Therapeutic applications of CRISPR type V systems

Abstract

The present disclosure provides methods and compositions for therapeutic use, including Type V CRISPR systems with RNA guides comprising ribonucleotide bases and at least one deoxyribonucleotide base, used to perform therapeutic genome editing in somatic cells, induced pluripotent stem cells (iPSCs), and germ or embryonic cells of animals for organ and tissue xenotransplantation.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 330,695, filed April 13, 2022, and U.S. Provisional Patent Application No. 63 / 332,173, filed April 18, 2022, all of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable.

[0003] The present disclosure relates generally to the field of cell therapy utilizing cells modified with regularly interspaced short palindromic repeats (CRISPR) systems, and more specifically, CRISPR-Cas12 systems. [Background technology]

[0004] Regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been found in many prokaryotic genomes and confer adaptive immunity to viruses. A current description and classification of various CRISPR-Cas systems, including those found in their natural reservoirs (Class 1 Type I, Class 2 Type II and Type V), those targeting RNA (Class 2 Type VI), and those targeting both DNA and RNA (Class 1 Type III), can be found in Makarova et al. (Nat. Rev. Microbiol., 2020, 18:67-83). Of particular interest are Type V systems, including various subtypes such as Type VA, Type VB, Type VC, Type VD, Type VE, Type VF, Type VG, Type VH, Type VI, Type VJ, Type VK, and Type VU. The Type VA subtype encodes the Cas12a protein (formerly known as Cpf1). Cas12a has a RuvC-like nuclease domain that is homologous to the corresponding domain in Cas9, but lacks the HNH nuclease domain.

[0005] The V-type strains include Parcubacteria bacterium GWC2011_GWC2_44_17 (PbCpf1), Lachnospiraceae bacterium MC2017 (Lb3 Cpf1), Butyrivibrio proteoclasticus (BpCpf1), Peregrinibacteria bacterium GW2011_GWA_33_10 (PeCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), and Porphyromonas macacae. macacae (PmCpf1), Lachnospira bacterium ND2006 (LbCpf1), Porphyromonas crevioricanis (PcCpf1), Prevotella disiens (PdCpf1), Moraxella bovoculi 237 (MbCpf1), Smithella sp. SC_K08D17 (SsCpf1), Leptospira inadai (LiCpf1), Lachnospira bacterium MA2020 (Lb2Cpf1), Franciscella It has been identified so far in several bacteria, such as Candida novicida U112 (FnCpf1), Candidatus methanoplasma termitum (CMtCpf1), and Eubacterium eligens (EeCpf1).

[0006] The CRISPR-Cas system provides a powerful tool for site-specific genome editing by deleting, inserting, mutating, or replacing specific nucleic acid sequences. These modifications can be gene-specific or site-specific. Genome editing can use site-specific nucleases, such as Cas proteins, and their corresponding polynucleotides to cleave target nucleic acids, thereby generating the modification site. In certain cases, this cleavage can introduce a double-strand break (DSB) into the target DNA sequence. DSBs can be repaired, for example, by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR). HDR relies on the presence of a template for repair. In some examples of this genome editing, a donor polynucleotide or a portion thereof can be inserted into the break. Summary of the Invention

[0007] This genome editing process, utilizing a V-type CRISPR-Cas protein, such as Cas12a, in combination with a CRISPR hybrid RNA-DNA guide (chRDNA), is particularly useful for generating genetically modified cells useful for therapeutic applications.

[0008] In some embodiments, the invention is a method of treating a disease or condition characterized by aberrant expression of a gene, the method comprising introducing into somatic cells of a patient suffering from the disease or condition: (a) a first nucleoprotein complex comprising a Cas12a protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the first target nucleic acid, and the first CRISPR guide molecule comprises at least one deoxyribonucleotide; and (b) a donor polynucleotide comprising a coding sequence of a target gene that is aberrantly expressed in an individual suffering from the disease or condition; Cleavage by the Cas12a protein results in insertion of a coding sequence into the genome of the somatic cell, wherein the introduction is by contacting the somatic cell with lipid nanoparticles comprising a first nucleoprotein complex and a donor polynucleotide, and the target gene is selected from Table 3. In some embodiments, in the CRISPR guide molecule, the activation region, the targeting region, or both comprise at least one deoxyribonucleotide. In some embodiments, the method further comprises introducing into the somatic cell a second nucleoprotein complex comprising a Cas12a protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule has a targeting region capable of binding to a second target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the second target nucleic acid, and the coding sequence is inserted between the cleavage sites in the first target nucleic acid and the second target nucleic acid cleaved by the Cas12a protein. In some embodiments, the second CRISPR guide molecule comprises at least one deoxyribonucleotide.

[0009] In some embodiments, insertion of the coding sequence into the genome of the somatic cell results in increased expression of the gene in the somatic cell. In some embodiments, the lipid nanoparticles comprise one or more cationic lipids, and the pK a is 6.1 to 6.7. In some embodiments, the lipid nanoparticles comprise a neutral lipid. In some embodiments, the lipid nanoparticles comprise a sterol. In some embodiments, the lipid nanoparticles comprise one or more lipids selected from the group consisting of DSPC, DPPC, POPC, DOPE, SM, PEG-DMA, PEG-DMG, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, GL67A-DOPE-DMPE-PEG, 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, PEG-CerC14, and PEG-CerC20.

[0010] In some embodiments, introduction into somatic cells occurs ex vivo, hi some embodiments, introduction into somatic cells is by systemic intravenous administration, administration into the portal vein, or intraocular administration.

[0011] In some embodiments, the invention is a therapeutic composition for treating a disease or condition characterized by aberrant gene expression, the composition comprising: (a) a first nucleoprotein complex comprising a Cas12a protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the first target nucleic acid, and the first CRISPR guide molecule comprises at least one deoxyribonucleotide; and (b) a donor polynucleotide comprising a coding sequence of a target gene that is aberrantly expressed in an individual suffering from the disease or condition, wherein the first nucleoprotein complex and the donor polynucleotide are present in a lipid nanoparticle, and the target gene is selected from Table 3. In some embodiments, in the CRISPR guide molecule, the activation region, the targeting region, or both comprise at least one deoxyribonucleotide.

[0012] In some embodiments, the composition further comprises a second nucleoprotein complex comprising a Cas12a protein and a second CRISPR guide molecule, the second CRISPR guide molecule having a targeting region capable of binding to a second target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, the Cas12a protein being capable of cleaving the second target nucleic acid. In some embodiments of the composition, the second CRISPR guide molecule comprises at least one deoxyribonucleotide. In some embodiments of the composition, the lipid nanoparticle comprises one or more cationic lipids, and the pK ais 6.1 to 6.7. In some embodiments of the composition, the lipid nanoparticles comprise a neutral lipid. In some embodiments of the composition, the lipid nanoparticles comprise a sterol. In some embodiments of the composition, the lipid nanoparticles comprise one or more lipids selected from the group consisting of DSPC, DPPC, POPC, DOPE, SM, PEG-DMA, PEG-DMG, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, GL67A-DOPE-DMPE-PEG, 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, PEG-CerC14, and PEG-CerC20. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0013] In some embodiments, the invention is a method of treating a disease or condition characterized by aberrant gene expression with differentiated, genetically modified induced pluripotent stem cells (iPSCs), the method comprising: (1) introducing into the iPSCs a first nucleoprotein complex comprising a Cas12a protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the first target nucleic acid, and wherein the first CRISPR guide molecule comprises at least one deoxyribonucleotide, wherein cleavage by the Cas12a protein results in modification of a target gene selected from Table 4 or Table 5; (2) differentiating the iPSCs into a cell type affected by the disease or condition in an individual affected by the disease or condition; and (3) administering the differentiated iPSCs to a patient affected by the disease or condition. In some embodiments, in a CRISPR guide molecule, the activation region, the targeting region, or both, comprise at least one deoxyribonucleotide.

[0014] In some embodiments, the method further comprises introducing into the iPSCs a second nucleoprotein complex comprising a Cas12a protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule has a targeting region capable of binding to a second target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the second target nucleic acid, and wherein a coding sequence is inserted between the cleavage sites in the first target nucleic acid and the second target nucleic acid cleaved by the Cas12a protein. In some embodiments of the method, the second CRISPR guide molecule comprises at least one deoxyribonucleotide. In some embodiments, the method further comprises introducing into the iPSCs a donor polynucleotide comprising a coding sequence of a target gene selected from Table 4. In some embodiments of the method, cleavage by the Cas12a protein results in insertion of the coding sequence into the genome of the iPSCs. In some embodiments of the method, insertion of the coding sequence into the genome of the iPSCs results in increased expression of the gene in the iPSCs. In some embodiments of the method, cleavage by the Cas12a protein results in disruption of the coding sequence of a target gene, as set forth in Table 5, in the genome of the iPSC. In some embodiments of the method, disruption of the genome of the iPSC results in reduced expression of the gene in the iPSC. In some embodiments of the method, the iPSC is generated by reprogramming a somatic cell. In some embodiments of the method, the reprogramming is performed by inducing expression of one or more genes in the somatic cell. In some embodiments of the method, the reprogramming is performed by introducing mRNA into the somatic cell to induce gene expression. In some embodiments of the reprogramming, the one or more genes are selected from Oct4, Sox2, Klf4, c-Myc, NANOG, Sox1, Sox3, Sox15, Sox18, Klf1, Klf2, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, LIN28, and Wnt.In some embodiments of reprogramming, the one or more genes consist of a combination of Oct4, Sox2, Klf4, and c-Myc. In some embodiments of reprogramming, the one or more genes consist of a combination of Oct4, Sox2, and NANOG. In some embodiments of the method, reprogramming is achieved by the use of MEK inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (−)-depudecin), HC toxin, nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyric acid (e.g., sodium phenylbutyrate), and valproic acid ((VP A) and other short-chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MG The method further includes contacting the iPSCs with one or more of CD0103, NVP-LAQ-824, CBHA (m-carboxycinnamic bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50. In some embodiments of the method, the iPSCs are differentiated into neurons.In some embodiments of the method, iPSCs are differentiated into neurons by incubating them in the presence of one or more of a GSK-3 inhibitor, a TGF-β receptor or TGF-β inhibitor, an ALK inhibitor, dorsomorphin, Compound E, FGF, EGF, all-trans retinoic acid, sonic hedgehog protein, purmorphamine, SAG dihydrochloride, CNTF, and GDNF. In some embodiments of the method, differentiation of iPSCs into neurons is assessed by measuring the expression of one or more of Sox1, Pax6, Nestin, HB9, MAP2, NeuroFilament, Tuj1, and Olig2 after the differentiation process. In some embodiments of the method, differentiation of iPSCs into neurons is assessed by measuring the electrical activity of the cells after the differentiation process.

[0015] In some embodiments of the method, the iPSCs are differentiated into muscle cells. In some embodiments of the method, the iPSCs are differentiated into muscle cells by incubating the iPSCs in the presence of one or more of a GSK-3 inhibitor and a Wnt-dependent phosphorylation inhibitor. In some embodiments of the method, differentiation of the iPSCs into muscle cells is assessed after the differentiation process by measuring the expression of one or more of TBX5, TNNT2, MYH6, and MYL7.

[0016] In some embodiments, the invention provides a composition for treating a disease or condition characterized by aberrant gene expression with differentiated, genetically modified induced pluripotent stem cells (iPSCs), the composition comprising iPSCs, the iPSCs comprising a first nucleoprotein complex comprising a Cas12a protein and a first CRISPR guide molecule, the first CRISPR guide molecule having a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, the Cas12a protein being capable of cleaving the first target nucleic acid, the first CRISPR guide molecule comprising at least one deoxyribonucleotide, wherein cleavage by the Cas12a protein results in modification of a target gene selected from Table 4 or Table 5, and the iPSCs are capable of differentiating into a cell type affected by the disease or condition in an individual affected by the disease or condition. In some embodiments of the composition, the CRISPR guide molecule, the activation region, the targeting region, or both comprise at least one deoxyribonucleotide. In some embodiments, the composition further comprises introducing into the iPSCs a second nucleoprotein complex comprising a Cas12a protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule has a targeting region capable of binding to a second target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the second target nucleic acid, and wherein a coding sequence is inserted between the cleavage sites in the first target nucleic acid and the second target nucleic acid cleaved by the Cas12a protein. In some embodiments of the composition, the second CRISPR guide molecule comprises at least one deoxyribonucleotide. In some embodiments, the composition further comprises a donor polynucleotide comprising a coding sequence of a target gene selected from Table 4. In some embodiments of the composition, cleavage by the Cas12a protein results in insertion of the coding sequence into the genome of the iPSCs. In some embodiments of the composition, insertion of the coding sequence into the genome of the iPSCs results in increased expression of the gene in the iPSCs.In some embodiments of the composition, cleavage by the Cas12a protein results in disruption of the coding sequence of a target gene, as set forth in Table 5, in the genome of the iPSC. In some embodiments of the composition, disruption of the genome of the iPSC results in reduced expression of the gene in the iPSC. In some embodiments of the composition, the iPSC is generated by reprogramming a somatic cell. In some embodiments of the composition, reprogramming is achieved by inducing expression of one or more genes in the somatic cell. In some embodiments of the composition, reprogramming is achieved by introducing mRNA into the somatic cell to induce gene expression. In some embodiments of the composition, the one or more genes are selected from Oct4, Sox2, Klf4, c-Myc, NANOG, Sox1, Sox3, Sox15, Sox18, Klf1, Klf2, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, LIN28, and Wnt. In some embodiments of the composition, the one or more genes consist of a combination of Oct4, Sox2, Klf4, and c-Myc. In some embodiments of the composition, the one or more genes consist of a combination of Oct4, Sox2, and NANOG.In some embodiments of the compositions, reprogramming is achieved using an inhibitor such as MEK inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyric acid (e.g., sodium phenylbutyrate), and valproic acid ((VP A) and other short-chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD010 3, further comprising contacting the iPSCs with one or more of NVP-LAQ-824, CBHA (m-carboxycinnamic acid bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50.

[0017] In some embodiments of the composition, iPSCs are differentiated into neurons. In some embodiments of the composition, iPSCs are differentiated into neurons by incubating them in the presence of one or more of a GSK-3 inhibitor, a TGF-β receptor or TGF-β inhibitor, an ALK inhibitor, dorsomorphin, Compound E, FGF, EGF, all-trans retinoic acid, sonic hedgehog protein, purmorphamine, SAG dihydrochloride, CNTF, and GDNF. In some embodiments of the composition, differentiation of iPSCs into neurons is assessed by measuring the expression of one or more of Sox1, Pax6, nestin, HB9, MAP2, NeuroFilament, Tuj1, and Olig2 after the differentiation process. In some embodiments of the composition, differentiation of iPSCs into neurons is assessed by measuring the electrical activity of the cells after the differentiation process.

[0018] In some embodiments of the composition, the iPSCs are differentiated into muscle cells. In some embodiments of the composition, the iPSCs are differentiated into muscle cells by incubating the iPSCs in the presence of one or more of a GSK-3 inhibitor and a Wnt-dependent phosphorylation inhibitor. In some embodiments of the composition, differentiation of the iPSCs into muscle cells is assessed after the differentiation process by measuring the expression of one or more of TBX5, TNNT2, MYH6, and MYL7.

[0019] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0020] In some embodiments, the invention is a method of producing differentiated genetically modified induced pluripotent stem cells (iPSCs) for treating a disease or condition characterized by aberrant gene expression, the method comprising: (1) introducing into the iPSCs a first nucleoprotein complex comprising a Cas12a protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the first target nucleic acid, and the first CRISPR guide molecule comprises at least one deoxyribonucleotide, wherein cleavage by the Cas12a protein results in modification of a target gene selected from Table 4 or Table 5; (2) differentiating the iPSCs into a cell type affected by the disease or condition in an individual affected by the disease or condition; and (3) administering the differentiated iPSCs to a patient affected by the disease or condition. In some embodiments of the method, the CRISPR guide molecule, the activation region, the targeting region, or both comprise at least one deoxyribonucleotide. In some embodiments, the method further comprises introducing into the iPSCs a second nucleoprotein complex comprising a Cas12a protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule has a targeting region capable of binding to a second target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, and the Cas12a protein is capable of cleaving the second target nucleic acid, and a coding sequence is inserted between the cleavage sites in the first target nucleic acid and the second target nucleic acid cleaved by the Cas12a protein. In some embodiments of the method, the second CRISPR guide molecule comprises at least one deoxyribonucleotide. In some embodiments, the method further comprises introducing into the iPSCs a donor polynucleotide comprising a coding sequence of a target gene selected from Table 4. In some embodiments of the method, cleavage by the Cas12a protein results in insertion of the coding sequence into the genome of the iPSCs.In some embodiments of the method, the insertion of a coding sequence into the genome of the iPSC results in increased expression of the gene in the iPSC. In some embodiments of the method, cleavage by the Cas12a protein results in the disruption of the coding sequence of the target gene shown in Table 5 in the genome of the iPSC. In some embodiments of the method, disruption of the genome of the iPSC results in decreased expression of the gene in the iPSC. In some embodiments of the method, the iPSC is generated by reprogramming a somatic cell. In some embodiments of the method, the reprogramming is performed by inducing expression of one or more genes in the somatic cell. In some embodiments of the method, the reprogramming is performed by introducing mRNA into the somatic cell to induce gene expression. In some embodiments of reprogramming, the one or more genes are selected from Oct4, Sox2, Klf4, c-Myc, NANOG, Sox1, Sox3, Sox15, Sox18, Klf1, Klf2, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, LIN28, and Wnt. In some embodiments of reprogramming, the one or more genes consist of a combination of Oct4, Sox2, Klf4, and c-Myc. In some embodiments of reprogramming, the one or more genes consist of a combination of Oct4, Sox2, and NANOG.In some embodiments of the method, reprogramming is achieved by the use of MEK inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (−)-depudecin), HC toxin, nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyric acid (e.g., sodium phenylbutyrate), and valproic acid ((VP A) and other short-chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD010 3, further comprising contacting the iPSCs with one or more of NVP-LAQ-824, CBHA (m-carboxycinnamic acid bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50.

[0021] In some embodiments of the method, iPSCs are differentiated into neurons. In some embodiments of the method, iPSCs are differentiated into neurons by incubating them in the presence of one or more of a GSK-3 inhibitor, a TGF-β receptor or TGF-β inhibitor, an ALK inhibitor, dorsomorphin, compound E, FGF, EGF, all-trans retinoic acid, sonic hedgehog protein, purmorphamine, SAG dihydrochloride, CNTF, and GDNF. In some embodiments of the method, differentiation of iPSCs into neurons is assessed by measuring the expression of one or more of Sox1, Pax6, nestin, HB9, MAP2, NeuroFilament, Tuj1, and Olig2 after the differentiation process. In some embodiments of the method, differentiation of iPSCs into neurons is assessed by measuring the electrical activity of the cells after the differentiation process.

[0022] In some embodiments of the method, the iPSCs are differentiated into muscle cells. In some embodiments of the method, the iPSCs are differentiated into muscle cells by incubating the iPSCs in the presence of one or more of a GSK-3 inhibitor and a Wnt-dependent phosphorylation inhibitor. In some embodiments of the method, differentiation of the iPSCs into muscle cells is assessed after the differentiation process by measuring the expression of one or more of TBX5, TNNT2, MYH6, and MYL7.

[0023] In some embodiments, the present invention provides a method for producing a transgenic animal for xenotransplantation, the method comprising: (1) introducing into a cell of the animal a first nucleoprotein complex comprising a Cas12a protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, the Cas12a protein being capable of cleaving the first target nucleic acid, the first CRISPR guide molecule comprising at least one deoxyribonucleotide, and cleavage by the Cas12a protein resulting in modification of a target gene selected from Table 6; and (2) introducing the cell into a foster mother female animal. In some embodiments of the method, the animal cell is an oocyte, egg, or zygote. In some embodiments of the method, the animal cell is a somatic cell, and the method further comprises, after step (1), transferring the nucleus of the cell into an enucleated egg or zygote. In some embodiments of the method, the animal is a pig. In some embodiments of the method, in the CRISPR guide molecule, the activation region, the targeting region, or both comprise at least one deoxyribonucleotide. In some embodiments, the method further comprises introducing into the iPSCs a second nucleoprotein complex comprising a Cas12a protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule has a targeting region capable of binding to a second target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, wherein the Cas12a protein is capable of cleaving the second target nucleic acid, and wherein a coding sequence is inserted between the cleavage sites in the first target nucleic acid and the second target nucleic acid cleaved by the Cas12a protein. In some embodiments of the method, the second CRISPR guide molecule comprises at least one deoxyribonucleotide.In some embodiments, the method further comprises introducing into the cell a donor polynucleotide comprising a coding sequence of a target gene selected from one or more of A20, HO-1, FAT-1, TNF-α receptor, CD39, hirudin, TFPI, EPCR, TBM, CD46, DAF (CD55), CD59, CR1, CTLA4, CD47, and class I HLA. In some embodiments of the method, cleavage by the Cas12a protein results in insertion of the coding sequence into the genome of the cell. In some embodiments of the method, insertion of the coding sequence into the genome of the cell results in increased expression of the gene in the cell. In some embodiments of the method, cleavage by the Cas12a protein results in disruption of the coding sequence in the genome of the cell of a target gene selected from one or more of GGTA1, b4GalNT2, CMAH, GT (α(1,3)-galactosyltransferase), GHR, and class I SLA. In some embodiments of the method, disruption of the genome of the cell results in decreased expression of the gene in the cell.

[0024] In some embodiments, the present invention provides a composition for generating a transgenic animal for xenotransplantation, the composition comprising an animal cell, the animal cell comprising a first nucleoprotein complex comprising a Cas12a protein and a first CRISPR guide molecule, the first CRISPR guide molecule having a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, the Cas12a protein capable of cleaving the first target nucleic acid, the first CRISPR guide molecule comprising at least one deoxyribonucleotide, and cleavage by the Cas12a protein results in modification of a target gene selected from Table 6. In some embodiments of the composition, the animal cell is an oocyte, egg, or zygote. In some embodiments of the composition, the animal cell is an egg or zygote obtained by transferring the nucleus of a somatic cell into an enucleated egg or zygote. In some embodiments of the composition, the animal is a pig. In some embodiments of the composition, the CRISPR guide molecule, the activation region, the targeting region, or both comprise at least one deoxyribonucleotide. In some embodiments, the composition further comprises introducing into the iPSCs a second nucleoprotein complex comprising a Cas12a protein and a second CRISPR guide molecule, wherein the second CRISPR guide molecule has a targeting region capable of binding to a second target nucleic acid sequence and an activation region capable of forming a nucleoprotein complex with the Cas12a protein, the Cas12a protein is capable of cleaving the second target nucleic acid, and a coding sequence is inserted between the cleavage sites in the first target nucleic acid and the second target nucleic acid cleaved by the Cas12a protein. In some embodiments of the composition, the second CRISPR guide molecule comprises at least one deoxyribonucleotide. In some embodiments, the composition further comprises a donor polynucleotide comprising a coding sequence for a target gene selected from one or more of A20, HO-1, FAT-1, TNF-α receptor, CD39, hirudin, TFPI, EPCR, TBM, CD46, DAF (CD55), CD59, CR1, CTLA4, CD47, Class I HLA.In some embodiments of the composition, cleavage by the Cas12a protein results in the insertion of a coding sequence into the genome of the cell. In some embodiments of the composition, insertion of the coding sequence into the genome of the cell results in increased expression of a gene in the cell. In some embodiments of the composition, cleavage by the Cas12a protein results in the disruption of the coding sequence of a target gene selected from one or more of GGTA1, b4GalNT2, CMAH, GT (α(1,3)-galactosyltransferase), GHR, and class I SLA in the genome of the cell. In some embodiments of the composition, disruption of the genome of the cell results in decreased expression of the gene in the cell.

[0025] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, in which: The drawings are not drawn to proportion and are not to scale. The location of display elements is approximate. [Brief explanation of the drawings]

[0026] [Figure 1A] An example of a V-type CRISPR-Cas12a guide RNA is shown. [Figure 1B] An example of a V-type CRISPR-Cas12a guide RNA is shown. [Figure 1C] An example of a V-type CRISPR-Cas12a guide RNA is shown. [Figure 2] Cleavage of a target polynucleotide by the Cas12a chRDNA guide / nucleoprotein complex. [Figure 3] Figures 3A-3I show various canonical and non-canonical nucleotides for use in Cas12 chRDNA guides. [Figure 4] Cleavage of a target polynucleotide by the Cas12a chRDNA guide / nucleoprotein complex. [Figure 5] Cas12a crRNA guide is shown. [Figure 6] A Cas12a chR DNA guide containing DNA bases in the activation region and target binding sequence is shown. [Figure 7] 1 shows a Cas12a chRDNA guide containing DNA bases and chemically modified nucleic acids in the activation region and target binding sequence. [Figure 8] 1 shows the formation of the Cas12 chRDNA guide / nucleoprotein complex and binding of the target polynucleotide. [Figure 9] 1 shows the generation of insertions or deletions (indels) in the target polynucleotide by the Cas12 chRDNA guide / nucleoprotein complex. [Figure 10] 1 shows insertion of a donor polynucleotide sequence in a target polynucleotide by a Cas12 chRDNA guide / nucleoprotein complex. [Figure 11] Nicking of the target polynucleotide by the Cas12 chRDNA guide / nucleoprotein complex. [Figure 12] 1 shows tandem nicking of a target polynucleotide by two Cas12 chRDNA guide / nucleoprotein complexes and insertion of a donor polynucleotide sequence in the target polynucleotide. DETAILED DESCRIPTION OF THE INVENTION

[0027] definition The following definitions are helpful in understanding this disclosure.

[0028] As used herein, the terms "guide" and "guide polynucleotide" refer to one or more polynucleotides that form a nucleoprotein complex with a Cas protein, and this nucleoprotein complex preferentially binds to the nucleic acid target sequence of the polynucleotide (compared to a polynucleotide that does not contain a nucleic acid target sequence). Such guides can include ribonucleotide bases (e.g., RNA), deoxyribonucleotide bases (e.g., DNA), combinations of ribonucleotide bases and deoxyribonucleotide bases (e.g., RNA / DNA), nucleotide analogs, modified nucleotides, etc., as well as synthetic, naturally occurring, and non-naturally occurring modified backbone residues or linkages. Many such guides are known, including, but not limited to, single-stranded guide RNAs (including truncated single-stranded guide RNAs), crRNAs, and dual-guide RNAs, such as, but not limited to, crRNA / tracrRNA molecules, and their uses vary depending on the particular Cas protein. For example, a "type V CRISPR-Cas12-associated guide" is a guide that specifically associates with a corresponding Cas12 protein to form a nucleoprotein complex.

[0029] As used herein, a "CRISPR polynucleotide" is a polynucleotide sequence that comprises a portion of a guide molecule. In some embodiments, a CRISPR polynucleotide comprises a targeting region and / or an activation region.

[0030] As used herein with respect to guide molecules, the terms "spacer," "spacer sequence," "spacer element," or "targeting region" refer to a polynucleotide sequence capable of specifically hybridizing to a target nucleic acid sequence. The targeting region interacts with the target nucleic acid sequence through hydrogen bonding between complementary base pairs (i.e., paired bases). The targeting region binds to a selected nucleic acid target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell, whether in vitro, ex vivo (e.g., when generating CAR-T cells), or in vivo (e.g., when administering a composition directly to a subject). The targeting region determines the location of site-specific binding and nucleolytic cleavage of the Cas12 protein. Variability in the functional length of a targeting region is known in the art.

[0031] With respect to a guide molecule, the term "activation region" refers to a portion of a polynucleotide that can associate with or bind to a Cas12 polypeptide, such as a Cas12a polypeptide.

[0032] As used herein, the terms "base analog," "non-standard base," and "chemically modified base" refer to compounds that have structural similarity to standard purine or pyrimidine bases present in DNA or RNA. Base analogs may contain modified sugars and / or modified nucleobases compared to the purine or pyrimidine bases naturally occurring in DNA or RNA. In some embodiments, the base analog is inosine or deoxyinosine, e.g., 2'-deoxyinosine. In other embodiments, the base analog is a 2'-deoxyribonucleoside, 2'-ribonucleoside, 2'-deoxyribonucleotide, or 2'-ribonucleotide, and the nucleobase comprises a modified base (e.g., xanthine, uridine, oxanine (oxanosine), 7-methylguanosine, dihydrouridine, 5-methylcytidine, a C3 spacer, 5-methyl dC, 5-hydroxybutynl-2'-deoxyuridine, 5-nitroindole, 5-methyl iso-deoxycytosine, isodeoxyguanosine, deoxyuridine, isodeoxycytidine, other 0-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deazahypoxanthine, other 7-deazapurines, and 2-methylpurines). In some embodiments, the base analog may be selected from the group consisting of 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidite-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. The term "base analog" also includes, for example, 2'-deoxyribonucleosides, 2'-ribonucleosides, 2'-deoxyribonucleotides, or 2'-ribonucleotides, where the nucleobase is a substituted hypoxanthine. For example, the substituted hypoxanthine may be substituted with a halogen, such as fluorine or chlorine. In some embodiments, the base analog can be fluoroinosine or chloroinosine, for example, 2-chloroinosine, 6-chloroinosine, 8-chloroinosine, 2-fluoroinosine, 6-fluoroinosine, or 8-fluoroinosine.In other embodiments, the base analog is deoxyuridine. In other embodiments, the base analog is a nucleic acid mimic (e.g., artificial nucleic acids and xenonucleic acids (XNAs)).

[0033] As used herein, the term "CRISPR hybrid RNA / DNA guide" (chRDNA) refers to a polynucleotide guide molecule that contains a targeting region, where the polynucleotide comprises RNA but is designed to also contain DNA.

[0034] As used herein, the term "Cas12-chRDNA guide nucleoprotein complex" refers to the complexation of a chRDNA guide molecule with a Cas12 protein to form a nucleoprotein complex that is capable of site-specific binding to a nucleic acid target sequence that is complementary to a nucleic acid target binding sequence present in the chRDNA guide molecule.

[0035] "Linker element nucleotide sequence," "linker nucleotide sequence," and "linker polynucleotide" are used interchangeably herein and refer to a sequence of one or more nucleotides covalently attached to a first nucleic acid sequence (5'-linker nucleotide sequence-first nucleic acid sequence-3'). In some embodiments, the linker nucleotide sequence links two separate nucleic acid sequences to form a single polynucleotide (e.g., 5'-first nucleic acid sequence-linker nucleotide sequence-second nucleic acid sequence-3').

[0036] As used herein, the term "corresponding" typically refers to a Cas12 protein (e.g., Cas12a) and one or more type V CRISPR-Cas12 associated guides (e.g., Cas12 chRDNA guides) that can form a nucleoprotein complex capable of site-specific binding to a nucleic acid target sequence that is complementary to a nucleic acid target binding sequence present in one of the one or more guides.

[0037] The terms "engineered," "genetically engineered," "genetically modified," "recombinant," "modified," "non-naturally occurring," and "non-naturally occurring" refer to the deliberate manipulation by humans of the genome of an organism or cell. These terms encompass genome editing, as defined herein, as well as methods of genome modification, including techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis, gene shuffling, codon optimization, and the like. Methods of genetic engineering are known in the art.

[0038] As used herein, a Cas12 protein is said to "target" a polynucleotide when the Cas12 guide / nucleoprotein complex binds to or cleaves the polynucleotide at a nucleic acid target sequence within the polynucleotide.

[0039] As used herein, "protospacer adjacent motif" or "PAM" refers to a double-stranded nucleic acid sequence that includes a binding recognition sequence for a Cas12 protein, with amino acids of the Cas12 protein directly interacting with the recognition sequence (e.g., a Cas12a protein interacts with PAM 5'-TTTN-3' or PAM 5'-TTTV-3'). The PAM sequence can be present on the non-target strand and located 5' or 3' to the target-complementary sequence (e.g., in the CRISPR-Cas12a system, the PAM 5'-TTTN-3' or PAM 5'-TTTV-3' sequence is present on the non-target strand and located 5' to the target-complementary sequence).

[0040]

[0013] The terms "target," "target sequence," "nucleic acid target sequence," "target nucleic acid sequence," and "on-target sequence" are used interchangeably herein to refer to a nucleic acid sequence that is wholly or partially complementary to a nucleic acid target binding sequence (e.g., a targeted region) of a Cas12 polynucleotide. Typically, the nucleic acid target binding sequence is selected to be 100% complementary to the nucleic acid target sequence to which the Cas12 nucleoprotein complex is intended to bind, although a lower percentage of complementarity can be used to weaken binding to the nucleic acid target sequence.

[0041] The terms "donor polynucleotide," "donor oligonucleotide," "donor template," "non-viral donor," and "non-viral template" are used interchangeably herein and may be a double-stranded polynucleotide (e.g., DNA), a single-stranded polynucleotide (e.g., DNA or RNA), or a combination thereof. The donor polynucleotide may include homologous arms flanking the insertion sequence (e.g., a DSB in DNA). The homologous arms on each side may be of various lengths to ensure the desired level of hybridization under the conditions used.

[0042] As used herein, "homologous recombination repair" (HDR) refers to DNA repair that occurs in cells, for example, in repairing DSBs in DNA. HDR requires nucleotide sequence homology and uses a donor polynucleotide to repair a sequence where a DSB (e.g., a DSB within a target DNA sequence) has occurred. For example, a donor polynucleotide can be used to repair a break in a target DNA sequence, which results in the introduction of genetic information (e.g., a polynucleotide sequence) from the donor polynucleotide at or adjacent to the break site in the DNA. Thus, new genetic information (e.g., a polynucleotide sequence) can be inserted or replicated into the target DNA sequence.

[0043] As used herein, "homology-independent targeted integration" (HITI) refers to DNA repair that occurs in cells, for example, in repairing DSBs in DNA. Unlike HDR, HITI does not require nucleotide sequence homology and uses a donor polynucleotide to repair the sequence where the DSB occurred (e.g., within the target DNA sequence). HITI, for example, introduces genetic information from a donor polynucleotide into the target DNA sequence. Thus, new genetic information (e.g., a polynucleotide sequence) may be inserted or replicated into the target DNA sequence.

[0044] A "genomic region" is a segment of a chromosome within the genome of a host cell that flanks a nucleic acid target sequence site or that also contains a portion of the nucleic acid target sequence site. The homologous arms of a donor polynucleotide have sufficient homology to undergo homologous recombination with the corresponding genomic region.

[0045] As used herein, " non-homologous end joining " (NHEJ) refers to the repair of DSB in DNA by directly ligating one cut end with another cut end without the need for donor polynucleotide.NHEJ is the DNA repair pathway that cells can use to repair DNA without using repair template.In the absence of donor polynucleotide, NHEJ often results in random insertion or deletion of nucleotides at DSB site.

[0046] "Microhomology-mediated end joining" (MMEJ) is a pathway for repairing DSBs in DNA. MMEJ involves deletion of the DSB flanking regions and alignment of microhomology sequences within the break site prior to joining. MMEJ is genetically defined and requires the activity of, for example, CtIP, poly(ADP-ribose) polymerase 1 (PARP1), DNA polymerase theta (Pol θ), DNA ligase 1 (Lig 1), or DNA ligase 3 (Lig 3). Additional genetic components are known in the art. See, for example, Sfeir et al. (Trends in Biochemical Sciences, 2015, 40:701-714).

[0047] As used herein, "DNA repair" encompasses any process by which cellular mechanisms repair damage to DNA molecules contained in cells. The repaired damage can include single-strand breaks or double-strand breaks (DSBs). There are at least three mechanisms for repairing DSBs: HDR, NHEJ, and MMEJ. "DNA repair" is also used herein to refer to DNA repair by human manipulation, whereby a target gene locus is modified, for example, by nucleotide insertion, deletion, or substitution, all of which represent forms of genome editing.

[0048] As used herein, the terms "regulatory sequence," "regulatory element," and "control element" are interchangeable and refer to polynucleotide sequences that are upstream (5' non-coding sequences), within, or downstream (3' untranslated sequences) of a target polynucleotide to be expressed. Regulatory sequences influence, for example, the timing, amount or level of transcription, RNA processing or stability, and / or translation of associated structural nucleotide sequences. Regulatory sequences can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, transcription start sites, repressor binding sequences, stem-loop structures, translation initiation sequences, internal ribosome entry sites (IRES), translation leader sequences, transcription termination sequences (e.g., polyadenylation signals and polyU sequences), translation termination sequences, primer binding sites, etc.

[0049] As used herein, the term "operably linked" refers to polynucleotide sequences or amino acid sequences arranged in a functional relationship to each other. For example, a regulatory sequence (e.g., a promoter or enhancer) is "operably linked" to a polynucleotide encoding a gene product if the regulatory sequence regulates or contributes to the regulation of the transcription of the polynucleotide. An operably linked regulatory element is usually contiguous with the coding sequence. However, an enhancer can function even when it is several kilobases or more away from the promoter. Thus, some regulatory elements may be operably linked to a polynucleotide sequence but not contiguous with that polynucleotide sequence. Similarly, translational regulatory elements contribute to the regulation of protein expression from a polynucleotide.

[0050] As used herein, the term "modulate" refers to a change in the number, degree, or amount of a function. For example, the Cas12-guide / nucleoprotein complexes disclosed herein can modulate the activity of a promoter sequence by binding to a nucleic acid target sequence at or near the promoter. Depending on the effect occurring after binding, the Cas12-guide / nucleoprotein complex can induce, enhance, repress, or inhibit transcription of a gene operably linked to the promoter sequence. Thus, "modulation" of gene expression includes both gene activation and gene repression.

[0051] "Adoptive cells" refer to cells that can be or have been genetically modified for use in cell therapy treatments.

[0052] "Stem cells" refer to cells capable of self-renewal, i.e., undergoing numerous cell division cycles while remaining undifferentiated. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells are embryonic stem cells, fetal stem cells, amniotic fluid stem cells, adult stem cells, or induced pluripotent stem cells.

[0053] "Induced pluripotent stem cells" (iPSCs) refer to a type of pluripotent stem cell that is artificially derived from a non-pluripotent cell, usually a somatic cell. Pluripotent stem cells can be edited with a Cas12 chRDNA guide / nucleoprotein complex before or after differentiation. iPSCs can be further modified by introducing a foreign gene or sequence, such as a sequence encoding a CAR, into their genome before or after differentiation.

[0054] "Hematopoietic stem cells" refer to undifferentiated cells that have the ability to differentiate into hematopoietic cells such as lymphocytes.

[0055] "Lymphocyte" refers to a leukocyte (white blood cell) that is part of the immune system of a vertebrate. The term "lymphocyte" also encompasses hematopoietic stem cells that give rise to lymphoid cells. Lymphocytes include T cells, e.g., CD4+ and / or CD8+ cytotoxic T cells, alpha / beta T cells and gamma / delta T cells, regulatory T cells such as Treg cells, for cell-mediated cytotoxic adaptive immunity, natural killer (NK) cells that function in cell-mediated cytotoxic innate immunity, and B cells, NK / T cells, cytokine-induced killer cells (CIK cells), and antigen-presenting cells (APCs) such as dendritic cells for humoral antibody-driven adaptive immunity. Lymphocytes can be mammalian cells, such as human cells.

[0056] Tumor-infiltrating lymphocytes (TILs) are also encompassed by the term "lymphocytes" as used herein. TILs are immune cells that have infiltrated the tumor microenvironment. The term "lymphocytes" also encompasses genetically modified T cells and NK cells (CAR-T cells and CAR-NK cells).

[0057] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to humans and other primates, as well as other mammals, livestock, domestic mammals, and laboratory animals. In some embodiments, cells are derived from a subject (e.g., lymphocytes, stem cells, progenitor cells, or tissue-specific cells). In some embodiments, the subject is a non-human subject.

[0058] The term "effective amount" or "therapeutically effective amount" of a composition or agent, such as the genetically engineered adoptive cells provided herein, refers to a sufficient amount of the composition or agent to bring about a desired response. Preferably, an effective amount prevents, avoids, or eliminates one or more adverse side effects. Such a response will vary depending on the particular disease at issue. For example, in a patient being treated for cancer using adoptive cell therapy, the desired response may include, prevent, avoid, or eliminate one or more of the following: treatment or prevention of the effects of graft-versus-host disease (GvHD), host-versus-graft rejection, cytokine release syndrome (CRS), cytokine storm, and reduction of cancer transformation of the administered genetically engineered cells. The precise therapeutic amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the condition being treated, as well as the particular engineered lymphocytes used, the method of administration, etc. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art through routine experimentation.

[0059] "Treatment" of or "treating" a particular disease, such as a cancerous condition or GvHD, includes preventing the disease, e.g., preventing the disease from occurring or causing the disease to occur at a milder severity in a subject who may be predisposed to the disease but who does not experience or show symptoms of the disease; inhibiting the disease, e.g., reducing the rate of progression, halting progression, or causing remission from the pathological state; and / or alleviating the symptoms of the disease, e.g., reducing the number of symptoms experienced by a subject.

[0060] This disclosure is described in, for example, Sambrook, Joseph. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 2001; EA Greenfield (Antibodies: A Laboratory Manual, 2014, Second edition, Cold Spring Harbor Laboratory Press, ISBN 978-1-936113-81-1); Specialized Applications, 2016, 7th Edition, Wiley-Blackwell, ISBN 978-1118873656), JM Walker (Methods in Molecular Biology (Series), Humana Press, ISSN 1064-3745), Green et al. (Molecular Cloning: A Laboratory Manual, 2012, Fourth Edition, Cold Spring Harbor Laboratory Press, ISBN The present invention relies on those skilled in the art to be familiar with conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant polynucleotides, as taught by standard publications such as (978-1605500560).

[0061] CRISPR V type system Regularly interspaced short palindromic repeats (CRISPRs) and associated CRISPR-associated proteins (Cas proteins) comprise the CRISPR-Cas system. Classification of CRISPR-Cas systems has been repeatedly attempted. Makarova et al. (Nat. Rev. Microbiol., 2020, 18:67-83) proposed a classification scheme that takes into account the signature cas genes unique to each type and subtype of CRISPR-Cas system. This classification also considered sequence similarity among multiple common Cas proteins, the phylogeny of the most conserved Cas proteins, the genetic organization, and the structure of the CRISPR array. This approach provided a classification scheme that divides CRISPR-Cas systems into two distinct classes: Class 1 and Class 2.

[0062] In class 2 type V systems, Cas12 is involved in binding of the crRNA and the target and also in cleaving the target nucleic acid. For example, the RuvC-like nuclease domain of Cas12a cleaves both strands of the target nucleic acid in an asymmetric fashion, creating 5' overhangs, as opposed to the blunt ends generated by Cas9 cleavage. These 5' overhangs can facilitate DNA insertion via homologous recombination.

[0063] Other proteins involved in the binding and cleavage of type V crRNA and targets include Cas12b (formerly C2c1) and Cas12c (formerly C2c3). Cas12b and Cas12c proteins are similar in length to the CRISPR class 2 type II Cas9 protein and CRISPR class 2 type V Cas12a protein, ranging from approximately 1,100 to approximately 1,500 amino acids. Furthermore, C2c1 and C2c3 proteins contain a RuvC-like nuclease domain and have a similar structure to Cas12a. The C2c1 protein is similar to the Cas9 protein in that it requires crRNA and tracrRNA for target binding and cleavage, but its optimal cleavage temperature is 50°C. Like Cas12a, the C2c1 protein targets the AT-rich PAM located 5' of the target sequence. See, for example, Shmakov et al. (Molecular Cell, 2015, 60(3):385-397).

[0064] CRISPR type V subtypes include Cas12 proteins and exhibit a wide range of sequence and size diversity, but Cas12 subtypes share a common evolutionary origin with the TnpB nuclease encoded by IS605-like transposons. Due to the low sequence similarity of Cas12 proteins and the likelihood of evolution through multiple independent recombination events, the classification of Cas12 proteins into their respective subtypes has led to multiple nomenclatures. Table 1 presents the classification and names of type V Cas12 proteins, as well as their approximate sizes, guide requirements, preferred target polynucleotides, and representative organisms of origin. [Table 1-1] [Table 1-2]

[0065] Cas12 homologs can be identified using sequence similarity search methods known to those skilled in the art. Typically, a Cas12 protein can interact with a corresponding Cas12 guide to form a Cas12 guide / nucleoprotein complex capable of binding to a target nucleic acid sequence. In some embodiments of the present disclosure, the Cas12 protein or a homolog thereof is a Cas12a protein or a homolog thereof.

[0066] Cas12a proteins are found in Parkinobacteria bacterium GWC2011_GWC2_44_17 (PbCpf1), Lachnospira bacterium MC2017 (Lb3 Cpf1), Butyrivibrio proteoclasticus (BpCpf1), Peregrinibacteria bacterium GW2011_GWA_33_10 (PeCpf1), Acidaminococcus species (Acidaminococcus spp.) BV3L6 (AsCpf1), Porphyromonas macacae (PmCpf1), Lachnospira bacterium ND2006 (LbCpf1), Porphyromonas creviolicanis (PcCpf1), Prevotella diciens (PdCpf1), Moraxella boehmculi 237 (MbCpf1), Smithella species SC_K08D17 (SsCpf 1), Leptospira inadae (LiCpf1), Lachnospira bacterium MA2020 (Lb2Cpf1), Francisella novicida U112 (FnCpf1), Candidatus methanoplasma thermitum (CMtCpf1), and Cas12a from Eubacterium erythrocytes (EeCpf1).

[0067] In type V systems, binding of the nucleic acid target sequence typically involves a Cas12 protein and a crRNA, which are also involved in cleavage of the nucleic acid target sequence. In type V systems, the RuvC-like nuclease domain of the Cas12 protein sequentially cleaves both strands of the nucleic acid target sequence (see Swarts et al. (Mol. Cell, 2017, 66:221-233)), generating 5' overhangs, as opposed to the blunt ends generated by cleavage by the Cas9 protein.

[0068] The cleavage activity of the Cas12 protein in type V systems can be tracrRNA-independent (e.g., type VA). Some type V systems require only a single crRNA with a stem-loop structure that forms an internal duplex. The Cas12 protein binds to the crRNA in a sequence- and structure-specific manner by recognizing the stem-loop and adjacent sequences, particularly the 5' nucleotide of the spacer sequence that hybridizes to the nucleic acid target sequence. This stem-loop structure typically ranges from 15 to 22 nucleotides in length. Substitutions that disrupt this stem-loop duplex abolish cleavage activity, but other substitutions that do not disrupt the stem-loop duplex do not. Some type V systems, such as type V-F1, type VG, type VC, type VE (CasX), type VK, and type VB, require hybridization between the crRNA and tracrRNA. See, for example, Yan et al. (Science, 2019, 363(6422):88-91).

[0069] Cas12 Guide CRISPR Cas12 chRDNA guides capable of forming nucleoprotein complexes with a corresponding Cas12 protein, such as a Cas12a protein, are described in International Patent Application No. PCT / US21 / 55394, "DNA-containing polynucleotides and guides for CRISPR Type V systems and methods of making and using the same," filed October 18, 2021. The complexes described therein are capable of targeting sequences complementary to a spacer sequence.

[0070] Figure 1A shows an example of an Acidaminococcus sp. BV316 Cas12a guide molecule that includes an activation region (Figure 1A, 101) with a stem-loop duplex (Figure 1A, 102) and a spacer sequence (Figure 1A, 103) that includes a target binding sequence (Figure 1A, 104). Figure 1B shows another Cas12a guide molecule that includes an activation region (Figure 1B, 105) with a stem-loop duplex (Figure 1B, 106) and a spacer sequence (Figure 1B, 107) that includes a target binding sequence (Figure 1B, 108) and a 3' extension (Figure 1B, 109). The 3' extension (Figure 1B, 109) can be linked to the spacer sequence (Figure 1B, 107) via a linker sequence. Figure 1C shows another Cas12a guide molecule that includes an activation region (Figure 1C, 110) that includes a stem-loop duplex (Figure 1C, 111) and linker nucleotides (Figure 1C, 114) and a 5' extension (Figure 1C, 115), and a spacer sequence (Figure 1C, 112) that includes a target binding sequence (Figure 1C, 113).

[0071] In the Cas12 chRDNA guide molecules of the present disclosure, the targeting and activating regions, together or separately, may comprise DNA, RNA, or a mixture of DNA and RNA. In certain embodiments, the targeting and activating regions may also comprise other base analogs, modified nucleotides, abasic sites, etc., as well as synthetic, naturally occurring, and non-naturally occurring modified backbone residues or linkages, or combinations thereof.

[0072] In some embodiments, the activating region is 10-25 bases in length, including the optional abasic site.

[0073] In some embodiments, the targeting region is 10-30 bases in length, including optional abasic sites.

[0074] Figure 2 shows a Cas12a protein (Figure 2, 206) bound to a corresponding Cas12a chRDNA guide molecule (Figure 2, 204) containing a target binding sequence (Figure 2, 205). The Cas12a chRDNA guide / nucleoprotein complex unwinds a target polynucleotide containing the target sequence, and the target binding sequence of the Cas12 chRDNA guide molecule (Figure 2, 205) is bound to the target sequence (Figure 2, 207) by hydrogen bonds (Figure 2, indicated by vertical lines between polynucleotides). In Figure 2, the target polynucleotide comprises a target strand (Figure 2, 201) containing the target sequence (Figure 2, 207) and a non-target strand (Figure 2, 202) containing a PAM sequence (Figure 2, 203). The PAM sequence (Figure 2, 203) typically occurs upstream (i.e., 5') of the target sequence (Figure 2, 207) on the non-target strand (Figure 2, 202). The formation of hydrogen bonds between the target binding sequence (Figure 2, 205) and the target sequence (Figure 2, 207) of the Cas12a chRDNA guide molecule results in staggered cleavage (Figure 2, 208) of the target strand (Figure 2, 201) and the non-target strand (Figure 2, 202).

[0075] Figures 3A-3I show various standard and non-standard nucleotides for use in the Cas12 chRDNA guide molecules of the present disclosure. Table 2 shows a set of labels used in Figures 3A-3I. [Table 2-1] [Table 2-2]

[0076] Figure 4 shows a Cas12a protein (Figure 4, 406) bound to a corresponding Cas12a chRDNA guide molecule (Figure 4, 404) containing a target binding sequence (Figure 4, 405), which includes non-RNA nucleotides (Figure 4, 409), such as the standard and non-standard nucleotides presented in Figures 3B-3I. The Cas12a chRDNA guide / nucleoprotein complex unwinds a target polynucleotide containing the target sequence, and the target binding sequence of the Cas12 chRDNA guide molecule (Figure 4, 405) is bound to the target sequence (Figure 4, 407) by hydrogen bonds (Figure 4, indicated by vertical lines between the polynucleotides). In Figure 4, the target polynucleotide includes a target strand (Figure 4, 401) containing the target sequence (Figure 4, 407) and a non-target strand (Figure 4, 402) containing a PAM sequence (Figure 4, 403). The PAM sequence (Figure 4, 403) typically occurs upstream (i.e., 5') of the target sequence (Figure 4, 407) on the non-target strand (Figure 4, 402). Formation of hydrogen bonds between the target binding sequence (Figure 4, 405) of the chRDNA guide molecule and the target sequence (Figure 4, 407) results in staggered cleavage (Figure 4, 408) of the target strand (Figure 4, 401) and the non-target strand (Figure 4, 402).

[0077] Figure 5 shows an example of an Acidaminococcus species (BV3L6 strain) Cas12a crRNA guide molecule that contains an activation region (Figure 5, 501) that includes a stem-loop duplex (Figure 5, 502) and a spacer (Figure 5, 503) that includes a target binding sequence (Figure 5, 504). Each nucleotide position in the activation region (Figure 5, 501) and the spacer (Figure 5, 503) is labeled from the 5' end of the guide molecule, and the activation region and target binding region each contain RNA.

[0078] Figure 6 shows an example of an Acidaminococcus species (BV3L6 strain) Cas12a chRDNA guide molecule that includes an activation region (Figure 6, 601) that includes a stem-loop duplex (Figure 6, 602) and a spacer (Figure 6, 603) that includes a target binding sequence (Figure 6, 604). Each nucleotide position in the activation region (Figure 6, 601) and spacer (Figure 6, 603) is labeled from the 5' end of the guide molecule, where the activation region includes a mixture of RNA (white fill) and DNA (gray fill), and the target binding sequence includes a mixture of RNA (white fill) and DNA (gray fill).

[0079] Figure 7 shows an example of an Acidaminococcus species (BV3L6 strain) Cas12a chR DNA guide molecule that contains an activation region (Figure 7, 701) that includes a stem-loop duplex (Figure 7, 702) and a spacer (Figure 7, 703) that includes a target binding sequence (Figure 7, 704). Each nucleotide position in the activation region (Figure 7, 701) and spacer (Figure 7, 703) is labeled from the 5' end of this guide molecule, and the activation region contains a mixture of RNA (white fill) and DNA (gray fill). Cas12a chRDNA guide molecules further include other non-standard nucleotides, such as chemically modified sugar nucleotides (Figure 7, 705), abasic ribonucleotides (Figure 7, 706), deoxyribonucleotides with chemically modified backbones (Figure 7, 707), ribonucleotides with chemically modified backbones (Figure 7, 708), and abasic deoxyribonucleotides (Figure 7, 709).

[0080] Figure 8 shows the formation of a Cas12 chRDNA guide / nucleoprotein complex, in which the Cas12 protein (Figure 8, 801) binds to the Cas12 chRDNA guide molecule (Figure 8, 802) to form a Cas12 chRDNA guide / nucleoprotein complex (Figure 8, 803). The Cas12 chRDNA guide / nucleoprotein complex (Figure 8, 803) binds to a target polynucleotide (Figure 8, 804), which contains a target sequence complementary to the target binding sequence of the Cas12 chRDNA guide molecule, resulting in hydrogen bonds being formed between the target binding sequence of the Cas12 chRDNA guide molecule and the target sequence (Figure 8, 805).

[0081] Figure 9 shows the generation of insertions or deletions (indels) in a target polynucleotide by the Cas12 chRDNA guide / nucleoprotein complex, in which the Cas12 protein (Figure 9, 901) complexed with the Cas12 chRDNA guide molecule (Figure 9, 902) binds to a target polynucleotide (Figure 9, 903) containing a PAM (Figure 9, 904), and the Cas12 chRDNA guide / nucleoprotein complex cleaves the target polynucleotide (Figure 9, 905). After targeting occurs, the Cas12 chRDNA guide / nucleoprotein complex dissociates from the target polynucleotide (Figure 9, 906), leaving the target polynucleotide with an upstream (i.e., 5') strand (Figure 9, 907) and downstream (i.e., 3') strand (Figure 9, 908) relative to the PAM (Figure 9, 904). The cell's DNA repair machinery repairs the target polynucleotide by inserting or deleting (Figure 9, 910) a sequence around the cut site in the target polynucleotide. The upstream strand (Figure 9, 911) and downstream strand (Figure 9, 912) are rejoined, and the edited target polynucleotide (Figure 9, 914) contains an indel (Figure 9, 913) at the cut site, such that the edited target polynucleotide has a different sequence from the unedited target polynucleotide. In some embodiments, generation of an insertion or deletion (indel) in the target polynucleotide by the Cas12 chRDNA guide / nucleoprotein complex occurs intracellularly.

[0082] Figure 10 illustrates the integration of a donor polynucleotide sequence into a target polynucleotide, where a Cas12 protein (Figure 10, 1001) complexed with a Cas12 chRDNA guide molecule (Figure 10, 1002) binds to a target polynucleotide (Figure 10, 1003) containing a PAM (Figure 10, 1004), and the target polynucleotide is cleaved by the Cas12 chRDNA guide / nucleoprotein complex (Figure 10, 1005). After targeting occurs, the Cas12 chRDNA guide / nucleoprotein complex dissociates from the target polynucleotide (Figure 10, 1006), leaving the target polynucleotide containing an upstream (i.e., 5') strand (Figure 10, 1007) and downstream (i.e., 3') strand (Figure 10, 1008) relative to the PAM (Figure 10, 1004), providing a donor polynucleotide (Figure 10, 1009). The cell's DNA repair machinery uses the donor polynucleotide (Figure 10, 1011) to repair the target polynucleotide (Figure 10, 1010). The resulting edited target polynucleotide (Figure 10, 1010) comprises the donor sequence (Figure 10, 1011) at the target site. In some embodiments, integration of the donor polynucleotide sequence into the target polynucleotide occurs intracellularly.

[0083] Figure 11 shows nicking of a target polynucleotide, where a Cas12 protein (Figure 11, 1101) complexed with a Cas12 chRDNA guide molecule (Figure 11, 1102) containing DNA bases in its target binding sequence (Figure 11, 1106) binds to a target polynucleotide (Figure 11, 1103) containing a PAM (Figure 11, 1104), and the Cas12 chRDNA guide / nucleoprotein complex nicks the target polynucleotide on only one strand of the target polynucleotide (Figure 11, 1105).

[0084] Figure 12 illustrates the use of two nicking Cas12 chRDNA guide / nucleoprotein complexes to generate staggered double-stranded breaks in a target polynucleotide, where a first Cas12 chRDNA guide / nucleoprotein complex binds to a target sequence upstream (i.e., in the 5' direction) of the target polynucleotide (Figure 12, 1201), forming a first nick in the target polynucleotide (Figure 12, 1202), and a second Cas12 chRDNA guide / nucleoprotein complex binds to a target sequence downstream (i.e., in the 3' direction) of the target polynucleotide (Figure 12, 1203), forming a second nick in the target polynucleotide (Figure 12, 1204). After tandem nicking occurs, the cleaved target polynucleotide contains an upstream (i.e., in the 5' direction) strand with a 5' overhang (Figure 12, 1205) and a downstream (i.e., in the 3' direction) strand (Figure 12, 1206). A donor polynucleotide is provided, and the cell's DNA repair machinery uses the donor polynucleotide (Figure 12, 1208) to repair the target polynucleotide (Figure 12, 1207). The resulting edited target polynucleotide (Figure 12, 1209) contains the donor sequence (Figure 12, 1210) at the tandemly nicked sites. In some embodiments, the use of two nicking Cas12chRDNA guide / nucleoprotein complexes to generate staggered DSBs in the target polynucleotide occurs intracellularly.

[0085] Methods for designing specific Cas12 chRDNA guide molecules, in which deoxyribonucleotides and, optionally, additional modifications (e.g., base analogs, modified nucleotides, abasic sites, modified backbone residues or linkages, or combinations thereof) can be designed, are described, for example, in PCT / US21 / 55394. Briefly, to design a Cas12 guide, the genomic sequence of the gene to be targeted is first identified. The exact region of the gene selected for targeting will vary depending on the specific application. For example, to activate or repress a target gene, Cas12 can be targeted to the promoter driving expression of that gene or to a constitutively expressed exon 5' to reduce the likelihood of the target region being removed from the mRNA by alternative splicing. Other exons in the N-terminal region can be targeted, as frameshift mutations in these regions result in nonfunctional protein products. Exons encoding essential protein domains can also be targeted. For gene editing using HDR, the target sequence should be near the desired location of the edit. In this case, the location where editing is desired is identified and a target sequence near it is selected.

[0086] In some embodiments, the Cas12 chRDNA guide may be designed to bind outside the cleavage site of the Cas12 protein, so that the target nucleic acid can be separated from the Cas12 nucleoprotein complex. In some embodiments, the Cas12 chRDNA guide may be designed to bind inside the cleavage site of the Cas12 protein. In this case, the target nucleic acid can be bound to the Cas12 nucleoprotein complex.

[0087] In some embodiments of the Cas12 chRDNA guide molecule, the targeting region, the activation region, or both comprise deoxyribonucleotides or modified nucleotides. In some embodiments, the Cas12a chRDNA guide comprises one or more deoxyribonucleotides, e.g., 23 or fewer deoxyribonucleotides. In some embodiments, all of the deoxyribonucleotides in the targeting region of the chRDNA form standard base pairs with the target sequence. In some embodiments, at least one of the deoxyribonucleotides in the targeting region of the chRDNA does not form standard base pairs with the target sequence or forms a non-standard base pair with the target sequence.

[0088] Cas12 protein The Cas12 proteins of the present disclosure include, but are not limited to, wild-type Cas12 proteins, modified Cas12 proteins, Cas12 protein mutants, Cas12 orthologs, and combinations thereof from type V CRISPR-Cas systems. In some embodiments, the Cas12 protein is a wild-type Cas12a protein, a modified Cas12a protein, a Cas12a protein mutant, a Cas12a ortholog, or a combination thereof.

[0089] Cas12 proteins typically consist of six domains: REC1, REC2, PAM-interacting (PI), Nuclease (Nuc), Wedge (WED), and RuvC. See, e.g., Yamano et al. (Cell, 2016, 165(4):949-962). The WED and RuvC domains may have a tripartite structure with other domain sequences interposed between them. For example, the WED domain sequence of Acidaminococcus sp. Cas12a contains REC1, REC2, and PI domain sequences. Furthermore, certain subtypes of Cas12 proteins contain a bridge helix domain adjacent to or located between the RuvC domain sequences.

[0090] To generate modified Cas12 proteins, regions of the Cas12 protein can be modified to modulate the activity of the Cas12 protein. For example, regions corresponding to residues in the PI domain (598-718) and WED domain (526-597 and 719-883) of the Acidaminococcus sp. (BV3L6 strain) Cas12a protein can be modified to alter PAM specificity. See, e.g., Toth et al. (Nucleic Acid Research, 2020, 48(7):3722-3733). Regions corresponding to residues in the REC1 (24-319) and REC2 (320-526) domains of the Acidaminococcus sp. (BV3L6 strain) Cas12a protein can be modified to alter target binding and cleavage kinetics. Regions of the REC1 (226-304) and REC2 (368-435) domains interact directly with the target binding sequence and the PAM-distal end of the target sequence and can be engineered to modify the cleavage efficiency of the target sequence. Regions of the Nuc domain (1066-1261) and RuvC domain (940-956, 957-1065, and 1261-1307) can be engineered to alter the cleavage efficiency of the target strand, non-target strand, or target and non-target strands of the target sequence. Engineering these regions can include introducing mutations, substitution with corresponding regions from other Cas12 orthologs, deletions, insertions, etc.

[0091] Modified Cas12 proteins can be used in combination with a Cas12 chRDNA guide molecule to alter the activity or specificity of the Cas12 protein. In some cases, the Cas12 protein can be modified to enhance its activity or specificity when complexed with a Cas12 chRDNA guide molecule, where Cas12 modifications are made in the REC1, REC2, RuvC, WED, and / or Nuc domains. In some cases, the Cas12 protein can be modified to enhance its activity or specificity when complexed with a Cas12 chRDNA guide molecule, where Cas12a modifications are made in the following regions: 226-304, 368-435, 940-956, 978-1158, 1159-1180, and 1181-1298 (numbering based on the Acidomicocus spp. Cas12a sequence).

[0092] In some embodiments, the Cas12 protein is an nCas12 protein. The nCas12 protein is a nuclease-deficient mutant, also referred to as a "nicking Cas12" or "Cas12 nickase." Such molecules lack some endonuclease activity and are therefore capable of nicking only one strand of a target nucleic acid. See, for example, Jinek et al. (Science, 2012, 337:816-821). This can be achieved, for example, by introducing mutations into the RuvC nuclease domain. Non-limiting examples of such modifications include D917A, E1006A, and D1225A to the RuvC nuclease domain of the F. novicida Cas12a protein. It is understood that mutations of other catalytic residues to reduce the activity of the RuvC nuclease domain can also be performed by those skilled in the art. The resulting nCas12 protein is unable to cleave double-stranded DNA, but retains the ability to form a complex with a guide molecule, bind to a target DNA sequence, and nick only one strand of the target DNA. Targeting specificity is determined by Cas12 protein binding to the PAM sequence and complementary base pairing of the guide molecule to the genomic locus. In some embodiments of the present disclosure, the nCas12 protein is an nCas12a protein.

[0093] In some embodiments, the Cas12 protein is a dCas12 protein. dCas12 proteins are nuclease-inactivated mutants, also referred to as "catalytically inactive Cas12 proteins," "enzymatically inactive Cas12," "catalytically abolished Cas12," or "abolished Cas12." Because such molecules lack endonuclease activity, they can be used for RNA-guided gene regulation. See, for example, Jinek et al. (Science, 2012, 337:816-821). Mutation of catalytic residues to abolish the activity of the RuvC domain can be performed by those skilled in the art. The resulting dCas12 protein is unable to cleave double-stranded DNA but retains the ability to form a complex with a guide molecule and bind to the target DNA sequence. Targeting specificity is determined by Cas12 protein binding to the PAM sequence and complementary base pairing of the guide molecule to the genomic locus. In some embodiments of the present disclosure, the dCas12 protein is a dCas12a protein.

[0094] Certain Cas12 protein subtypes lack nuclease activity due to either inactivation of the RuvC-like nuclease domain or the partial or complete absence of the RuvC-like nuclease domain. One such subtype, the VK type and the related protein Cas12k, is instead related to the Tn7-like transposable elements tnsB, tnsC, and tniQ. See, e.g., Strecker et al. (Science, 2019, 364(6448):48-53). Cas12k retains the ability to bind to target DNA sequences in complex with guide molecules, and the associated Tn7-like protein facilitates RNA-guided transposition of DNA sequences. In some embodiments of the present disclosure, the Cas12 chRDNA guide / nucleoprotein complex is a Cas12k chRDNA guide / nucleoprotein complex.

[0095] Other amino acid modifications may include glycosylated forms of amino acids, aggregation complexes with other molecules, and covalent complexes with unrelated chemical moieties (e.g., pegylated molecules). Covalent variants may be created by linking functional groups to groups found on the amino acid chain or the N- or C-terminal residues. In some cases, mutated site-directed polypeptides may also include allelic and species variants.

[0096] In certain embodiments, the Cas12 protein can be a fusion or chimeric protein that includes a first domain from the Cas12 protein and a second domain from a different protein, such as a Csy4 protein. Fusion modifications to the Cas12 protein can confer additional activity to the modified Cas12 protein. Such activities can include nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, reverse transcriptase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, and / or myristoylation or demyristoylation activity that modify polypeptides (e.g., histones) associated with nucleic acid target sequences.

[0097] In certain embodiments, a Cas12 protein can include one or more NLS sequences (e.g., added to and / or inserted within the Cas12 protein sequence). The NLS sequences can be located, for example, at the N-terminus, C-terminus, or internally within the Cas12 protein (e.g., Cas12a protein), or can include combinations thereof (e.g., one or more NLSs at the N-terminus and one or more NLSs at the C-terminus). NLS sequences can be derived from SV40 large T antigen, nucleoplasmin, 53BP1, VACM-1 / CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrp1, cMyc(1), cMyc(2), mouse c-able IV, Matα2, and MINIYO.

[0098] The NLS sequence may be covalently linked (e.g., to the Cas12 protein, to another NLS sequence, or to a fusion peptide sequence linked to the Cas12 protein) either directly or via a linker polypeptide. The length of the linker sequence can be optimized depending on the structural features of the particular Cas12 protein (e.g., solvent accessibility of the termini, presence of other important functional peptide sequences at the termini, etc.) to ensure accessibility of the NLS sequence for binding and transport of the corresponding importin protein.

[0099] In some embodiments, the linker sequence comprises at least one glycine, serine, and / or threonine residue. In some embodiments, the linker sequence comprises at least one glycine residue and at least one serine residue. In some embodiments, the linker sequence comprises multiple glycine residues and at least one serine residue. In some embodiments, the linker sequence consists of or comprises a GS sequence.

[0100] Genome editing in cells using a Cas12-chRDNA-guided nucleoprotein complex Delivery of the disclosed Cas12 chRDNA guide molecules, Cas12 proteins, and Cas12-chRDNA guide nucleoprotein complexes into cells in vitro, ex vivo, or in vivo can be achieved by several methods known to those skilled in the art. Non-limiting methods for introducing these components into cells include viral vector delivery, sonoporation, cell squeezing, electroporation, nucleofection, lipofection, particle gun technology, microprojectile bombardment, or chemicals (e.g., cell-penetrating peptides).

[0101] In some embodiments, electroporation can be used to deliver the Cas12 chRDNA guide molecule of the present disclosure into cells. Electroporation can also be used to deliver the Cas12 chRDNA guide / nucleoprotein complex of the present disclosure. In these methods, the chRDNA guide molecule or the Cas12 chRDNA guide / nucleoprotein complex is mixed with target cells in an electroporation buffer to form a suspension. An electric pulse at an optimal voltage is then applied to this suspension, which causes transient pores to form in the phospholipid bilayer of the cell membrane, allowing charged molecules (such as nucleic acids and proteins) to pass through the pores into the cells. Reagents and equipment for performing electroporation are commercially available.

[0102] In some embodiments, delivery of the Cas12 chRDNA guide, Cas12 protein, and Cas12-chRDNA guide nucleoprotein complex is achieved by packaging these components into a compartment. The compartment containing the components can be administered in vivo (e.g., into the cells of an organism, provided that in some embodiments, the organism is a non-human organism). In some embodiments, the compartment is a biological compartment, such as a virus (lentivirus, adenovirus) or a liposome. In some embodiments, the compartment is a non-biological compartment selected from nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vesicles, polyethylene glycol particles, hydrogels, and micelles.

[0103] In some embodiments, the compartment is a lipid nanoparticle (LNP).

[0104] When a Cas12 chRDNA guide molecule is introduced into a cell along with a Cas12 protein, thereby forming a Cas12-chRDNA guide nucleoprotein complex, the Cas12-chRDNA guide nucleoprotein complex can be used to cleave or bind to a target nucleic acid. The nucleoprotein complex can hybridize to a target nucleic acid containing a PAM. The nucleoprotein complex includes a Cas12 chRDNA guide having a targeting region complementary to a nucleic acid target sequence. Optionally, a second Cas12-chRDNA guide nucleoprotein complex including a Cas12 chRDNA guide having a second targeting region complementary to a second nucleic acid target is also introduced into the cell.

[0105] The step of binding to the nucleic acid target sequence can be performed in vitro (e.g., in a biochemical reaction or in cultured cells), in vivo (e.g., in cells of an organism or patient), or ex vivo (e.g., in cells removed from a subject or patient for return to the subject or patient).

[0106] In another embodiment, a Cas12 protein-chRDNA guide nucleoprotein complex can be used to introduce a donor polynucleotide into a cell, facilitating the integration of at least a portion of the donor polynucleotide into the genomic DNA of the cell. Typically, the donor polynucleotide is brought into proximity to the site-specific target nucleic acid cleavage site by binding to a Cas12 protein (e.g., Cas12a) that generates double-strand breaks. This proximity enhances the insertion (e.g., homologous recombination) of the donor polynucleotide into the double-strand break site.

[0107] Therapeutic compositions, uses, and methods The disclosed Cas12 chRDNA guide molecules and Cas12-chRDNA guide nucleoprotein complexes can be used to generate modified cells for therapeutic purposes, which can be used in adoptive cell therapy, such as adoptive immunotherapy, as disclosed in PCT / US21 / 55394.

[0108] Lymphocytes can be isolated from a subject, such as a human subject, by techniques well known in the art, e.g., from blood or solid tumors, such as in the case of TILs, or from lymphoid organs, such as the thymus, bone marrow, lymph nodes, and mucosa-associated lymphoid tissue. Once isolated, lymphocytes can be characterized in terms of specificity, frequency, and function, e.g., by ELISPOT assays, which measure the frequency of T cell responses. Isolated lymphocytes can optionally be activated using techniques well known in the art to promote proliferation and differentiation into specialized effector lymphocytes.

[0109] In some embodiments, isolated lymphocytes can be modified using the Cas12-chRDNA-guided nucleoprotein complex of the present disclosure, for example, by inserting a gene encoding a chimeric antigen receptor (CAR). In some embodiments, the Cas12-chRDNA-guided nucleoprotein complex is used to inactivate an endogenous T cell receptor, such as the TRAC gene. The resulting lymphocytes form CAR-T cells or CAR-NK cells for use in adoptive immunotherapy.

[0110] Additionally, the Cas12-chRDNA nucleoprotein complex can be used to protect the survival of adoptive cells in a host. In some embodiments, this protective modification involves inactivation of an immune checkpoint protein, such as the PD-1 protein encoded by the PDCD1 gene. Other immune checkpoint proteins that can be inactivated include cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152), LAG3 (also known as CD223), Tim3 (also known as HAVCR2), BTLA (also known as CD272), BY55 (also known as CD160), TIGIT (also known as IVSTM3), LAIR1 (also known as CD305), SIGLEC10, 2B4 (also known as CD244), PPP2CA, PPP2CB, PTPN6, PTPN22, and CD19. 96, CRTAM, SIGLEC7, SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3. In some embodiments, the Cas12-chRDNA guide nucleoprotein complex is used to inactivate one or more immune checkpoint molecules.

[0111] In some embodiments, the protective modification comprises inactivation of β2-microglobulin (B2M), a component of MHC class I molecules present on nucleated cells, hi some embodiments, the protective modification comprises insertion of an HLA-E gene into the inactivated B2M locus.

[0112] In some embodiments, the present invention is a method of treating or alleviating a disease or condition by administering cells modified with a V-type CRISPR system comprising a chRDNA of the present disclosure. Table 3 lists diseases or conditions with genes targeted with a V-type CRISPR system comprising a chRDNA. [Table 3-1] [Table 3-2] [Table 4] [Table 5] [Table 6]

[0113] In some embodiments, the Type V CRISPR system comprising the chRDNA generates a genomic modification that results in the expression or elimination in the modified cell of one or more genes set forth in Table 3. In some embodiments, aberrant expression of the gene results in a disease or condition set forth in Table 3, and elimination or expression of the gene by genomic modification alleviates the disease or condition.

[0114] In some embodiments, the exogenous nucleic acid is inserted into the genome of the cell. According to the present disclosure (see Figures 10 and 12), a donor polynucleotide is provided that includes one or more copies of an exogenous gene. The exogenous gene includes a protein-encoding sequence under the control of its promoter, another promoter active in the target cell, or a constitutive promoter. The exogenous gene is selected from the list presented in Table 3.

[0115] In some embodiments, in the cases of heart failure, spinal cord injury, SMA, and ALS, a V-type CRISPR system containing chRDNA generates a genomic modification that eliminates expression of genes that cause transplant rejection. This genomic modification allows for the transplantation of foreign neurons (in the case of spinal cord injury, SMA, or ALS) or foreign cardiomyocytes (in the case of heart failure) without rejection of the foreign neurons or foreign cardiomyocytes by the recipient's immune system. In some embodiments, the present invention is a method of treating a disease or condition of the central nervous system (CNS), the method comprising administering to a patient a gene encoding an HLA-binding protein, including motor neurons. - / CIITA - In some embodiments, the present invention provides a method for treating heart failure, the method comprising administering a gene modification by a V-type CRISPR system containing chRDNA, which results in the formation of neurons. - / CIITA - The present invention includes genetic modifications achieved by a V-type CRISPR system containing chRDNA, which result in the formation of cardiomyocytes. In some embodiments, the V-type CRISPR system containing chRDNA causes inactivation of one or more MHC class I genes selected from HLA-A, HLA-A2, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, and further causes inactivation of the class II transactivator (CIITA) gene.

[0116] Lipid nanoparticles (LNPs) In some embodiments, components of the Type V CRISPR system, including chRDNA, are encapsulated within microscopic lipid droplets, sometimes referred to as lipid nanoparticles (LNPs). Examples of LNPs are described, for example, in Sharma et al. (2014), "Next generation delivery system for proteins and genes of therapeutic purpose: why and how?" Biomed Res Int. 2014:327950, and further in U.S. Patent Application Publication No. 2019 / 0136231, "Lipid nanoparticle formulations for CRISPR / Cas components," U.S. Patent Application Publication No. 2016 / 0317676, "Methods and compositions for delivery of nucleic acids," U.S. Patent Application Publication No. 2019 / 0022247, "Lipids and lipid nanoparticle compositions for delivery of nucleic acids," and U.S. Patent Application Publication No. 2021 / 0251898, "Lipid nanoparticles for mRNA vaccines."

[0117] In some embodiments, the LNPs used herein have a diameter of about 100 nm to about 1 μm, preferably less than 100 nm. In some embodiments, the LNPs comprise one or more cationic lipids. When combined, the cationic lipids have a pK aThe cationic lipids may be selected so that the measured value of β is 6.1 or greater and 6.7 or less, e.g., 6.2 to 6.6, or 6.3 to 6.5. The cationic lipids may have a head group, one or more hydrophobic tails, and a linker between the head group and one or more tails. The head group may include an amine, which is the site of a positive charge. The amine may be a primary, secondary, or tertiary amine, or a quaternary amine. The one or more hydrophobic tails may include two hydrophobic chains, which may be the same or different. The tails may be aliphatic chains, fatty acid chains, or other hydrophobic chains. The linker may include, for example, a glyceride linker, an acyclic glyceride analog linker, or a cyclic linker. The linker may include a functional group such as an ether, ester, phosphate, phosphonate, phosphorothioate, sulfonate, disulfide, acetal, ketal, imine, hydrazone, or oxime. Cationic lipids contain one or more amine groups that are positively charged. Preferred cationic lipids are ionizable so that they can exist positively or neutrally depending on the pH. The ionization of cationic lipids can affect the surface charge of lipid nanoparticles (LNPs), which can affect the absorption of plasma proteins, blood clearance, tissue distribution, and ability to fuse with cell membranes.

[0118] In some embodiments, the LNP further comprises a neutral lipid. The neutral lipid may be selected from DSPC, DPPC, POPC, DOPE, or SM. The lipid capable of reducing aggregation may be a PEG lipid. In some embodiments, the lipid particle further comprises a sterol. In some embodiments, the molar ratio of all cationic lipids in the particle is about 20% to about 60%, the neutral lipid may be present in a molar ratio of about 5% to about 25%, the sterol may be present in a molar ratio of about 25% to about 55%, and the PEG lipid may be PEG-DMA, PEG-DMG, or a combination thereof, and may be present in a molar ratio of about 0.5% to about 15%.

[0119] Other examples of lipids used to make LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC cholesterol, DOTAP cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Other examples of PEG-modified lipids include PEG-CerC14 and PEG-CerC20.

[0120] In some embodiments, the LNPs further comprise a lipid that can reduce aggregation, eg, aggregation of the LNPs.

[0121] In some embodiments, the surface of the LNP is further modified with a polymer or lipid (e.g., chitosan, a cationic polymer, or a cationic lipid) or conjugated to a targeting molecule (an antibody specific for a cell surface receptor or a natural ligand of a cell surface receptor) to target the nanoparticle to the appropriate cell type and increase the likelihood of cellular uptake, as described in Jian et al. (2012) Cationic core shell liponanoparticles for ocular gene delivery, Biomaterials 33(30):7621-30).

[0122] Delivery of LNP In some embodiments, the present invention involves delivering a V-type CRISPR system comprising chRDNA as described herein to cells and tissues of a patient. In some embodiments, delivery is achieved by lipid nanoparticles (LNPs) as described herein. Delivery may be to patient cells in vitro, ex vivo, or in vivo. In some embodiments, LNPs comprising a V-type CRISPR system comprising chRDNA are administered systemically to a patient (i.e., intravenously into the systemic circulation). In some embodiments, a target nucleic acid is expressed (or misexpressed) in a specific organ, and LNPs comprising a V-type CRISPR system comprising chRDNA are administered to that organ. In some embodiments, LNPs comprising a V-type CRISPR system comprising chRDNA are contacted with patient cells ex vivo, and the treated cells are administered to a patient.

[0123] In some embodiments, the target nucleic acid is expressed (or aberrantly expressed) in the liver. In such embodiments, LNPs containing a V-type CRISPR system containing chRDNA are administered systemically or into the hepatic circulation (e.g., the portal vein or another hepatic blood vessel or blood vessels leading to the liver).

[0124] In some embodiments, the target nucleic acid is expressed (or aberrantly expressed) in the liver, more specifically in hepatocytes. In such embodiments, a patient's own (autologous) hepatocytes or a donor's (allogeneic) hepatocytes are treated ex vivo with LNPs containing a V-type CRISPR system that includes chRDNA, and the treated hepatocytes are administered systemically to the patient or into the patient's hepatic circulation (e.g., the portal vein or another hepatic blood vessel or blood vessels that feed into the liver).

[0125] In some embodiments, the target nucleic acid is expressed (or aberrantly expressed) in liver sinusoidal endothelial cells or hematopoietic cells throughout the body (e.g., the Factor VIII gene, deficiency of which causes hemophilia A). In such embodiments, target cells for in vivo or ex vivo administration of a Type V CRISPR system comprising a chRDNA include liver sinusoidal endothelial cells, progenitor cells that differentiate into liver sinusoidal endothelial cells, hemogenic endothelial cells, or progenitor cells that differentiate into hemogenic endothelial cells.

[0126] In some embodiments, the target nucleic acid is expressed (or aberrantly expressed) in cells of the eye. In some embodiments, LNPs containing a V-type CRISPR system containing chRDNA are delivered to the eye (intraocular delivery). In some embodiments, delivery is intravitreal. In some embodiments, delivery is directly to the retina to reach the retinal pigment epithelium.

[0127] In some embodiments, the administration is in combination with a pharmaceutically acceptable carrier.

[0128] Induced pluripotent stem cells (iPSCs) In some embodiments, the invention includes a method of ex vivo cell-based therapy comprising editing the genome of induced pluripotent stem cells (iPSCs) using a type V CRISPR system containing chRDNA, differentiating the edited iPSCs into cells of a desired lineage, and transplanting the differentiated cells into a patient.

[0129] In some embodiments, the iPSCs are patient-derived (autologous). In some embodiments, somatic cells are taken from a subject or patient, reprogrammed into induced pluripotent stem cells (iPSCs), genome-edited using the V-type CRISPR system containing chRDNA described herein, redifferentiated into cells of the desired cell type, and administered to the same subject or patient.

[0130] In some embodiments, the iPSCs are donor-derived or cell line-derived. In some embodiments, differentiation of iPSCs is artificially induced in vitro or ex vivo by administration of specific stimuli.

[0131] Reprogramming cells into iPSCs In some embodiments, reprogramming of differentiated cells to iPSCs is artificially induced in vitro or ex vivo by administration of an exogenous agent. In some embodiments, reprogramming of differentiated cells to iPSCs involves restoring one or more inherited nucleic acid modification patterns, such as methylation. In some embodiments, reprogramming of differentiated cells to iPSCs is achieved by expressing specific genes in the differentiated cells. In some embodiments, the genes are introduced into the cells using a plasmid or viral expression vector. In some embodiments, the genes are introduced as mRNA that can be translated within the cell. In some embodiments, the genes that induce reprogramming are one or more of Oct4, Sox2, Klf4, and c-Myc, as described in Takahashi et al. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell 126(4):663-76. In some embodiments, the gene that induces reprogramming is one or more or all three of Oct4, Sox2, and NANOG, as described in Budniatzky et al. (2014) Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming, Stem Cells Transl Med. 3(4):448-57, and references cited therein. In some embodiments, the gene that induces reprogramming is one or more of Sox1, Sox3, Sox15, Sox18, Klf1, Klf2, Klf5, NR5A2, c-Myc, l-Myc, n-Myc, Rem2, Tert, and LIN28 or Wnt.In some embodiments, reprogramming of somatic cells to iPSCs can be achieved using a variety of inhibitors, including MEK inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyric acid (e.g., sodium phenylbutyrate), and valproic acid ((VP A) and other short-chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0 The activity can be enhanced by incorporating one or more of the following: 103, NVP-LAQ-824, CBHA (m-carboxycinnamic bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50.

[0132] In some embodiments, reprogramming of differentiated cells to iPSCs is assessed by detecting or measuring the expression of markers associated with iPSCs. In some embodiments, reprogramming is assessed by detecting or measuring the expression of one or more genes selected from SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In some embodiments, reprogramming to motor neurons is assessed by detecting or measuring the expression of a combination of Sox1, Pax6, Nestin, HB9, MAP2, NeuroFilament, Tuj1, and Olig2. In some embodiments, expression is assessed or measured by detecting the mRNA of interest by Southern blotting or PCR, such as reverse transcription PCR (RT-PCR), real-time PCR (rtPCR), and digital droplet PCR (ddPCR), or nucleic acid sequencing. In some embodiments, expression is confirmed by detecting the protein in question by immunological methods selected from Western blot, flow cytometry, immunochemical methods, and immunocytochemical methods.

[0133] iPSC modification In some embodiments, the invention includes methods of genetically modifying iPSCs to result in expression of a gene, the aberrant expression of which is associated with a disease or condition in a patient. The genetically modified iPSCs are then differentiated into a cell type characterized by the aberrant expression of the gene in the patient, and the differentiated cells are administered to the patient to alleviate symptoms of the disease or condition.

[0134] In some embodiments, iPSCs are cultured in a suitable medium (e.g., mTeSR-plus medium (STEMCELL Technologies, Cambridge, Mass.) with one or more supplements) prior to nucleofection with a V-type CRISPR system containing chRDNA. In some embodiments, cells are dispersed with Accetase (STEMCELL Technologies, Cambridge, Mass.) prior to nucleofection. In some embodiments, cells are counted to achieve the desired number of cells in the nucleofection well. In some embodiments, 4×10 3 ~2×10 4 Cells are present in one well of a 96-well plate. The Cas12a guide / nucleoprotein complex is added and nucleofection is performed according to the manufacturer's recommendations. In some embodiments, Nucleocuvette™ plates and the Nucleofector™ device are used (Lonza, Allendale, NJ).

[0135] iPSC differentiation In some embodiments, the method includes differentiating iPSCs genetically modified using a type V CRISPR system containing chRDNA according to the methods of the invention. In some embodiments, the iPSCs are differentiated into neurons, including motor neurons, cells of the central nervous system (CNS), such as retinal cells or glial cells, or cells of the cardiovascular system, such as endothelial cells or cardiomyocytes. In some embodiments, the iPSCs are differentiated into hepatocytes or mesenchymal stem cells. In some embodiments, the iPSCs are differentiated into ocular (non-neuronal) cells, such as corneal, scleral, or choroidal cells.

[0136] In some embodiments, iPSCs are differentiated into neurons. In some embodiments, the method includes preparing a fresh culture of confluent iPSCs and dissociating the confluent culture of iPSCs before seeding them into neural induction medium containing one or more of serum replacement, non-essential amino acids, glutamine or glutamine substitute, vitamins, a GSK-3 inhibitor, a TGF-β receptor or TGF-β inhibitor, an ALK inhibitor, dorsomorphin, and compound E. In some embodiments, one or more medium changes are performed. In some embodiments, the subsequent medium contains growth factors such as FGF and EGF. In some embodiments, for the differentiation and formation of motor neurons, the subsequent medium is MN induction medium and Neurobasal Medium. In some embodiments, the medium is further supplemented with one or more of all-trans retinoic acid, sonic hedgehog protein, purmorphamine, SAG dihydrochloride, CNTF, and GDNF. In some embodiments, to assess differentiation into neurons, the cells are evaluated by fluorescence microscopy. In some embodiments, cells are fixed in formaldehyde or paraformaldehyde, permeabilized with, for example, Triton-X and / or Tween-20, and stained with a primary antibody capable of specifically binding to one or more of Sox1, Pax6, Nestin, HB9, MAP2, NeuroFilament, Tuj1, and Olig2. To further assess differentiation into motor neurons, calcium activity is assessed, for example, by imaging using Oregon Green 488 BAPTA-2 calcium indicator. In some embodiments, electrical activity of the cells is measured, for example, using a MultiClamp 700B microelectrode amplifier (Molecular Devices, San Jose, Calif.).

[0137] In some embodiments, iPSCs are differentiated into muscle cells, such as cardiomyocytes. In some embodiments, iPSCs are freshly expanded so that the cultures reach 60-70% confluency, treated with one or more of a GSK-3 inhibitor and a Wnt-dependent phosphorylation inhibitor, and incubated in insulin-containing medium. In some embodiments, cells are assessed by measuring gene expression of cardiomyocyte-specific markers (e.g., TBX5, TNNT2, MYH6, and MYL7) and, optionally, by measuring the decrease in expression of pluripotency markers (NANOG, POUF5F1).

[0138] xenograft In some embodiments, the invention is a method of producing a transgenic non-human mammal for xenotransplantation by modifying the animal's genome using a type V CRISPR system containing chRDNA to eliminate the expression of one or more genes that cause immunological incompatibility and / or to introduce one or more genes that establish immunological compatibility between a non-human donor and a human recipient or reduce the likelihood of rejection by the human immune system.

[0139] In some embodiments, the transgenic non-human mammal is a pig, and the method comprises delivering a Type V CRISPR system containing chRDNA into an oocyte, egg, or zygote of the pig, followed by implanting the genetically modified oocyte, egg, or zygote into a foster mother female.

[0140] In some embodiments, the transgenic non-human mammal is a pig, and the method comprises delivering a type V CRISPR system comprising a chRDNA into a somatic cell of the pig, and further comprises transferring the nucleus of the somatic cell into an enucleated egg or zygote, and subsequently transferring the resulting egg or zygote into a foster mother female.

[0141] In some embodiments, the genomic modification is the insertion of a functional copy of a gene that results in the production of a foreign protein. In some embodiments, the foreign protein has cytoprotective, anticoagulant, complement inhibitory, or immunosuppressive properties. In some embodiments, the foreign gene is a human gene. In some embodiments, the foreign protein has cytoprotective properties and the gene is selected from A20, HO-1, FAT-1, and TNF-α receptor. In some embodiments, the foreign protein has anticoagulant properties and the gene is selected from CD39, hirudin, TFPI, EPCR, and TBM. In some embodiments, the foreign protein has complement inhibitory properties and the gene is selected from CD46, DAF (CD55), CD59, and CR1. In some embodiments, the foreign protein has immunosuppressive properties and the gene is selected from CTLA4 and CD47.

[0142] In some embodiments, the genomic modification is a disruption of an endogenous gene that results in the reduction or elimination of expression of an endogenous protein. In some embodiments, the disrupted endogenous gene is part of the porcine major histocompatibility complex (SLA complex) and is selected from SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, SLA-8, SLA-9, SLA-11, and SLA-12. In some embodiments, the disruption of one or more of the porcine class I SLA genes is accompanied by an insertion of one or more of the human class I HLA genes selected from HLA-A, HLA-A2, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0143] In some embodiments, the disrupted endogenous genes are one or both of α(1,3)-galactosyltransferase (GT) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). Reducing or eliminating expression of GT and CMAH inhibits glycosylation of surface proteins in porcine cells, thereby reducing the immunogenicity of porcine xenografts. GT catalyzes the addition of galactose-α-1,3-galactose residues to glycoproteins. CMAH catalyzes the conversion of N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc), which, along with other porcine enzymes, forms immunogenic glycoproteins found on the surface of porcine cells but not human cells. [Example]

[0144] Example 1. Cloning, expression, production, and assembly of Cas12a-guide nucleoprotein complexes A non-limiting example of successful preparation of a functional Cas12a-guide nucleoprotein complex is described in International Patent Application No. PCT / US2021 / 055394, filed October 18, 2021.

[0145] Briefly, the catalytically active Acidaminococcus species (BV3L6 strain) Cas12a protein (AsCas12a) sequence can be codon-optimized for expression in E. coli cells and linked to a nuclear localization sequence (NLS) via a linker, e.g., a glycine-serine linker. Several NLS sequences, including the nucleoplasmin (NLP) NLS and the SV40 large T antigen NLS, have been validated for Cas12a. The DNA sequence encoding NLS-Cas9 can be cloned into a suitable bacterial expression vector using standard cloning methods.

[0146] Essentially, for example, as described in Swarts et al. (Molecular Cell, 2017, 66:221-233), the AsCas12a protein can be expressed in E. coli using an expression vector and purified using affinity chromatography, ion exchange, and size exclusion chromatography.

[0147] Cas12a guides can be generated by linking a targeting region to a specific Cas12a guide activation region. The targeting region, or spacer, preferably contained a 20-nucleotide target-binding sequence. The target-binding sequence was complementary to the target sequence downstream (3' direction) of the 5'-TTTV or 5'-TTTN PAM. Cas12a guides (e.g., crRNA and chRDNA) can be synthesized by commercial manufacturers or generated by in vitro transcription (e.g., T7 Quick High Yield RNA Synthesis Kit, New England Biolabs, Ipswich, Mass.).

[0148] Nucleoprotein complexes can be formed, for example, at a concentration of 80 pmol Cas12a protein:240 pmol guide. The Cas12a protein and each of the guide components (e.g., crRNA or chRDNA) are adjusted to the desired total concentration, incubated at 95°C for 2 minutes, removed from the thermocycler, and allowed to equilibrate to room temperature. The Cas12a protein is diluted to the appropriate concentration with binding buffer (60 mM Tris-acetate, 150 mM potassium acetate, 30 mM magnesium acetate, pH 7.9) to a final volume of 1.5 μl, mixed with 1 μl of the guide component, and then incubated at 37°C for 10 minutes.

[0149] Example 2. Tiling of target genes for Cas12a cleavage To target a selected gene, all 20 nucleotides downstream (3' direction) of the PAM motif (e.g., 5'-TTTV) can be used for targeting. Target selection criteria include, but are not limited to, homology to other regions in the genome, GC content, melting temperature, and the presence of homopolymers within the spacer.

[0150] The identified 20-nucleotide sequence can be added downstream (3') of the AsCas12a guide activation region sequence to create the desired guide.

[0151] Optionally, the guide can be designed to include one or more deoxyribonucleotides (DNA) among the ribonucleotides (RNA) of the targeting or activation region.

[0152] Further optionally, the guide can be designed to contain one or more chemically modified nucleotides. Chemical modifications can include backbone modifications, nitrogenous base modifications, and sugar modifications. One example is phosphorothioate modification of the nucleic acid backbone.

[0153] Example 3. (Prophetic) Transfection of human induced pluripotent stem cells with Cas12a-chRDNA nucleoprotein complexes. This example describes nucleofection of iPSCs using a Cas12a guide / nucleoprotein complex. Cas12a (AsCas12a) is prepared as described herein. Guides are prepared against target sequences of the genes listed in Table 3.

[0154] Cells are prepared as follows: iPSCs are cultured in mTeSR-plus medium (STEMCELL Technologies, Cambridge, Mass.) supplemented with Rho-associated coiled-coil-containing protein kinase inhibitor ("ROCKi," MilliporeSigma, Burlington, Mass.) at a final concentration of 10 μM for 3 hours at 37°C prior to transfection. The mTeSR-plus / ROCKi medium is removed, and the iPSCs are washed with 10 mL of PBS. 3 mL of Accetase (STEMCELL Technologies, Cambridge, Mass.) is then added, and the cells are incubated at 37°C for 5-10 minutes. Next, 7 mL of mTeSR-pulse and ROCKi are added to the cells, and the cells are mixed and counted. The cells are then centrifuged, the medium is removed, and the cells are washed with 10 mL of PBS, centrifuged again, and the PBS is removed. Cells are then seeded into Nucleofector™ P4 or P3 (Lonza, Allendale, NJ) solution at 2 x 10 per sample. 5 ~10 6 The cells were resuspended to a density of 100 cells / ml. Next, 20 μl of cell suspension was added to each well containing 2.5 μl of Cas12a guide / nucleoprotein complex, and the entire volume from each well was transferred to a well of a 96-well Nucleocuvette™ plate (Lonza, Allendale, NJ). The plate was loaded onto a Nucleofector™ 96-well Shuttle, and the cells were nucleofected using the CA137 Nucleofector™ program. After nucleofection, 77.5 μl of ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL) was added to each well, and the entire volume of the transfected cell suspension was transferred to a 96-well cell culture plate containing 100 μl of prewarmed ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL). Plates were transferred to a tissue culture incubator and maintained at 37°C in 5% CO2 for 48 hours before downstream analysis.

[0155] Example 4. (Prophetic) Differentiation of Nucleofected iPSCs into Motor Neurons This example describes the differentiation of nucleofected iPSCs into motor neurons. Briefly, we use the protocol described by Bianchi, F. et al. ((2018) Rapid and efficient differentiation of functional motor neurons from iPSCs for neural injury, Stem Cell Research 32:126). After nucleofection, iPSCs are allowed to grow until confluent. Confluent iPSCs were dissociated using Accetase and plated at 0.5 × 10 cells onto MG-coated 6-well plates in neural induction medium (NIM) consisting of a 1:1 mixture of neurobasal medium (NBM) and KO-DMEM / F:12 supplemented with 10% KnockOut serum replacement, 1% non-essential amino acids (NEAA) (all from ThermoFisher Scientific), and 1% GlutaMAX, 0.1 mM L-ascorbic acid, 3 μM CHIR99021 (both from Sigma-Aldrich), 2 μM SB431542 (CellGuidance Systems), 1 μM dorsomorphin, and 1 μM Compound E (all from StemCell). 6 Cells were seeded at 1000 cells / well. 1% RevitaCell (ThermoFisher Scientific) was added for the first 24 hours only. The NIM was replaced daily for 6 days, after which cells were dissociated with activase and seeded in NPC growth medium consisting of a 1:1 mixture of NBM and KO-DMEM:F12 supplemented with 1% P / S, 1% B27, 1% N2, 1% NEAA, 1% GlutaMAX, 0.1 mM L-AA, 10 ng / mL bFGF, and 10 ng / mL EGF. Optionally, one-fifth of the cells were retained at each passage for marker analysis.

[0156] For motor neuron differentiation and formation, NPCs are cultured for 6 days in MN induction medium consisting of a 1:1 mixture of Neurobasal Medium and KO-DMEM:F12 supplemented with 1% P / S, 1% B27, 1% N2, 1% non-essential amino acids, 1% GlutaMAX, 0.1 mM L-ascorbic acid, 10 μM all-trans retinoic acid, 100 ng / ml recombinant SHH, 1 μM purmorphamine (Abcam), and 1 mM SAG dihydrochloride (Sigma-Aldrich). After 7 days, cells are dissociated using Actase and replated in maturation medium consisting of 1:1 NBM and KO-DMEM:F12 supplemented with 1% P / S, 1% B27, 1% N2, 1% NEAA, 1% GlutaMAX, 0.1 mM L-AA, 10 ng / mL CNTF, 10 ng / mL BDNF, 10 ng / mL NT-3, and 10 ng / mL GDNF.

[0157] cell analysis To assess differentiation, cells are fixed in 3.75% paraformaldehyde solution in phosphate-buffered saline (PBS) and blocked and permeabilized with 0.1% Triton-X, 0.1% Tween-20, and 2.5% BSA in PBS. Primary antibodies against Sox1, Pax6, Nestin, HB9, MAP2, NeuroFilament, Tuj1, and Olig2 (e.g., from AbCam) are added. Cells are then counterstained with AlexaFluor-labeled secondary antibodies. Cell nuclei are labeled, for example, with NucBlue. Cells are imaged using a fluorescence microscope, for example, an inverted fluorescence microscope.

[0158] To further assess differentiation into motor neurons, calcium activity is imaged using Oregon Green 488 BAPTA-2 calcium indicator. Cells are incubated with the dye solution in imaging medium, e.g., FluoroBrite-DMEM imaging medium, for 30 minutes, washed twice with PBS, and further incubated in fresh FBDMEM for 30 minutes. Cells are imaged using a standard FITC filter, and the fluorescence intensity of individual segmented cells is recorded over time. To further assess differentiation into motor neurons, electrical activity of the cells is measured using a MultiClamp 700B microelectrode amplifier (Molecular Devices, San Jose, Calif.).

[0159] Example 5 (Prophetic) Differentiation of Nucleofected iPSCs into Cardiomyocytes. This example describes the differentiation of nucleofected iPSCs into cardiomyocytes. Briefly, we use the protocol described by Balafkan, N. et al. ((2020) A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates, Nature 10:18498). After nucleofection, iPSCs are allowed to settle and grow until confluent. Wells of a 96-well plate are coated with Advanced DMEM / F-12 containing Geltrex (both Thermo Fisher Scientific). Before seeding, human iPSC colonies are homogenized and plated at 2.4 × 10 cells per well using Essential 8 Medium (Thermo Fisher Scientific). 4 cells / cm 2The cells are seeded at a density of 1000 kJ / ml and incubated for 3 days with daily medium changes so that the cultures reach 60-70% confluency. The cells are then treated with a GSK-3 inhibitor (e.g., CHIR99021) in medium (e.g., RPMI 1640). After 24 hours, the medium is changed to one without the GSK-3 inhibitor and the cells are left for 48 hours (days 1-2). On day 3, the cells are treated with 5 μM of a Wnt-dependent phosphorylation inhibitor (e.g., IWP2) and incubated for an additional 48 hours (days 3-4). On day 5, the medium is changed to one without the Wnt-P inhibitor and the cells are left for 48 hours (days 5-6). On day 7, the medium is changed to one containing insulin. The cells are evaluated by measuring gene expression of cardiomyocyte-specific markers (e.g., TBX5, TNNT2, MYH6, and MYL7) and by measuring the reduction in expression of pluripotency markers (NANOG, POUF5F1).

[0160] Example 6. Cultivation of immortalized mouse hepatocytes This example demonstrates the culture of the immortalized mouse hepatocyte cell line H2.35 (ATCC, Manassas, VA).

[0161] H2.35 cells were removed from liquid nitrogen storage and thawed in a 37°C water bath for 3 minutes. The cells were diluted in calcium- and magnesium-free phosphate-buffered saline (PBS, Thermo Scientific, Wilmington, DE) to a final volume of 10 mL and centrifuged at 300 g for 5 minutes. The PBS was aspirated, and the cells were resuspended in 10 mL of prewarmed H2.35 medium containing Dulbecco's Modified Eagle Medium (DMEM, Thermo Scientific, Wilmington, DE) containing 1 g / mL glucose supplemented with 4% fetal bovine serum and 200 nM dexamethasone (Merck / Millipore-Sigma, Munich, Germany). The cells were counted using a Countess® 3 automated cell counter (Life Technologies, Grand Island, NY). The cells were then counted at 10,000 cells / cm. 2The cells were cultured in flat-bottomed adherent flasks at a density of 1000 kJ / ml and incubated at 32°C in 10% CO2.

[0162] When cells reach 60-70% confluency, they are routinely passaged by aspirating the medium, washing with enough PBS to cover the bottom of the flask, and gently rocking back and forth. Next, the PBS is aspirated, and room temperature (RT) Accetase (Thermo Scientific, Wilmington, DE) is added to cover the bottom of the flask. The flask is then gently rocked back and forth and incubated for 3 minutes at room temperature. The flask is gently tapped 5-10 times against the palm of your hand to dislodge the cells, and 2.5 volumes of H2.35 medium per Accetase volume are added to the flask and mixed using a serological pipette. The cells are centrifuged at 300g for 5 minutes, the medium is decanted, and the cells are counted using a Countess® 3 automated cell counter at 10,000-20,000 cells / cm in H2.35 medium. 2 Seed new flasks at a density of 1000 x g.

[0163] Example 7. Cloning, expression, production, and assembly of Cas12a guide / nucleoprotein complexes This example describes methods for cloning, expressing, and purifying the Cas12a guide / nucleoprotein complex, as well as methods for generating the Cas12a guide components.

[0164] A. Cloning of Cas12 Protein The catalytically active Cas12a protein sequence (SEQ ID NO: 1) from Acidaminococcus sp. (BV3L6 strain) was codon-optimized for expression in E. coli cells. A glycine-serine linker and a nuclear localization sequence (NLS) (SEQ ID NO: 2) were added to the C-terminus. Oligonucleotide sequences encoding the Cas12a-NLS protein (referred to as AsCas12a and Cas12a protein in the following examples) were provided to a commercial manufacturer for synthesis. The DNA sequences were then cloned into a suitable bacterial expression vector using standard cloning methods.

[0165] B. Expression and Purification of Cas12a Protein Essentially, the AsCas12a protein was expressed in E. coli using an expression vector and purified using affinity chromatography, ion exchange, and size exclusion chromatography, e.g., as described in Swarts et al. (Molecular Cell, 2017, 66:221-233).

[0166] C. Creation of Cas12a guide components Cas12a guides were generated by linking a targeting region to a specific Cas12a guide activation region. The targeting region, or spacer, preferably contained a 20-nucleotide target binding sequence. The target binding sequence was complementary to the target sequence downstream (3' direction) of the 5'-TTTV or 5'-TTTN PAM. Exemplary Cas12a guide activation region sequences are SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10 for the Cas12a species Acidaminococcus sp., L. bacterium, and F. novicida, respectively.

[0167] Cas12a guide sequences (e.g., crRNA and chRDNA) were provided to commercial manufacturers for synthesis.

[0168] The guide RNA component (e.g., crRNA) can be generated by in vitro transcription from a double-stranded (ds)DNA template by incorporating a T7 promoter at the 5' end of the dsDNA template sequence (e.g., T7 Quick High Yield RNA Synthesis Kit, New England Biolabs, Ipswich, MA).

[0169] D. Assembly of the Cas12a guide / nucleoprotein complex Acidaminococcus species Cas12a (AsCas12a) tagged with a C-terminal nuclear localization sequence was recombinantly expressed in E. coli and purified using chromatographic methods. Unless otherwise specified, nucleoprotein complexes were formed at a concentration of 80 pmol Cas12a protein:240 pmol guide. Prior to assembly with Cas12a protein, each guide component (e.g., crRNA or chRDNA) was adjusted to the desired total concentration (240 pmol) in a final volume of 1 μl, incubated at 95°C for 2 minutes, removed from the thermocycler, and allowed to equilibrate to room temperature. Cas12a protein was diluted to the appropriate concentration with binding buffer (60 mM Tris-acetate, 150 mM potassium acetate, 30 mM magnesium acetate, pH 7.9) to a final volume of 1.5 μl, mixed with 1 μl of guide component, and subsequently incubated at 37°C for 10 minutes. The Cas12a guide / nucleoprotein complex was used immediately or stored frozen at -20°C until needed.

[0170] Example 8. Electroporation of immortalized mouse hepatocytes with Cas12a guide / nucleoprotein complexes. This example demonstrates electroporation of the immortalized mouse hepatocyte cell line H2.35 with a Cas12a guide / nucleoprotein complex for gene editing.

[0171] The Cas12a guide / nucleoprotein complex from Example 7 was transfected into H2.35 cells using the Nucleofector™ 96-well Shuttle System (Lonza, Allendale, NJ). The Cas12a guide / nucleoprotein complex was dispensed into individual wells of a 96-well plate in a final volume of 2.5 μl. H2.35 cells were removed from culture flasks in a manner similar to the process described in Example 1. After counting the H2.35 cells, 100,000 cells per transfection condition were resuspended in 17.5 μL of CTS™ Xenon™ electroporation buffer (Thermo Scientific, Wilmington, DE), mixed with 2.5 μL of Cas12a guide / nucleoprotein complex, and transferred to wells of a 96-well Nucleocuvette™ plate (Lonza, Allendale, NJ). Plates were loaded onto a Nucleofector™ 96-well Shuttle (Lonza, Allendale, NJ) and cells were nucleofected using the EH-110 Nucleofector™ program (Lonza, Allendale, NJ).

[0172] After nucleofection, 80 μl of H2.35 medium was added to each well, and the entire volume of the transfected cell suspension was transferred to a 96-well cell culture plate containing 100 μl of prewarmed H2.35 medium. The plate was transferred to a tissue culture incubator and maintained at 32°C in 10% CO for 72 hours before downstream analysis.

[0173] Example 9. Tiling of mouse genes with Cas12a guide / nucleoprotein complexes This example describes the design and use of Cas12a guide / nucleoprotein complexes to target genes encoding mouse proprotein convertase subtilisin / kexin type 9 (PCSK9), transthyretin (TTR), and angiopoietin-like 3 (ANGPTL3) in the immortalized mouse hepatocyte cell line H2.35.

[0174] A. Design of AsCas12a crRNA guide A collection of 20 nucleotide sequences downstream (3' direction) of the 5'-TTTV PAM motif in the coding regions of the genes encoding mouse PCSK9, TTR, and ANGPLT3 were selected for targeting (SEQ ID NOS: 5-94). Target selection criteria included, but were not limited to, homology to other regions in the genome, GC content, melting temperature, and the presence of homopolymers within the spacer.

[0175] The identified 20-nucleotide sequence was added downstream (3' direction) of the AsCas12a activation region sequence (SEQ ID NO: 3).

[0176] The sequences were provided to a commercial manufacturer for synthesis. Individual Cas12a guide / nucleoprotein complexes were then prepared as described in Example 7 and transfected into primary H2.35 cells as described in Example 3.

[0177] B. Determining Genome Editing Efficiency (1) Generation of target dsDNA sequences for deep sequencing Seventy-two hours after transfection, gDNA was isolated from nucleofected H2.35 cells using Cas12a guide / nucleoprotein complexes and 50 μL per well of QuickExtract™ DNA extraction solution (Epicentre, Madison, WI), followed by incubation at 37° C. for 10 minutes, 65° C. for 30 minutes, and 95° C. for 3 minutes to terminate the reaction. The isolated gDNA was diluted with 50 μL of sterile water, and samples were stored at −20° C.

[0178] The isolated gDNA was used to perform a first round of PCR using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA) at a 1x concentration. Primers designed to amplify regions surrounding the Cas12a target were used at 0.5 μM each, and 3.75 μL of gDNA was used in a final volume of 10 μL. Amplification consisted of an initial cycle of 98°C for 1 minute, followed by 35 cycles of 98°C for 10 seconds, 60°C for 20 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 2 minutes. The PCR reaction was diluted 1:100 with water.

[0179] A unique set of index primers for barcoding PCR was used to facilitate multiplex sequencing of each sample. Barcoding PCR was performed using a reaction mixture containing 1x Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA), 0.5 μM of each primer, and 1 μL of first-round PCR diluted 1:100 in a final volume of 10 μL. The reaction mixture was amplified at 98°C for 1 minute, followed by 12 cycles of 98°C for 10 seconds, 60°C for 20 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 2 minutes.

[0180] (2) SPRIselect cleanup PCR reactions were pooled and transferred to a single microtube for SPRIselect (Beckman Coulter, Pasadena, CA) bead-based cleanup of amplification products for sequencing.

[0181] 0.9x volume of SPRIselect beads was added to the amplified product, mixed, and incubated for 10 minutes at room temperature. The microtube was placed on a magnetic tube stand until the solution cleared. The supernatant was removed and discarded, and the remaining beads were washed with 1 volume of 85% ethanol. The beads were then incubated for 30 seconds at room temperature. After incubation, the ethanol was aspirated, and the beads were air-dried for 10 minutes at room temperature. The microtube was removed from the magnetic stand, and 0.25x volume of Qiagen EB buffer (Qiagen, Venlo, Netherlands) was added to the beads, mixed vigorously, and incubated for 2 minutes at room temperature. The microtube was returned to the magnetic stand and incubated until the solution cleared. The supernatant containing the purified amplified product was then dispensed into a clean microtube. Purified amplification products were quantified using a Nanodrop™ 2000 System (Thermo Scientific, Wilmington, DE), and library quality was analyzed using a Fragment Analyzer™ System (Advanced Analytical Technologies, Ames, IA) and a DNF-910 dsDNA Reagent Kit (Advanced Analytical Technologies, Ames, IA).

[0182] (3) Deep sequencing setup The pooled amplification products were normalized to a concentration of 4 nM, calculated from the Nanodrop™ 2000 System values ​​and the average size of the amplification products. Libraries were analyzed on a MiSeq Sequencer (Illumina, San Diego, CA) using the MiSeq Reagent Kit v2 (Illumina, San Diego, CA) for 300 cycles with two 151-cycle paired-end runs and two 8-cycle index reads.

[0183] (4) Deep sequencing data analysis The identity of products in the sequencing data was determined based on the index barcode sequences applied to the amplified products in the barcoding PCR. Computer scripts were used to process the MiSeq data, performing, for example, the following tasks: a. Reads were aligned to the mouse genome (build GRCm38 / mm10) using Bowtie (bowtie-bio.sourceforge.net / index.shtml) software. b. The aligned reads were compared to the predicted wild-type genomic locus sequence, and reads that did not align to any part of the wild-type locus were discarded. c. Reads matching the wild-type sequence were tallied. d. Reads with indels (insertion or deletion of bases) were classified by indel type and tabulated. e. The percentage of mutant reads was calculated by dividing the total number of indel reads by the sum of the number of wild-type and indel reads.

[0184] The genome editing efficiency obtained for the Cas12a guide / nucleoprotein complex was determined by identifying indel sequences in the region targeted by the Cas12a guide / nucleoprotein complex. The results of this intracellular editing experiment are shown in Table 7 below. [Table 7-1] [Table 7-2] [Table 7-3]

[0185] The data presented in Table 7 above demonstrate that the Cas12a crRNA / nucleoprotein complex is capable of on-target editing of multiple genes in mouse H2.35 cells. Other genes, such as those described elsewhere herein, can be targeted in a similar manner using AsCas12a or other Cas12a proteins (e.g., L. bacterium or F. novicida).

[0186] Example 10. Engineering a Cas12a chRDNA guide molecule with DNA in the activation region sequence. The following examples describe engineering AsCas12a chRDNA guide molecules to include DNA bases in the activation region sequence.

[0187] A. In silico Cas12a chRDNA guide design The 20-nucleotide activation region sequence of the AsCas12a guide (SEQ ID NO: 03) was selected for engineering and designed to contain DNA bases in place of RNA at positions 1, 3, 7, 10, 12, 14, 15, and 19 (counting from 5' to 3' along the guide).

[0188] Nine target sequences were selected from the list of targets shown in Table 7 in Example 9 and engineered to include DNA bases in the activation region sequence, as well as the Cas12a crRNA control sequence, and were provided to a commercial manufacturer for synthesis.

[0189] B. Cell Transfection and Analysis Individual Cas12a guide / nucleoprotein complexes for screening were prepared essentially as described in Example 7. The nucleoprotein complexes were transfected into H2.35 cells as described in Example 8, and the resulting genome editing efficiencies of the Cas12a guide / nucleoprotein complexes were determined as described in Example 9. The results of this intracellular editing experiment are shown in Table 8 below. [Table 8]

[0190] The data presented in Table 8 above demonstrate that AsCas12a guides containing DNA in their activation region sequences enable on-target editing rates comparable to all-RNA guides (e.g., compare SEQ ID NO:148 with SEQ ID NO:149, SEQ ID NO:153 with SEQ ID NO:154, and SEQ ID NO:160 with SEQ ID NO:161). Other guides, such as those described elsewhere herein, can be engineered using AsCas12a or other Cas12a proteins (e.g., L. bacterium or F. novicida) to include DNA in their activation region sequences in a similar manner.

[0191] Example 11. Engineering a Cas12a chRDNA guide molecule with DNA in the guide repeat sequence. The following examples describe engineering AsCas12a chRDNA guide molecules to include DNA bases in the target binding sequence.

[0192] A. In silico Cas12a chRDNA guide design One target sequence in the genes encoding mouse PCSK9 (PCSK9-tgt9), TTR (TTR-tgt5), and ANGPTL3 (ANGPLT3-tgt18) was selected for engineering. Cas12a chR DNA guide and Cas12a crRNA control sequences for each target, containing individual DNA bases at positions corresponding to the target binding sequence, were provided to a commercial manufacturer for synthesis (SEQ ID NO:74, SEQ ID NO:92, SEQ ID NO:22, and SEQ ID NOs:95-130).

[0193] B. Cell Transfection and Analysis Individual Cas12a guide / nucleoprotein complexes for screening were prepared essentially as described in Example 7. The nucleoprotein complexes were transfected into H2.35 cells as described in Example 8, and the resulting genome editing efficiencies of the Cas12a guide / nucleoprotein complexes were determined as described in Example 9. The results of this intracellular editing experiment are shown in Table 9 below. [Table 9-1] [Table 9-2]

[0194] The editing results in Table 9 above demonstrate that Cas12a chRDNA guide molecules containing DNA in the spacer enable editing rates comparable to crRNA across multiple targets (compare SEQ ID NO:148 to SEQ ID NO:103, SEQ ID NO:152 to SEQ ID NO:109, and SEQ ID NO:156 to SEQ ID NO:130). The editing rates of the chRDNA guide designs in Table 3 were normalized to the editing rate of crRNA for each target and used to determine which positions within the target binding sequence for selected targets could be engineered as Cas12a guide DNA.

[0195] Example 12. Cas12a chRDNA guide molecules with multiple DNA bases in the target binding sequence. This example describes the design and testing of Cas12a chRDNA guide molecules with multiple DNA bases in the target binding sequence.

[0196] A. In silico design of Cas12a chRDNA guide Three 20-nucleotide sequences were selected for editing: three targets in the gene encoding human B2M (B2M-tgt12, B2M-tgt1, B2M-intron-tgt12), one in the gene encoding human TRAC (TRAC-tgt12), and one in the gene encoding human DNA methyltransferase 1 (DNMT1-tgt1). For each target, one to seven DNA nucleotides were designed to be included in the target-binding sequence of each AsCas12a guide. Design criteria for DNA base positions included, but were not limited to, previous single-position screening data (see Example 10), prior consensus on DNA-tolerant positions, the distance between individual DNA bases in the target-binding sequence, and known locations of mismatches in off-target sequences. Cas12a chR DNA guide designs and a control sequence without DNA in the target-binding sequence ("V3'" in Table 10) were provided to a commercial manufacturer for synthesis.

[0197] B. Cell Transfection and Analysis Individual Cas12a guide / nucleoprotein complexes for screening were prepared essentially as described in Example 7. The Cas12a guide / nucleoprotein complexes were transfected into primary T cells as described in Example 8, and the resulting genome editing efficiencies of the Cas12a guide / nucleoprotein complexes were determined as described in Example 9. The results of this intracellular editing experiment and the positions of the DNA bases in the target binding sequence of each Cas12a chRDNA guide are shown in Table 10 below. [Table 10]

[0198] The editing results in Table 10 above demonstrate that Cas12a chRDNA guide molecules containing multiple DNA bases in the target binding sequence enable editing rates comparable to crRNA across multiple targets (compare SEQ ID NO:136 with SEQ ID NO:131, SEQ ID NO:141 with SEQ ID NO:139, or SEQ ID NO:147 with SEQ ID NO:142).

[0199] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various modifications can be made within the scope of the invention. Accordingly, the scope of the invention should be limited not by the embodiments described herein, but by the claims set forth below.

Claims

1. A pharmaceutical composition for treating a disease or condition characterized by abnormal gene expression, (a) A first nuclear protein complex comprising a Cas12a protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nuclear protein complex with the Cas12a protein, the Cas12a protein is capable of cleaving the first target nucleic acid, and the first CRISPR guide molecule contains at least one deoxyribonucleotide, (b) a donor polynucleotide containing the coding sequence of a target gene abnormally expressed in an individual suffering from the disease or condition, A pharmaceutical composition wherein the first nuclear protein complex and the donor polynucleotide are present in lipid nanoparticles, and the target gene is selected from Table 3.

2. The pharmaceutical composition according to claim 1, wherein the CRISPR guide molecule comprises at least one deoxyribonucleotide in the activation region, the targeting region, or both thereof.

3. The lipid nanoparticles comprise one or more cationic lipids, and the pK of the lipids or a combination of two or more lipids a The pharmaceutical composition according to claim 1, wherein the coefficient is 6.1 to 6.

7.

4. The pharmaceutical composition according to claim 1, wherein the lipid nanoparticles contain neutral lipids.

5. The pharmaceutical composition according to claim 1, wherein the lipid nanoparticles contain sterols.

6. The lipid nanoparticles are one or more selected from the group consisting of DSPC, DPPC, POPC, DOPE, SM, PEG-DMA, PEG-DMG, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, GL67A-DOPE-DMPE-PEG, 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, PEG-CerC14, and PEG-CerC20. A pharmaceutical composition according to claim 1, comprising lipids.

7. The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier.

8. A method for creating transgenic non-human animals for xenotransplantation, (1) Introducing a first nuclear protein complex comprising Cas12a protein and a first CRISPR guide molecule into a non-human animal cell, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nuclear protein complex with the Cas12a protein, the Cas12a protein is capable of cleaving the first target nucleic acid, the first CRISPR guide molecule contains at least one deoxyribonucleotide, and the first nuclear protein complex is present in lipid nanoparticles. The cleavage by the Cas12a protein results in the introduction of a target gene selected from Table 6, which leads to modification of the target gene. (2) Introducing the cells into a female non-human animal that serves as a surrogate mother. Methods that include...

9. The method according to claim 8, wherein the non-human animal cell is an oocyte, egg, or zygote.

10. The method according to claim 8, wherein the cells of the non-human animal are somatic cells, and the method further comprises, after step (1), transplanting the nuclei of the cells into an enucleated oocyte or zygote.

11. The method according to claim 8, wherein the non-human animal is a pig.

12. The method according to claim 8, wherein the CRISPR guide molecule comprises at least one deoxyribonucleotide in the activation region, the targeting region, or both thereof.

13. The method according to claim 8, further comprising introducing a donor polynucleotide into the cells, which contains the coding sequence of a target gene selected from one or more of the following: A20, HO-1, FAT-1, TNF-α receptor, CD39, hirudin, TFPI, EPCR, TBM, CD46, DAF (CD55), CD59, CR1, CTLA4, CD47, and class I HLA.

14. The method according to claim 13, wherein the cleavage by the Cas12a protein results in the insertion of the coding sequence into the genome of the cell.

15. The method according to claim 8, wherein the cleavage by the Cas12a protein results in the disruption of the coding sequence of a target gene selected from one or more of GGTA1, b4GalNT2, CMAH, GT (α(1,3)-galactosyltransferase), GHR, and class I SLA in the genome of the cell.

16. A composition for producing transgenic non-human animals for xenotransplantation, comprising non-human animal cells, wherein the non-human animal cells are The first nuclear protein complex comprises a Cas12a protein and a first CRISPR guide molecule, wherein the first CRISPR guide molecule has a targeting region capable of binding to a first target nucleic acid sequence and an activation region capable of forming a nuclear protein complex with the Cas12a protein, the Cas12a protein is capable of cleaving the first target nucleic acid, the first CRISPR guide molecule contains at least one deoxyribonucleotide, and the first nuclear protein complex is present in lipid nanoparticles. The cleavage by the Cas12a protein modifies the target gene selected from Table 6. A composition that is added by dripping.

17. The composition according to claim 16, wherein the CRISPR guide molecule comprises at least one deoxyribonucleotide in the activation region, the targeting region, or both thereof.

18. The composition according to claim 16, further comprising a donor polynucleotide containing the coding sequence of a target gene selected from one or more of the following: A20, HO-1, FAT-1, TNF-α receptor, CD39, hirudin, TFPI, EPCR, TBM, CD46, DAF (CD55), CD59, CR1, CTLA4, CD47, and class I HLA.

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