent; for example, the gRNA is transcribed from a plasmid or viral vector such as AAV or lentivirus. Since these genes do not encode protein products and gRNA cannot act alone, the transcription of these genes is not physiologically important. The second component, an RNA-inducible nuclease molecule, is delivered transiently, for example, as mRNA or as a protein, ensuring that only the complete RNA-inducible nuclease molecule / gRNA complex is present and is short-lived in activity.

[0349] Furthermore, the components may be delivered in different molecular forms or different delivery vectors that complement each other to enhance safety and tissue specificity.

[0350] The use of differential delivery modes can enhance performance, safety, and / or efficacy, for example, by reducing the possibility of final off-target modifications. Since peptides from bacterial Cas enzymes are presented on the cell surface by MHC molecules, delivery of immunogenic components such as the Cas9 molecule via a less persistent mode may reduce immunogenicity. Two-part delivery systems can mitigate these drawbacks.

[0351] Using differential delivery modes, components can be delivered to different but overlapping target regions. The formed active complex is minimized outside the overlap of the target regions. Thus, in certain embodiments, a first component, such as gRNA, is delivered by a first delivery mode, resulting in a first spatial distribution, such as tissue distribution. A second component, such as an RNA-induced nuclease molecule, is delivered by a second delivery mode, resulting in a second spatial distribution, such as tissue distribution. In certain embodiments, the first mode includes a first element selected from liposomes, nanoparticles such as polymer nanoparticles, and nucleic acids such as viral vectors. The second mode includes a second element selected from the group. In certain embodiments, the first delivery mode includes a first targeting element, such as a cell-specific receptor or antibody, and the second delivery mode does not include that element. In certain embodiments, the second delivery mode includes a second targeting element, such as a second cell-specific receptor or a second antibody.

[0352] When RNA-induced nuclease molecules are delivered in viral delivery vectors, liposomes, or polymer nanoparticles, targeting only a single tissue may be desirable, but there is potential for delivery to multiple tissues and therapeutic activity within them. Two-part delivery systems can solve this problem and enhance tissue specificity. If the gRNA and RNA-induced nuclease molecules are packaged in separate delivery vehicles with distinct but overlapping tissue tropisms, a fully functional complex will be formed only within the tissues targeted by both vectors.

[0353] Exemplary, non-limiting embodiments A. In certain non-limiting embodiments, the subject matter disclosed herein provides isolated CRISPRs from Prevotella and Francisella 1 (Cpf1) RNA-induced nucleases, including nuclear localization signals (NLS).

[0354] A1. Cpf1 RNA-induced nuclease is the Cpf1 RNA-induced nuclease described above as A, which contains an NLS at or near the N-terminus of the nuclease.

[0355] A2. Cpf1 RNA-induced nucleases are the Cpf1 RNA-induced nucleases described above as A, which contain an NLS at or near the C-terminus of the nuclease.

[0356] A3. Cpf1 RNA-induced nuclease is the Cpf1 RNA-induced nuclease described in A1 above, which contains two NLS sequences at or near the N-terminus of the nuclease.

[0357] A4. Cpf1 RNA-induced nuclease is the Cpf1 RNA-induced nuclease described in A2 above, which contains two NLS sequences at or near the C-terminus of the nuclease.

[0358] A5. Cpf1 RNA-induced nucleases are Cpf1 RNA-induced nucleases of type A described above, which contain NLS at both the N-terminus and C-terminus or in the vicinity thereof.

[0359] A6. If a Cpf1 RNA-induced nuclease contains two or more NLS sequences, the NLS sequences are either identical or different, as described in A above.

[0360] A7. The NLS sequence or sequence group is selected from the group consisting of nucleoplasmin NLS (nNLS) (SEQ ID NO: 1) and Simianvirus 40 "SV40" NLS (sNLS) (SEQ ID NO: 2), and is the Cpf1 RNA-inducing nuclease of A mentioned above.

[0361] A8. The sequence of the Cpf1 RNA-inducing nuclease is selected from the group consisting of NLS (SEQ ID NO: 3); His-AsCpf1-sNLS (SEQ ID NO: 4); His-AsCpf1-sNLS-sNLS (SEQ ID NO: 5); His-sNLS-AsCpf1 (SEQ ID NO: 6); His-sNLS-sNLS-AsCpf1 (SEQ ID NO: 7); sNLS-sNLS-AsCpf1 (SEQ ID NO: 8); His-sNLS-AsCpf1-sNLS (SEQ ID NO: 9); and His-sNLS-sNLS-AsCpf1-sNLS-sNLS (SEQ ID NO: 10), and is the Cpf1 RNA-inducing nuclease of A mentioned above.

[0362] B. In certain non-limiting embodiments, the subject matter disclosed herein provides isolated Cpf1 RNA-induced nucleases comprising cysteine ​​amino acid deletions or substitutions.

[0363] B1.Cpf1 RNA-inducible nuclease is the Cpf1 RNA-inducible nuclease of B described above, which contains a deletion or substitution at C65, C205, C334, C379, C608, C674, C1025, or C1248 of the wild-type AsCpf1 amino acid sequence.

[0364] B2.Cpf1 RNA-inducible nuclease is the Cpf1 RNA-inducible nuclease of B1 described above, which includes substitutions selected from the group consisting of C65S / A, C205S / A, C334S / A, C379S / A, C608S / A, C674S / A, and C1025S / A, compared to the wild-type AsCpf1 amino acid sequence.

[0365] B3.Cpf1 RNA-inducible nuclease is the Cpf1 RNA-inducible nuclease of B1 described above, which contains a deletion or substitution at C334 and C674 or at any of C334, C379 and C674 in the wild-type AsCpf1 amino acid sequence.

[0366] B4. The Cpf1 RNA-inducible nuclease described above is the Cpf1 RNA-inducible nuclease of B3, which, compared to the wild-type AsCpf1 amino acid sequence, contains substitutions selected from the group consisting of (1) C334S / A and C674S / A; and (2) C334S / A, C379S / A and C674S / A.

[0367] B5.Cpf1 RNA-induced nuclease is the Cpf1 RNA-induced nuclease of B mentioned above, further containing NLS.

[0368] The sequence of B6.Cpf1 RNA-inducing nuclease is selected from His-AsCpf1-nNLSCys-less (SEQ ID NO: 11) and His-AsCpf1-nNLSCys-low (SEQ ID NO: 12), and is the same as the Cpf1 RNA-inducing nuclease B5 mentioned above.

[0369] C. In certain embodiments, the subject matter disclosed herein provides isolated nucleic acids encoding any of the aforementioned Cpf1 RNA-inducible nucleases A-A8 and B-B8.

[0370] D. In certain embodiments, the subject matter disclosed herein is Guide RNA (gRNA); and One of the aforementioned Cpf1 RNA-inducible nucleases A-A8 and B-B8, or a Cpf1 RNA-inducible nuclease encoded by the nucleic acid of C mentioned above. We provide genome editing systems that include [specific features / features].

[0371] E. In certain embodiments, the subject disclosed herein is a method for modifying a target sequence of interest within a cell, wherein the cell gRNA complementary to the target sequence of interest; and One of the aforementioned Cpf1 RNA-inducible nucleases A-A8 and B-B8, or a Cpf1 RNA-inducible nuclease encoded by the nucleic acid of C mentioned above. The present invention provides a method for modifying a target sequence of interest, comprising contacting the Cpf1 RNA-induced nuclease with the Cpf1 RNA-induced nuclease.

[0372] E1. The cells are T cells, hematopoietic stem cells (HSCs), or human umbilical cord blood-induced erythrocyte progenitor cells (HUDEP cells), as described in method E above.

[0373] E2.HSC is a CD34+ cell, CD34+CD90+ cell, CD34+CD38- cell, CD34+CD90+CD49f+CD38-CD45RA- cell, CD105+ cell, CD31+ or CD133+ cell, or CD34+CD90+CD133+ cell, as described in E1 above.

[0374] E3 T cells are CD8 + T cells, CD8 + Naive T cells, CD4 + central memory T cells, CD8 + central memory T cells, CD4 + Effector memory T cells, CD4 + Effector memory T cells, CD4 + T cells, CD4 + Stem cell memory T cell, CD8 + Stem cell memory T cell, CD4 + Helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, CD4+ naive T cells, TH17CD4 + T cells, TH1CD4 + T cells, TH2CD4 + T cells, TH9CD4 + T cells, CD4 + Foxp3 + T cells, CD4 + CD25 + CD127 - T cells or CD4 + CD25 + CD127 - Foxp3 + The method described above for T cells, E1.

[0375] E4. The Cpf1 RNA-induced nuclease modifies the target sequence of interest to achieve at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% editing, as described in method E above.

[0376] E5. The method of E described above, further including a second gRNA complementary to the second target sequence of interest.

[0377] E6. The method of E described above, further comprising a second RNA-inducible nuclease.

[0378] E7. The target sequence of interest is selected from the group consisting of a portion of the HBG1 gene sequence and a portion of the BCL11a gene sequence, by the method of E described above.

[0379] E8. Part of the HBG1 gene sequence is the -110nt promoter region of the HBG gene, as described in E7 above.

[0380] E9. A portion of the HBG1 gene sequence is the CAAT box in the -110nt promoter region of the HBG gene, as described in E8 above.

[0381] E10. A portion of the Bcl11a gene sequence is the +58DHS region of intron 2 of the BCL11a gene, as described in E7 above.

[0382] E11. A portion of the Bcl11a gene sequence is the GATA1 motif in the +58DHS region of intron 2 of the BCL11a gene, as described in E10 above.

[0383] E12. The target sequence of interest is selected from the group consisting of a portion of the FAS gene sequence; a portion of the BID gene sequence; a portion of the CTLA4 gene sequence; a portion of the PDCD1 gene sequence; a portion of the CBLB gene sequence; a portion of the PTPN6 gene sequence; a portion of the B2M gene sequence; a portion of the TRAC gene sequence; and a portion of the TRBC gene sequence, as described in method E above.

[0384] E13. The target sequence of interest is selected from the group consisting of a portion of the B2M gene sequence; a portion of the TRAC gene sequence; and a portion of the TRBC gene sequence, using the method described in E12 above.

[0385] E14. A portion of the B2M gene sequence is located within the first 500 bp of the B2M gene coding sequence, as described in E13 above.

[0386] E15. A portion of the B2M gene sequence is located between the 501st nucleotide and the last nucleotide of the B2M gene coding sequence, as described in E13 above.

[0387] E16. A portion of the TRAC gene sequence is located within the first 500 bp of the TRAC gene coding sequence, as seen in the aforementioned E12 cell.

[0388] E17. A portion of the TRBC gene sequence is located within the first 500 bp of the TRBC gene coding sequence, as seen in the aforementioned E12 cell.

[0389] F. In certain embodiments, the subject disclosed herein is a method for treating a subject, wherein cells from the subject are gRNA complementary to the target sequence of the target nucleic acid; and One of the aforementioned Cpf1 RNA-inducible nucleases A-A8 and B-B8 The present invention provides a method that includes bringing the object into contact with the object.

[0390] The F1.Cpf1 molecule forms a double-strand break in the target nucleic acid, as described by the F method.

[0391] The F2.Cpf1 molecule is selected from the group consisting of the Acidaminococcus strain BV3L6 Cpf1 molecule (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 molecule (LbCpf1), and Lachnospiraceae bacterium MA2020 (Lb2Cpf1), as described in the F or F1 method above.

[0392] F3. The subjects are those suffering from abnormal hemoglobin disorders, using one of the methods described in F to F2 above.

[0393] F4. Abnormal hemoglobin disorders are sickle cell disease or β-thalassemia, as described in F3 above.

[0394] F5. The cells are T cells, hematopoietic stem cells (HSCs), or human umbilical cord blood-induced erythrocyte progenitor cells (HUDEP cells), one of the methods F-F4 described above.

[0395] F6.T cells are CD8 + T cells, CD8 + Naive T cells, CD4 + central memory T cells, CD8 + central memory T cells, CD4 + Effector memory T cells, CD4 + Effector memory T cells, CD4 + T cells, CD4 + Stem cell memory T cell, CD8 + Stem cell memory T cell, CD4 + Helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, CD4+ naive T cells, TH17CD4 + T cells, TH1CD4 + T cells, TH2CD4 + T cells, TH9CD4 + T cells, CD4 + Foxp3 + T cells, CD4 + CD25 + CD127 - T cells or CD4 + CD25 + CD127 - Foxp3 + The aforementioned F5 method involves T cells.

[0396] F7.HSC cells are CD34 + cells, CD34 + CD90 + cells, CD34 + CD38 - cells, CD34 + CD90 + CD49f + CD38 - CD45RA - cells, CD105 +cells, CD31 + Alternatively, CD133 + Cells or CD34 + CD90 + CD133 + The method described above involves cells, specifically the F5 method.

[0397] F8. Contact is performed ex vivo using one of the methods F-F7 described above.

[0398] F9. The contact cells are returned to the target body using one of the methods described in F-F8 above.

[0399] G. In certain embodiments, the subject matter disclosed herein is (a) Any of the aforementioned Cpf1 RNA-induced nucleases A-A8 and B-B8, (b) gRNA complementary to the target sequence of the target nucleic acid; and (c) Cells from subjects that would benefit from one or more modifications of the target nucleic acid A reaction mixture containing the above is provided.

[0400] H. In certain embodiments, the subject matter disclosed herein is (a) one of the aforementioned Cpf1 RNA-induced nucleases A-A8 and B-B8, or a nucleic acid composition encoding a Cpf1 RNA-induced nuclease, and (b) gRNA complementary to the target sequence of the target nucleic acid or the nucleic acid composition of the gRNA We provide a kit that includes this.

[0401] I. In certain embodiments, the subject disclosed herein provides cells that include modifications to a target nucleic acid sequence introduced through the genome editing system described in D above.

[0402] I1. The modification is to the HBG1 gene sequence or the Bcl11a gene sequence, as described in I above.

[0403] I2. The modified HBG1 gene sequence is the -110nt promoter region of the HBG gene, as described in I1 above.

[0404] I3. The cell according to claim I1 above, wherein the modified HBG1 gene sequence is the CAAT box in the -110nt promoter region of the HBG gene.

[0405] I4. The cell according to claim I1 above, wherein the modified Bcl11a gene sequence is the +58DHS region of intron 2 of the BCL11a gene.

[0406] I5. The cell according to claim I1 above, wherein the modified Bcl11a gene sequence is the GATA1 motif in the +58DHS region of intron 2 of the BCL11a gene.

[0407] J. In certain embodiments, the subject matter disclosed herein is a method for evaluating CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of a target nucleic acid sequence by a test Cpf1 RNA-induced nuclease, (a) Determining the activity of a test Cpf1 RNA-induced nuclease with respect to editing and / or modifying the expression of a target nucleic acid sequence including a matching site target nucleic acid sequence; (b) With regard to editing and / or modifying the expression of target nucleic acid sequences including matching site target nucleic acid sequences, compare the activity of the test Cpf1 RNA-induced nuclease with the activity of the control RNA-induced nuclease. This provides a method that includes [something].

[0408] J1. The matching target nucleic acid sequence is selected from the group consisting of matching site 1 (sequence number 13), matching site 5 (sequence number 14), matching site 11 (sequence number 15), and matching site 18 (sequence number 16), as described in method J above.

[0409] J2. The Cpf1 RNA-induced nuclease and control RNA-induced nuclease were: (a) Having the same amino acid sequence; (b) The method of J described above, which is used to assay activity in different cell types.

[0410] J3. The Cpf1 RNA-induced nuclease and control RNA-induced nuclease were: (a) Having the same amino acid sequence; (b) The method of J described above, wherein the activity in different formulations is assayed.

[0411] J4. The Cpf1 RNA-induced nuclease and control RNA-induced nuclease were: (a) Having the same amino acid sequence; (b) The method of J described above, wherein the activity at different concentrations is assayed.

[0412] J5. The Cpf1 RNA-induced nuclease and control RNA-induced nuclease were: (a) Having the same amino acid sequence; (b) The method of J described above, wherein the activity is assayed after production via a different process.

[0413] J6. The Cpf1 RNA-induced nuclease and control RNA-induced nuclease were: (a) Having the same amino acid sequence; (b) The method of J described above, wherein the activity is assayed after delivery to cells via a different process.

[0414] J7. The Cpf1 RNA-inducible nuclease and the control RNA-inducible nuclease contained different amino acid sequences, as described in J above.

[0415] K. In certain embodiments, the subject disclosed herein provides cells comprising a CRISPR system capable of downregulating the gene expression of endogenous genes selected from the group consisting of BC11a and HBG1.

[0416] The K1.CRISPR system is the aforementioned K cell, which contains a gRNA complementary to a portion of the BC11a gene sequence.

[0417] A portion of the K2.BC11a gene sequence is the +58DHS region of intron 2 of the BCL11a gene, as described in the K1 cell.

[0418] A portion of the K3.BC11a gene sequence is the GATA1 motif in the +58DHS region of intron 2 of the BCL11a gene, as described in the K1 cell example.

[0419] The K4.CRISPR system is the aforementioned K cell, which contains a gRNA complementary to a portion of the HBG1 gene sequence.

[0420] A portion of the K5.HBG1 gene sequence is the -110nt promoter regi...

Claims

1. Isolated cells containing modifications to the HBG gene sequence or the BCL11a gene sequence, generated by delivery of an RNP complex comprising a CRISPR 1 (Cpf1) RNA-induced nuclease from the genera Prevotella and Francisella, and a gRNA molecule targeting the HBG gene sequence or the BCL11a gene sequence.

2. A population of CD34+ cells or hematopoietic stem cells (HSCs) having one or more cells containing a disruption of the cis-regulatory region of the HBG gene, wherein the disruption is generated using an RNP complex comprising a CRISPR / Cpf1 RNA-induced nuclease and a gRNA that targets the cis-regulatory region of the HBG gene.

3. The cell population according to claim 2, wherein the cis regulatory region includes the CAAT box of the HBG gene promoter.

4. A method for treating or alleviating symptoms of abnormal hemoglobinopathy in a subject in need thereof, comprising administering the cell population described in claim 2 or 3 to the subject.

5. The method according to claim 4, wherein the cell population has increased expression of fetal hemoglobin compared to the unmodified cell population, or results in increased expression of fetal hemoglobin following administration, and the increase in the expression of fetal hemoglobin is in an amount suitable for partially or completely alleviating the symptoms of the abnormal hemoglobin disorder.

6. Isolated T cells comprising modifications to a nucleic acid sequence, generated by delivery of a complex comprising a CRISPR 1 (Cpf1) RNA-induced nuclease from the genera Prevotella and Francisella and a gRNA molecule targeting a nucleic acid sequence, wherein the nucleic acid sequence is selected from the group consisting of a portion of the FAS gene sequence, a portion of the BID gene sequence, a portion of the CTLA4 gene sequence, a portion of the PDCD1 gene sequence, a portion of the CBLB gene sequence, a portion of the PTPN6 gene sequence, a portion of the B2M gene sequence, a portion of the TRAC gene sequence, a portion of the CIITA gene sequence, a portion of the TRBC gene sequence, and combinations thereof.

7. A T cell population comprising disruption of one or more genes selected from the group consisting of TRAC, TRBC, B2M, and CIITA, wherein the disruption is generated using one or more RNP complexes comprising a CRISPR / Cpf1 RNA-induced nuclease and a gRNA targeting a gene selected from the group consisting of TRAC, TRBC, CIITA, and B2M, and wherein at least 60% of the T cells in the T cell population do not contain detectable levels of MHC II receptor, TCR, or B2M on the surface of the T cells.

8. The T cell population according to claim 7, further comprising a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR) inserted into a disrupted TAC locus.

9. The T cells are CD8 ＋ T cells, CD8 ＋ Naive T cells, CD4 ＋ Central memory T cells, CD8 ＋ Central memory T cells, CD4 ＋ Effector memory T cells, CD4 ＋ Effector memory T cells, CD4 ＋ T cells, CD4 ＋ Stem cell memory T cells, CD8 ＋ Stem cell memory T cells, CD4 ＋ Helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, CD4+ naive T cells, TH17 CD4 ＋ T cells, TH1 CD4 ＋ T cells, TH2 CD4 ＋ T cells, TH9 CD4 ＋ T cells, CD4 ＋ Foxp3 ＋ T cells, CD4 ＋ CD25 ＋ CD127 － T cells or CD4 ＋ CD25 ＋ CD127 － Foxp3 ＋ The isolated T cell or T cell population according to any one of claims 6 to 8, which is a T cell.

10. CRISPR isolated from Prevotella and Francisella 1 (Cpf1) RNA-induced nucleases containing a nuclear localization signal (NLS), wherein the Cpf1 RNA-induced nuclease comprises one or more NLS sequences at or near the N-terminus of the nuclease, one or more NLS sequences at or near the C-terminus of the nuclease, or one or more NLS sequences at or near the N-terminus and C-terminus of the nuclease.

11. The isolated Cpf1 RNA-inducible nuclease according to claim 10, wherein the NLS sequence is selected from the group consisting of nucleoplasmin NLS (nNLS) (SEQ ID NO: 1) and Simianvirus 40 "SV40" NLS (sNLS) (SEQ ID NO: 2).

12. CRISPR isolated from Prevotella and Francisella 1 (Cpf1) RNA-inducible nucleases comprising a deletion or substitution of a cysteine ​​amino acid, wherein the Cpf1 RNA-inducible nuclease comprises a deletion or substitution at C65, C205, C334, C379, C608, C674, C1025, or C1248 of the wild-type AsCpf1 amino acid sequence, the substitution being selected from the group consisting of C65S / A, C205S / A, C334S / A, C379S / A, C608S / A, C674S / A, and C1025S / A.

13. An isolated nucleic acid encoding a Cpf1 RNA-induced nuclease according to any one of claims 10 to 12.

14. A method for modifying one or more target sequences of interest in a population of HSCs or T cells, wherein the cell population is: (a) a gRNA molecule complementary to the target sequence of interest; and (b) Cpf1 RNA-induced nuclease according to any one of claims 10 to 12 A method comprising contacting one or more RNP complexes containing a certain element ex vivo or in vitro, wherein the one or more RNP complexes modify one or more target sequences of interest within the cell population.

15. The method according to claim 14, wherein at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population contain productive indels.

16. The method according to claim 14 or 15, wherein the target nucleic acid sequence is selected from the group consisting of a part of the B2M gene sequence, a part of the TRAC gene sequence, a part of the CIITA gene sequence, a part of the TRBC gene sequence, and combinations thereof.

17. A method for administering to a cell population, wherein the cell population is generated by the delivery of a complex comprising a CRISPR 1 (Cpf1) RNA-induced nuclease from the genera Prevotella and Francisella, and a gRNA molecule targeting the HBG gene sequence or the BCL11a gene sequence, and includes modifications to the HBG gene sequence or the BCL11a gene sequence.

18. The method according to claim 17, wherein the subject is suffering from an abnormal hemoglobin disorder.

19. The method according to claim 17 or 18, wherein the cell population includes hematopoietic stem cells (HSCs) or human umbilical cord blood-induced erythrocyte progenitor (HUDEP) cells.

20. A method for administering a population of T cells, wherein the cell population is generated by delivery of a complex comprising Prevotella and Francisella 1 (Cpf1) RNA-induced nucleases and a gRNA molecule that targets a gene, and includes modifications to the gene selected from the group consisting of TRAC, TRBC, CIITA, and B2M.

21. The method according to claim 20, wherein the subject is suffering from cancer or an autoimmune disorder.

22. The method according to any one of claims 17 to 21, wherein at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the cell population contain productive indels.

23. A gRNA molecule of a CRISPR 1 (Cpf1) RNA-induced nuclease from the genera Prevotella and Francisella, comprising a first targeting domain complementary to a target sequence, wherein the target sequence is the HBG gene sequence or the BCL11a gene sequence, and the gRNA molecule is identical to the sequence provided in Figures 6, 7, 8, 46 and Table 19, or comprises a sequence differing by three or fewer nucleotides.

24. (a) The CRISPR / Cpf1 system comprising the gRNA molecule is introduced into a cell, and an indel is formed in or near the target sequence complementary to the first targeting domain of the gRNA molecule; and / or (b) When the CRISPR / Cpf1 system comprising the gRNA molecule is introduced into a cell, the deletion occurs in a sequence complementary to the first targeting domain of the gRNA within the HBG1 promoter region or the HBG2 promoter region, according to claim 23.

25. When the CRISPR system containing the aforementioned gRNA molecule is introduced into a cell population, (a) The expression of fetal hemoglobin is increased in the cell population or its offspring compared to the level of fetal hemoglobin expression in the cell population or its offspring population in which the gRNA molecule was not introduced; and / or (b) The gRNA molecule according to claim 23, which results in an increase in the expression of fetal hemoglobin in an amount suitable for partially or completely alleviating the symptoms of an abnormal hemoglobin disorder.

26. A composition comprising the gRNA molecule described in any one of claims 23 to 25.

27. The composition according to claim 26, further comprising CRISPR 1 (Cpf1) RNA-induced nucleases from the genera Prevotella and Francisella.

28. A composition comprising a ribonucleoprotein (RNP) complex containing the composition described in claim 27.

29. A gRNA molecule according to any one of claims 23 to 25 or a composition according to any one of claims 26 to 28, for use in the treatment of a subject suffering from an abnormal hemoglobin disorder.

30. A gRNA molecule of a CRISPR 1 (Cpf1) RNA-induced nuclease from the genera Prevotella and Francisella, comprising a first targeting domain complementary to the target sequence, wherein the target sequence is selected from the group consisting of a portion of the B2M gene sequence, a portion of the TRAC gene sequence, a portion of the CIITA gene sequence, a portion of the TRBC gene sequence, and combinations thereof, and the gRNA molecule is identical to the sequence provided in Tables 2 to 9, or contains a sequence that differs by three or fewer nucleotides.

31. (a) The CRISPR / Cpf1 system comprising the gRNA molecule is introduced into a cell, and an indel is formed in or near the target sequence complementary to the first targeting domain of the gRNA molecule; and / or (b) When the CRISPR / Cpf1 system comprising the gRNA molecule is introduced into a cell, the deletion occurs in a sequence complementary to the first targeting domain of the gRNA within the B2M gene sequence, the TRAC gene sequence, the CIITA gene sequence, or the TRBC gene sequence, according to claim 30.

32. A composition comprising the gRNA molecule described in claim 30 or 31.

33. The composition according to claim 32, further comprising CRISPR 1 (Cpf1) RNA-induced nucleases from the genera Prevotella and Francisella.

34. A gRNA molecule according to claim 30 or 31, or a composition according to claim 32 or 34, for use in the treatment of a subject suffering from cancer.

35. It is a genome editing system, (a) gRNA molecules containing sequences provided in Figures 6, 7, 8, 9, 10, 11, 12, 46 and Tables 2-9 and 19; and (b) Cpf1 RNA-induced nuclease according to any one of claims 10 to 12 A genome editing system comprising one or more RNP complexes including [specific RNP complexes].

36. An assay for evaluating CRISPR / Cpf1-mediated editing of a target nucleic acid sequence and / or modification of the expression of a target nucleic acid sequence by a test Cpf1 RNA-induced nuclease, (a) Determining the activity of the test Cpf1 RNA-induced nuclease with respect to editing and / or regulating the expression of a target nucleic acid sequence including a matching site target nucleic acid sequence; (b) With regard to the editing and / or expression adjustment of the target nucleic acid sequence including the matching site target nucleic acid sequence, compare the activity of the test Cpf1 RNA-induced nuclease with the activity of the control RNA-induced nuclease. Assays including

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