OMNI-103 CRISPR nuclease-RNA complex

Abstract

1. A composition comprising a non-naturally occurring RNA molecule, the RNA molecule comprising an RNA scaffold portion, the RNA scaffold portion having the structure: crRNA repeat sequence portion-tracrRNA portion, the RNA scaffold portion forming a complex with an OMNI-103 CRISPR nuclease and directing the OMNI-103 CRISPR nuclease to a DNA target site complementary to a guide sequence portion of the RNA molecule.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 286,855 (filed December 7, 2021), the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those referenced in parentheses. The disclosures of all publications mentioned in this application are incorporated by reference in their entireties into this application to fulfill the teachings of the present invention or to which the present invention pertains.

[0003] Sequence Listing Reference This application was created on November 9, 2022 on an IBM PC machine format using an operating system compatible with MS-Windows®, and incorporates by reference the nucleotide sequence in an XML file, 81 KB in size, filed as part of this application on December 6, 2022, entitled "221206_91822-A-PCT_Sequence_Listing_AWG.xml."

[0004] Technical Field The present invention relates, inter alia, to compositions and methods for genome editing. [Background technology]

[0005] Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in bacterial and archaeal adaptive immunity exhibit extreme diversity in protein composition and genomic locus structure. CRISPR systems have become important tools in research and genome engineering. Nevertheless, many details of CRISPR systems are unknown, and the application of CRISPR nucleases may be limited by sequence specificity, expression or delivery. Different CRISPR nucleases have diverse characteristics such as size, PAM site, on-target activity, specificity, cleavage patterns (e.g., blunt ends, sticky ends) and prominent patterns of indel formation after cleavage. Combinations of different properties may be useful for different applications. For example, some CRISPR nucleases are capable of targeting specific genomic loci, while another group of CRISPR nucleases cannot due to PAM site restrictions. In addition, some currently used CRISPR nucleases exhibit pre-immunity, which may limit their applicability in vivo. See Charlesworth et al., Nature Medicine (2019) and Wagner et al., Nature Medicine (2019). Thus, the discovery, utilization, and improvement of novel CRISPR nucleases, and the RNA molecules that activate and target them, is critical. Summary of the Invention

[0006] The present invention provides a composition comprising a non-naturally occurring RNA molecule, the RNA molecule comprising a crRNA repeat sequence portion and a guide sequence portion, the RNA molecule forming a complex with an OMNI-103 nuclease in the presence of a tracrRNA sequence to target the OMNI-103 nuclease to a DNA target site, the tracrRNA sequence being encoded by a tracrRNA portion of the RNA molecule or a tracrRNA portion of a second RNA molecule.

[0007] The present invention provides a composition comprising a non-naturally occurring RNA molecule, said RNA molecule comprising an RNA scaffold moiety, said RNA scaffold moiety having the structure: crRNA repeat sequence portion-tracrRNA portion wherein the RNA scaffold portion forms a complex with an OMNI-103 CRISPR nuclease and targets the OMNI-103 CRISPR nuclease to a DNA target site complementary to a guide sequence portion of the RNA molecule.

[0008] Disclosed herein are compositions and methods that can be used for genome engineering, epigenome engineering, genome targeting, cellular genome editing, and / or in vitro diagnostics using non-naturally occurring RNA molecules that include a scaffold portion that can specifically bind and activate OMNI-103 CRISPR nuclease to target a DNA target site based on a guide sequence portion of the RNA molecule, also referred to as an RNA spacer portion, and OMNI-103 CRISPR nuclease.

[0009] The disclosed compositions can be used to modify genomic DNA sequences. In this specification, genomic DNA refers to linear and / or chromosomal DNA and / or plasmid or other extrachromosomal DNA sequences present in a cell or cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments, the cell of interest is a prokaryotic cell. In some embodiments, the method creates a double-strand break (DSB) at a predetermined target site in the genomic DNA sequence, resulting in a mutation, insertion and / or deletion of the DNA sequence at the target site in the genome. [Brief description of the drawings]

[0010] [Figure 1A] Figure 1 is the predicted secondary structures of the sgRNAs listed in Table 3. Figure 1A: Scaffold V2. [Figure 1B] Figure 1B: Scaffold V2.1. [Figure 1C] Figure 1C: Scaffold V2.2. [Figure 1D] Figure 1D: Scaffold V2.3. [Figure 1E] Figure 1E: Scaffold V2.4. [Figure 1F] Figure 1F: Scaffold V2.5. [Diagram 2] OMNI-103 editing activity in HeLa cells with different sgRNA scaffolds (Table 3). HeLa cells were transfected with OMNI-103 and sgRNA plasmids targeting TRAC-s91 or PDCD-s40. Editing activity was calculated based on next-generation sequencing results (bars) and transfection efficiency was calculated based on FACS analysis of mCherry expression. The mean and standard deviation of three technical replicates are shown. [Diagram 3] Activity in U2OS. U2OS cells were electroporated with OMNI-103 and sgRNA (RNP) targeting TRACs35 and B2Ms12. Editing activity was calculated based on next generation sequencing (NGS) results. The average and standard deviation of three technical replicates are shown. [Figure 4] Activity in primary T cells. Primary T cells were isolated from PBMCs and activated according to the manufacturer's protocol (Miltenyi #130-096-535, #130-091-441). Activated T cells were electroporated with OMNI-103 and sgRNA (RNP) targeting TRAC-s35 and B2M-s12. After 8 days, the expression levels of TCR and B2M in cells were measured by flow cytometry. For analysis, only live and CD3 positive cells were counted. The results presented are representative and are one of three T cell donors that all showed similar results. [Diagram 5] T cell activation assay. Donor sample cells used in the cleavage activity assay were activated with beads for 72 hours and showed 85% primary T cell activation rate as measured by FACS (CD3+CD25+ cells). [Figure 6]Representative examples of RNA scaffolds. Representative RNA scaffold moieties include a crRNA moiety linked to a tracrRNA moiety by a tetraloop. The crRNA moiety includes a crRNA repeat sequence. The tracrRNA moiety includes a tracrRNA anti-repeat sequence and an additional tracrRNA segment. The RNA molecule may further include a guide sequence portion (i.e., an RNA spacer) linked to the crRNA repeat sequence such that the RNA molecule functions as a single guide RNA molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description The present invention provides a composition comprising a non-naturally occurring RNA molecule, the RNA molecule comprising a crRNA repeat sequence portion and a guide sequence portion, the RNA molecule complexing with an OMNI-103 protein in the presence of a tracrRNA sequence to target the OMNI-103 protein to a DNA target site, the tracrRNA sequence being encoded by the tracrRNA portion of the RNA molecule or the tracrRNA portion of a second RNA molecule. In some embodiments, the OMNI-103 is a nuclease, nickase or inactivated nuclease. In some embodiments, the OMNI-103 is encoded by the sequence shown in SEQ ID NO: 1 or a catalytic variant thereof. In some embodiments, the OMNI-103 is a nuclease capable of cleaving double-stranded DNA. In some embodiments, the OMNI-103 is a nickase capable of cleaving DNA at only one strand of double-stranded DNA.

[0012] In some embodiments, the length of the crRNA repeat sequence portion is up to 17 nucleotides, preferably 14-17 nucleotides.

[0013] In some embodiments, the crRNA repeat sequence portion has at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence set forth in SEQ ID NO: 22 or 23.

[0014] In some embodiments, the crRNA repeat sequence portion has at least 95% sequence identity to the sequence set forth in either SEQ ID NO: 22 or 23.

[0015] In some embodiments, the crRNA repeat sequence is other than the sequence set forth in SEQ ID NO:23.

[0016] In some embodiments, the RNA molecule comprising a crRNA repeat sequence portion and a guide sequence portion further comprises a tracrRNA portion.

[0017] In some embodiments, the crRNA repeat sequence portion is covalently linked to the tracrRNA portion via a polynucleotide linker portion.

[0018] In some embodiments, the composition comprises a second RNA molecule that comprises a tracrRNA portion.

[0019] In some embodiments, the OMNI-103 nuclease has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the OMNI-103 nuclease is a nickase having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and an amino acid substitution at a position selected from D12, E776, H988, D991, D856, H857, and N880. In some embodiments, the OMNI-103 nuclease is an inactivated nuclease having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, at least one amino acid substitution at a position selected from D12, E776, H988, and D991, and at least one amino acid substitution at a position selected from D856, H857, and N880.

[0020] In some embodiments, the length of the guide sequence portion is 17-30 nucleotides, preferably 22 nucleotides.

[0021] The present invention also provides a composition comprising a non-naturally occurring RNA molecule or a polynucleotide molecule encoding said RNA molecule, said RNA molecule comprising a tracrRNA portion, said RNA molecule forming a complex with and directing said OMNI-103 nuclease to a DNA target site in the presence of a crRNA repeat sequence and a guide sequence portion, wherein said crRNA repeat sequence and said guide sequence portion are encoded by said RNA molecule or a second RNA molecule.

[0022] In some embodiments, the length of the tracrRNA portion is less than 85 nucleotides, preferably 84-80, 79-75, 74-70, 69-65, or 64-60 nucleotides.

[0023] In some embodiments, the tracrRNA portion has at least 30-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the tracrRNA portion of any of SEQ ID NOs: 17-21.

[0024] In some embodiments, the tracrRNA portion has at least 95% sequence identity to the tracrRNA portion of a sequence set forth in any of SEQ ID NOs: 17-21.

[0025] In some embodiments, the tracrRNA portion is other than the tracrRNA portion of the sequence set forth in SEQ ID NO: 4 or 5.

[0026] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion up to 19 nucleotides in length, preferably 16-19 nucleotides.

[0027] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence set forth in either SEQ ID NO:24 or 25.

[0028] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having at least 95% sequence identity to the sequence set forth in either SEQ ID NO: 24 or 25.

[0029] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having a sequence other than that set forth in SEQ ID NO:25.

[0030] In some embodiments, the RNA molecule comprises a tracrRNA portion and further comprises a crRNA repeat sequence portion and a guide sequence portion.

[0031] In some embodiments, the tracrRNA portion is covalently linked to the crRNA repeat sequence via a polynucleotide linker portion.

[0032] In some embodiments, the polynucleotide linker portion is 4-10 nucleotides in length.

[0033] In some embodiments, the sequence of the polynucleotide linker is GAAA.

[0034] In some embodiments, the composition further comprises a second RNA molecule comprising a crRNA repeat sequence portion and a guide sequence portion.

[0035] In some embodiments, the OMNI-103 nuclease has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

[0036] In some embodiments, the length of the guide sequence portion is 17-30 nucleotides, preferably 22 nucleotides.

[0037] The present invention relates to a composition comprising a non-naturally occurring RNA molecule or a polynucleotide molecule encoding said RNA molecule, said RNA molecule comprising an RNA scaffold moiety, said RNA scaffold moiety having the structure: crRNA repeat sequence portion-tracrRNA portion wherein the RNA scaffold portion forms a complex with an OMNI-103 CRISPR protein and targets the OMNI-103 CRISPR protein to a DNA target site complementary to a guide sequence portion of the RNA molecule.

[0038] In some embodiments, OMNI-103 is a nuclease, nickase or inactivated nuclease. In some embodiments, OMNI-103 is encoded by the sequence set forth in SEQ ID NO: 1 or a catalytic variant thereof. In some embodiments, OMNI-103 is a nuclease capable of cleaving double-stranded DNA. In some embodiments, OMNI-103 is a nickase capable of cleaving DNA at only one strand of double-stranded DNA.

[0039] In some embodiments, the OMNI-103 nuclease has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

[0040] In some embodiments, the length of the RNA scaffold portion is between 110-105, 104-100, 99-95, 94-90, 89-85, 84-80, 79-75, or 74-70 nucleotides.

[0041] In some embodiments, the length of the RNA scaffold portion is 107, 101, 95, 85 or 79 nucleotides.

[0042] In some embodiments, the RNA scaffold portion has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 17-21.

[0043] In some embodiments, the length of the crRNA repeat sequence portion is up to 17 nucleotides, preferably 14-17 nucleotides.

[0044] In some embodiments, the crRNA repeat sequence portion has at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence set forth in SEQ ID NO: 22 or 23.

[0045] In some embodiments, the crRNA repeat sequence portion has at least 95% sequence identity to the sequence set forth in either SEQ ID NO: 22 or 23.

[0046] In some embodiments, the crRNA repeat sequence is other than the sequence set forth in SEQ ID NO:23.

[0047] In some embodiments, the length of the tracrRNA portion is less than 85 nucleotides, preferably 84-80, 79-75, 74-70, 69-65, or 64-60 nucleotides.

[0048] In some embodiments, the tracrRNA portion has at least 30-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the tracrRNA portion of any of SEQ ID NOs: 17-21.

[0049] In some embodiments, the tracrRNA portion has at least 95% sequence identity to the tracrRNA portion of a sequence set forth in any of SEQ ID NOs: 17-21.

[0050] In some embodiments, the tracrRNA portion is other than the tracrRNA portion of the sequence set forth in SEQ ID NO: 4 or 5.

[0051] In some embodiments, the RNA scaffold portion comprises an RNA scaffold structure comprising: crRNA repeat sequence - linker - tracrRNA The tracrRNA further comprises a linker portion between the crRNA repeat sequence portion and the tracrRNA portion, so that

[0052] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion, and the crRNA repeat sequence and the tracrRNA anti-repeat sequence portion are covalently linked via a linker portion.

[0053] In some embodiments, the linker moiety is a polynucleotide linker between 4 and 10 nucleotides in length.

[0054] In some embodiments, the sequence of the polynucleotide linker is GAAA.

[0055] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion up to 19 nucleotides in length, preferably 16-19 nucleotides.

[0056] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence set forth in either SEQ ID NO:24 or 25.

[0057] In some embodiments, the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having at least 95% sequence identity to the sequence set forth in either SEQ ID NO: 24 or 25.

[0058] In some embodiments, the tracrRNA anti-repeat sequence is other than the sequence set forth in SEQ ID NO:25.

[0059] In some embodiments, the tracrRNA portion comprises a first section of nucleotides linked to the tracrRNA anti-repeat portion, the first section of nucleotides having at least 95% sequence identity to any of SEQ ID NOs: 26-28.

[0060] In some embodiments, the tracrRNA portion comprises a second section of nucleotides linked to a first section of nucleotides, wherein the second section of nucleotides has at least 95% sequence identity to any of SEQ ID NOs:29-32.

[0061] In some embodiments, the RNA scaffold portion has at least 95% identity to a nucleotide sequence set forth in any of SEQ ID NOs:17-21.

[0062] In some embodiments, the RNA scaffold portion has the predicted structure of any of the V2, V2.1, V2.2, V2.3, V2.4, or V2.5 RNA scaffolds.

[0063] In some embodiments, the RNA scaffold portion has a sequence other than that shown in SEQ ID NO:4 or SEQ ID NO:5.

[0064] In some embodiments, the guide sequence portion is covalently linked to the crRNA repeat sequence portion of the RNA molecule and has the structure: Guide sequence - crRNA repeat sequence - tracrRNA A single guide RNA molecule is formed.

[0065] In some embodiments, the length of the guide sequence portion is 17-30 nucleotides, more preferably 20-23 nucleotides, more preferably 22 nucleotides.

[0066] In some embodiments, the composition further comprises an OMNI-103 CRISPR nuclease, wherein the OMNI-103 CRISPR nuclease has at least 95% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0067] In some embodiments, the RNA molecules are formed by in vitro transcription (IVT) or solid phase artificial oligonucleotide synthesis.

[0068] In some embodiments, the RNA molecule comprises modified nucleotides.

[0069] In some embodiments, the RNA molecule comprises a sequence set forth in any of SEQ ID NOs: 17 to 21. For example, the RNA molecule may be an sgRNA molecule having a scaffold of a sequence set forth in any of SEQ ID NOs: 17 to 21.

[0070] In some embodiments, the RNA molecule comprises the sequence shown in SEQ ID NO:18.

[0071] In some embodiments, the RNA molecule comprises the sequence shown in SEQ ID NO:19.

[0072] In some embodiments, the RNA molecule comprises a guide sequence portion and a sequence set forth in any one of SEQ ID NOs: 17 to 21. For example, in some embodiments, the RNA molecule is an sgRNA molecule comprising a guide sequence portion and a scaffold, and the sequence of the scaffold is set forth in any one of SEQ ID NOs: 17 to 21.

[0073] The present invention also provides a polynucleotide molecule encoding an RNA molecule according to any one of the above aspects.

[0074] The present invention also provides a method for modifying a nucleotide sequence at a DNA target site in the genome of a cell-free system or cell, comprising introducing into said system or cell a composition according to any one of claims 1 to 57 and a CRISPR nuclease having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:1.

[0075] In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell.

[0076] In some embodiments, the eukaryotic cell is a human cell or a plant cell.

[0077] The invention also provides a kit for modifying a nucleotide sequence at a DNA target site in the genome of a cell-free system or cell, comprising introducing into said system or cell a composition according to any one of the above aspects, a CRISPR nuclease having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, and instructions for delivering the RNA molecule and the CRISPR nuclease to said cell.

[0078] In some embodiments of the invention, the non-naturally occurring RNA molecule comprises a "spacer" or "guide sequence" portion. A "spacer portion" or "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize with a specific target DNA sequence, e.g., the guide sequence portion has a nucleotide sequence that is completely complementary to the targeted DNA sequence along the length of the guide sequence portion. In some embodiments, the length of the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or about 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 18-22, 19-22, 18-20, 17-20, or 21-22 nucleotides. Preferably, the entire length of the guide sequence portion is completely complementary to the targeted DNA sequence along the length of the guide sequence portion. The guide sequence portion is a portion of an RNA molecule having a "scaffold portion" that can form a complex with CRISPR nuclease and activate it, and the guide sequence portion of the RNA molecule serves as the DNA targeting portion of the CRISPR complex. When an RNA molecule having a scaffold portion and a guide sequence portion is present simultaneously with a CRISPR molecule, the RNA molecule can direct the CRISPR nuclease to a specific target DNA sequence. Each possibility is a separate aspect of this invention. The spacer portion of the RNA molecule can be specifically designed to target a desired sequence.

[0079] In one embodiment, the nucleotide sequence of the nuclease-binding RNA and the nucleotide sequence of the DNA-targeting RNA (e.g., the spacer or guide sequence portion) are on a single guide RNA molecule (sgRNA), which can form a complex with the OMNI-103 CRISPR nuclease and function as a DNA-targeting module.

[0080] In one embodiment, the nucleotide sequence of the nuclease-binding RNA is on a first RNA molecule and the nucleotide sequence of the DNA-targeting RNA is on a second RNA molecule, said first and second RNA molecules interacting by base pairing to form a complex with the CRISPR nuclease and functioning as a targeting module.

[0081] In some aspects of the invention, the disclosed methods include a method of modifying a nucleotide sequence at a target site in the genome of a cell-free system or a cell, comprising introducing into a cell a composition of any of the embodiments described herein.

[0082] The invention also provides the use of a composition or method of the invention to modify a nucleotide sequence at a DNA target site in a cell.

[0083] The invention provides a method for modifying a nucleotide sequence at a target site in the genome of a eukaryotic cell.

[0084] The invention provides methods for modifying a nucleotide sequence at a target site in the genome of a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0085] The invention provides a method for modifying a nucleotide sequence at a target site in the genome of a plant cell.

[0086] In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo. In some embodiments, some steps of the method are performed ex vivo and some steps are performed in vivo. In some embodiments, the mammalian cells are human cells.

[0087] The invention also provides modified cells or cells obtainable by the methods described herein. In some aspects, these modified cells or cells are capable of giving rise to progeny cells. In some aspects, these modified cells or cells are capable of giving rise to progeny cells after engraftment.

[0088] The invention also provides compositions comprising these modified cells and a pharma- ceutically acceptable carrier, as well as in vitro or ex vivo methods of preparing said compositions comprising combining cells with a pharma- ceutically acceptable carrier.

[0089] The nuclease-RNA guide complexes described herein can target a desired DNA target sequence with a guide RNA molecule. The nuclease-guide complexes also deliver molecules bound to the complex to the target site. Thus, this disclosure also contemplates fusion proteins comprising a CRISPR nuclease and a DNA modifying domain (e.g., a deaminase, nuclease, nickase, recombinase, methyltransferase, methylase, acetylase, acetyltransferase, transcriptional activator or transcriptional repressor domain) and the use of said fusion proteins in correcting mutations in a genome (e.g., the genome of a human subject) associated with a disease or in generating mutations in a genome (e.g., the human genome) to reduce or prevent expression of a gene.

[0090] In some embodiments, the CRISPR nuclease of the present application may be fused to a protein having an enzymatic activity. In some embodiments, the enzymatic activity is to modify the target DNA. In some embodiments, the enzymatic activity is a nuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, or a glycosylase activity. In some embodiments, the enzymatic activity is a nuclease activity. In some embodiments, the nuclease activity is to make a double-stranded break in the target DNA. In some embodiments, the enzymatic activity is to modify the target polypeptide associated with the target DNA. Optionally, the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, or a demyristoylating activity. Optionally, the target polypeptide is a histone and the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, or a deubiquitinating activity.

[0091] Thus, the CRISPR nucleases described herein, or versions of nucleases engineered to be nickases (i.e., with single-stranded DNA cleavage activity) or inactivated nucleases (i.e., unable to cleave DNA strands), may be fused (e.g., directly or via a linker) to another DNA modulating or modifying enzyme, including but not limited to deaminases, reverse transcriptases (e.g., see Anzaolone et al. (2019) for use in prime editing), enzymes that modify the methylation state of DNA (e.g., methyltransferases), or base editors such as modifiers of histones (e.g., histone acetyltransferases). Indeed, the OMNI-103 nucleases, nickases, and inactive nucleases described herein may be fused to a DNA modifying enzyme or its effector domain. Examples of DNA regulatory factors include, but are not limited to, deaminases, nucleases, nickases, recombinases, methyltransferases, methylases, acetylases, acetyltransferases, reverse transcriptases, helicases, integrases, ligases, transposases, demethylases, phosphatases, transcriptional activators, or transcriptional repressors. In some embodiments, the CRISPR nucleases of the present application are fused to a protein having an enzymatic activity. In some embodiments, the enzymatic activity is to modify a target DNA molecule. The CRISPR nucleases or fusion proteins thereof described herein may be used to correct or generate one or more mutations in a gene associated with a disease, or to increase, correct, decrease, or prevent the expression of a gene.

[0092] In some aspects of the invention, the disclosed methods include the use of the compositions described herein for the treatment of a subject suffering from a disease associated with a genomic mutation, including modifying a base sequence at a target site in the subject's genome.

[0093] In some aspects of the invention, the disclosed methods include methods of treating a subject having a mutational disorder comprising targeting an allele associated with the mutational disorder with a composition described herein.

[0094] In some aspects, the mutational disorder is associated with a disease or disorder selected from any of neoplasia, age-related macular degeneration, schizophrenia, neurological disorders, neurodegenerative diseases, movement disorders, fragile X syndrome, secretase-related disorders, prion-related disorders, ALS, addiction, autism, Alzheimer's disease, neutropenia, inflammation-related disorders, Parkinson's disease, blood and coagulation diseases and disorders, cellular dysregulation, tumor-related diseases and disorders, inflammation and immune-related diseases and disorders, metabolic, liver, kidney and protein diseases and disorders, muscular and skeletal diseases and disorders, skin diseases and disorders, neurological diseases and disorders, corneal diseases and disorders, retinal diseases and disorders, and ophthalmic diseases and disorders.

[0095] Disease and Treatment An embodiment of the present invention targets nucleases to specific loci associated with a disease or disorder as a form of gene editing, treatment or therapeutic method. For example, the compositions disclosed herein may be specifically targeted to pathogenic mutant alleles of genes using guide RNA molecules specifically designed to induce gene editing or knockout. It is preferable to design guide RNA molecules by first considering the PAM requirements of the nuclease, which also depends on the system in which gene editing is performed, as shown in the specification. For example, guide RNA molecules designed to target OMNI-103 nuclease to a target site are designed to include a spacer region complementary to the region adjacent to the OMNI-103 PAM sequence "NGG". Guide RNA molecules are preferably further designed to include a spacer region (i.e., the region of the guide RNA molecule complementary to the target allele) of sufficient and preferably optimal length to increase the specific activity of the nuclease and reduce off-target effects. For example, a guide RNA molecule designed to target the OMNI-103 nuclease to a target site may be designed to include a 22 nucleotide spacer to achieve high on-target cleavage activity.

[0096] As a non-limiting example, guide RNA molecules may be designed to target nucleases to specific regions of mutant alleles, for example, near the start codon, such that upon DNA damage by nucleases, non-homologous end joining (NHEJ) pathways are induced, resulting in silencing of the mutant allele by introducing frameshift mutations. This approach to design guide RNA molecules is particularly useful for altering the action of dominant-negative mutations, thereby treating subjects. As another non-limiting example, guide RNA molecules may be designed to target specific pathogenic mutations of mutated alleles, such that upon DNA damage by nucleases, homology-directed repair (HDR) pathways are induced, resulting in template-mediated correction of the mutant allele. This approach to design guide RNA molecules is particularly useful for altering the haploinsufficient action of mutant alleles, thereby treating subjects.

[0097] Non-limiting examples of genes that may be targeted for modification to treat disease or disorders are given below. Disease-associated genes and mutations that induce mutation disorders are described in the literature. Such mutations can be used to design DNA targeting RNA molecules that target the alleles of disease-associated genes, and CRISPR compositions that induce DNA damage to induce DNA repair pathways to modify the alleles, thereby treating the mutation disorder.

[0098] Mutations in the ELANE gene are associated with neutropenia. Accordingly, aspects of the invention that target ELANE may be used, without limitation, in methods of treating subjects suffering from neutropenia.

[0099] CXCR4 is a coreceptor in human immunodeficiency virus type 1 (HIV-1) infection. Accordingly, aspects of the invention that target CXCR4 may be used, without limitation, in methods of treating subjects with HIV-1 or conferring resistance to HIV-1 infection in a subject.

[0100] Disruption of programmed cell death protein 1 (PD-1) promotes CAR-T cell killing of tumor cells, making PD-1 a potential target for cancer therapy. Thus, embodiments of the present invention that target PD-1 may be used without restriction in methods of treating subjects with cancer. In one embodiment, the treatment is CAR-T cell therapy with T cells modified according to the present invention to be PD-1 deficient.

[0101] In addition, BCL11A is a gene involved in the suppression of hemoglobin production. By inhibiting BCL11A, globin production may be increased to treat diseases such as thalassemia and sickle cell anemia. See, for example, WO 2017 / 077394, U.S. Patent Application Publication No. 2011 / 0182867; Humbert et al. Sci. Transl. Med. (2019) and Canver et al. Nature (2015). Thus, aspects of the invention that target enhancers of BCL11A may be used without restriction in methods of treating subjects suffering from β-thalassemia or sickle cell anemia.

[0102] The present invention may be used to target disease-associated genes in the investigation, modification or treatment of diseases or disorders listed in Table A or Table B below. Indeed, disease-associated genes may be investigated, modified or targeted by the nucleases disclosed herein to treat diseases caused by the disease-associated genes. Non-limiting examples are listed in US Patent Application Publication No. 2018 / 0282762 and EP Patent No. 3079726 (B1).

[0103] [Table A]

[0104] [Table B-1]

[0105] [Table B-2]

[0106] [Table B-3]

[0107] Unless otherwise defined, all technical and / or scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described in the specification can be used in the practice or testing of embodiments of the present invention, representative methods and / or materials are described below. In case of conflict, the specification, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be necessarily limiting.

[0108] Unless otherwise stated in the discussion, adjectives such as "substantially" and "about" that modify the state or relationship of features of an embodiment of the invention are understood to mean that the state or relationship is defined within an acceptable range for operation of the embodiment for its intended use. Unless otherwise indicated, the term "or" in the specification and claims is considered an inclusive "or" rather than an exclusive "or," indicating at least one, and any combination, of the items it conjugates.

[0109] The term "a" or "an" as used herein should be understood to refer to "one or more" of the listed components. It will be apparent to one of ordinary skill in the art that the use of the singular includes the plural unless otherwise specified. Thus, the terms "a" and "at least one" have the same meaning in this application.

[0110] To better understand the teachings and in no way limit the scope of the teachings, unless otherwise specified, all numbers indicating quantities, percentages or ratios, and other numerical values ​​used in the specification and claims should be understood to be modified in all instances by the term "about." Thus, unless indicated to the contrary, the numerical values ​​set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, each numerical value should be interpreted taking into account the number of significant digits and applying ordinary rounding techniques.

[0111] When numerical ranges are stated in this specification, it is understood that the invention contemplates every integer between the upper and lower limits, inclusive, unless otherwise stated.

[0112] In this specification and the claims, the verbs "contain," "include," and "have," and each of their conjugations, are used to indicate that the object of the verb is not necessarily an exhaustive list of components, elements, or parts of the subject of the verb. Other terms in this specification are intended to have the meanings that are well known in the art.

[0113] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are synonymous. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA sequences, isolated RNA sequences, nucleic acid probes and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides or their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide elements. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0114] The term "nucleotide analog" or "modified nucleotide" refers to a nucleotide that contains one or more of a variety of chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U), adenine (A) or guanine (G)), in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analogs or open-ring sugar analogs), or in the phosphate moiety. Each of the RNA sequences described herein may contain one or more nucleotide analogs.

[0115] As used herein, the following nucleotide identifiers are used to represent nucleotide bases:

[0116] [Table C]

[0117] In this specification, the term "targeting sequence" or "targeting molecule" refers to a nucleotide sequence that can hybridize with a specific target sequence or a molecule that includes the nucleotide sequence, e.g., a targeting sequence has a nucleotide sequence that is at least partially complementary to the sequence to be targeted. The targeting sequence or targeting molecule may be a part of a targeting RNA molecule that can form a complex with a CRISPR nuclease, e.g., via a scaffolding portion, and the targeting sequence serves as a targeting portion (e.g., a spacer portion) of the CRISPR complex. When an RNA molecule having a targeting sequence is present simultaneously with a CRISPR nuclease, the RNA molecule, alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), can direct the CRISPR nuclease to a specific target sequence. As a non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single guide RNA molecule may serve as a targeting molecule. Each possibility is a separate aspect of this invention. The targeting sequence can be custom designed to target a desired sequence.

[0118] In this specification, the term "targeting" or "directing" refers to the preferential hybridization of a targeting molecule or targeting sequence with a nucleic acid having a target nucleotide sequence. The term "targeting" encompasses varying hybridization efficiency, and thus, the nucleic acid having a target nucleotide sequence is preferentially targeted, but it is understood that in addition to on-target hybridization, unintended off-target hybridization may also occur. When an RNA molecule targets a sequence, it is understood that the complex of the RNA molecule and the CRISPR nuclease molecule targets that sequence for nuclease activity.

[0119] The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize to a specific target DNA sequence, e.g., the guide sequence portion has a nucleotide sequence that is partially or completely complementary to the DNA sequence targeted along the guide sequence portion. In some embodiments, the length of the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or about 17-50, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-44, 17-45, 17-46, 17-47, 17-48, 17-49, 17-49, 17-50 ... 40, 17 to 39, 17 to 38, 17 to 37, 17 to 36, 17 to 35, 17 to 34, 17 to 33, 17 to 31, 17 to 30, 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 17 to 24, 17 to 22, 17 to 21, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 22, 18 to 20, 20 to 21, 21 to 22, or 17 to 20. Preferably, the entire length of the guide sequence portion is completely complementary to the target DNA sequence along the guide sequence portion. The guide sequence portion may be a part of an RNA molecule that can form a complex with a CRISPR nuclease, with the guide sequence portion serving as the DNA targeting portion of the CRISPR complex. When an RNA molecule having a guide sequence portion is present simultaneously with a CRISPR molecule, alone or in combination with one or more other RNA molecules (e.g., tracrRNA molecules), the RNA molecule can direct the CRISPR nuclease to a specific target DNA sequence. Thus, a CRISPR complex can be formed by directly binding an RNA molecule having a guide sequence portion to a CRISPR nuclease, or by binding an RNA molecule having a guide sequence portion to one or more RNA molecules to a CRISPR nuclease. Each possibility is a separate aspect of this invention. The guide sequence portion can be custom designed to target a desired sequence. Thus, a molecule that includes a "guide sequence portion" is a type of targeting molecule.Throughout this application, the terms "guide molecule," "RNA guide molecule," "guide RNA molecule," and "gRNA molecule" are synonymous with a molecule that comprises a guide sequence portion.

[0120] In the case of targeting a DNA sequence present in a plurality of cells, it is understood that targeting encompasses the hybridization of the guide sequence portion of the RNA molecule with a sequence in one or more cells, and also encompasses the hybridization of the RNA molecule with the target sequence in not all cells in the plurality of cells.Therefore, in the case of targeting a sequence in a plurality of cells, it is understood that the complex of the RNA molecule and the CRISPR nuclease hybridizes with the target sequence in one or more cells, and it is also understood that it may hybridize with the target sequence in not all cells.Therefore, it is understood that the complex of the RNA molecule and the CRISPR nuclease hybridizes with the target sequence in one or more cells, and may also hybridize with the target sequence in not all cells, and may also break the double strand.In this specification, the term "modified cell" refers to a cell in which double strand break is made by the complex of the RNA molecule and the CRISPR nuclease as a result of hybridization with the target sequence, i.e., on-target hybridization.

[0121] As used herein, the term "wild type" refers to a typical form of an organism, strain, gene, or trait that occurs in nature, as distinguished from a variant or mutant, a term of art that is understood by those of skill in the art. Thus, as used herein, when an amino acid or nucleotide sequence refers to a wild type sequence, a variant refers to a variant of that sequence, including, for example, a substitution, deletion, or insertion. In some embodiments of the invention, the modified CRISPR nuclease is a variant of a CRISPR nuclease that includes at least one amino acid modification (e.g., a substitution, deletion, and / or insertion) relative to the OMNI-103 CRISPR nuclease shown in Table 1.

[0122] The terms "non-natural," "non-naturally occurring," or "artificial" are used interchangeably and refer to human modification. When used with respect to a nucleic acid molecule or polypeptide, the term may mean that the nucleic acid molecule or polypeptide is at least substantially free from at least one component with which it is naturally associated in nature and which is found in nature.

[0123] As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and its D or I optical isomers, as well as amino acid analogs and peptidomimetics.

[0124] As used herein, "genomic DNA" refers to linear and / or chromosomal DNA and / or plasmid or other extrachromosomal DNA sequences present in a cell or cells of interest. In some embodiments, the cells of interest are eukaryotic cells. In some embodiments, the cells of interest are prokaryotic cells. In some embodiments, the method creates a double-strand break (DSB) at a predetermined target site in the genomic DNA sequence, resulting in a mutation, insertion and / or deletion of the DNA sequence at the target site in the genome.

[0125] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.

[0126] As used herein, the term "nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases may be isolated or derived from natural sources. The natural source may be any organism. Alternatively, nucleases may be modified or synthetic proteins that have phosphodiester bond cleavage activity.

[0127] As used herein, the term "PAM" refers to a nucleotide sequence in a target DNA that is located adjacent to the target DNA sequence and that is recognized by a CRISPR nuclease. The PAM sequence may vary depending on the nuclease.

[0128] As used herein, the term "mutation disorder" or "mutation disease" refers to a disorder or disease associated with a dysfunction of a gene caused by a mutation. A dysfunctional gene that manifests as a mutation disorder contains a mutation in at least one of its alleles and is called a "disease-associated gene". The mutation may be in any part of the disease-associated gene, for example, a regulatory part, a coding part, or a non-coding part. The mutation may be a substitution, an insertion, or a deletion mutation. The mutation of the disease-associated gene may manifest as a disorder or disease according to any mutation mechanism, such as a recessive, dominant negative, a gain-of-function, a loss-of-function, or a mutation that leads to haploinsufficiency of the gene product.

[0129] Those skilled in the art will understand that this embodiment of the invention discloses an RNA molecule that includes a scaffold portion that can form a complex with OMNI-103 CRISPR nuclease and activate OMNI-103 CRISPR nuclease to target a target DNA site of interest adjacent to a protospacer adjacent motif (PAM). OMNI-103 CRISPR nuclease targets said DNA site by a guide sequence portion (i.e., RNA spacer) that has complementarity to the target DNA site of interest. The nuclease then generates a double-strand break in the protospacer target site through cleavage of the target DNA.

[0130] The term "protein binding sequence" or "nuclease binding sequence" refers to a sequence that can bind to CRISPR nuclease to form a CRISPR complex. Those skilled in the art will understand that the scaffold RNA or tracrRNA that can bind to CRISPR nuclease to form a CRISPR complex includes a protein or nuclease binding sequence.

[0131] The "RNA binding portion" of a CRISPR nuclease refers to the portion of the CRISPR nuclease that may bind to an RNA molecule to form a CRISPR complex, for example, the nuclease binding sequence of the RNA scaffold portion of an sgRNA. The "active portion" of a CRISPR nuclease refers to the portion of the CRISPR nuclease that makes a double-stranded break in a DNA molecule, for example, when complexed with a DNA targeting RNA molecule.

[0132] The term "RNA scaffold" or "scaffold" refers to a portion of a non-naturally occurring molecule that includes a crRNA portion covalently linked to a tracrRNA portion. As used herein, a "crRNA portion" includes a crRNA repeat sequence. As used herein, a "tracrRNA portion" includes a tracrRNA anti-repeat sequence. The tracrRNA portion may further include another tracrRNA sequence linked to the tracrRNA anti-repeat sequence. Such sequences may include, but are not limited to, a nexus sequence, a hairpin sequence, or another tracrRNA sequence upstream or downstream of the nexus sequence, hairpin sequence, or tracrRNA anti-repeat sequence. Thus, the tracrRNA portion of the RNA scaffold includes an anti-repeat sequence, which is optionally linked to another tracrRNA section.

[0133] In this specification, an RNA molecule comprising an RNA scaffold portion and an RNA guide sequence portion (or an RNA spacer portion) functions as a single guide RNA (sgRNA) molecule. The RNA scaffold portion of the sgRNA specifically binds to and activates a CRISPR nuclease, and the RNA spacer portion of the sgRNA directs the CRISPR nuclease to a DNA target site. For example, an sgRNA molecule may be formed by covalent bonding between the crRNA repeat sequence portion and the guide sequence portion of the RNA scaffold.

[0134] Thus, in embodiments of the invention, RNA molecules may be designed as synthetic fusions of a scaffold portion and a spacer portion that together form a single guide RNA (sgRNA) capable of binding to and targeting the OMNI-103 CRISPR nuclease. See Jinek et al., Science (2012).

[0135] In some embodiments of the invention, separate crRNA and tracrRNA molecules may be used to form a CRISPR complex. In such embodiments, the crRNA molecule may hybridize to the tracrRNA molecule by at least partial hybridization between the crRNA repeat sequence portion of the crRNA molecule and the tracrRNA anti-repeat sequence portion of the tracrRNA molecule. Such partial hybridization may also include a typical bulge that separates the hybridized RNA nucleotides into an "upper" stem and a "lower" stem. Separate crRNA and tracrRNA molecules may be advantageous in certain applications of the invention described herein.

[0136] In embodiments of the invention, the scaffold portion of the RNA molecule may contain "nexus" regions and / or "hairpin" regions that may further specify the structure of the RNA molecule (see Briner et al., Molecular Cell (2014)).

[0137] As used herein, the term "direct repeat sequence" refers to two or more repeats of a particular amino acid or nucleotide sequence.

[0138] As used herein, an RNA sequence or molecule that can "interact with" or "bind to" a CRISPR nuclease refers to the ability of the RNA sequence or molecule to form a CRISPR complex with a CRISPR nuclease.

[0139] As used herein, the term "operably linked" refers to a relationship (i.e., fusion, hybridization) between two sequences or molecules that allows them to function in their intended manner. In embodiments of the invention, when an RNA molecule is operably linked to a promoter, the RNA molecule and the promoter can function in their intended manner.

[0140] As used herein, the term "heterologous promoter" refers to a promoter that is not naturally associated with the molecule being expressed or the pathway being promoted.

[0141] As used herein, a sequence or molecule has X% "sequence identity" with respect to a respective sequence or molecule if X% of the nucleotides or amino acids between the sequences of the molecules are the same and in the same relative positions. For example, a first nucleotide sequence that has at least 95% sequence identity with a second nucleotide sequence has at least 95% of the nucleotides in the same relative positions as the other sequence. As a non-limiting example, sequence identity may be determined by applying, for example, the Needleman-Wunsch algorithm to generate an alignment of the first and second nucleotide sequences.

[0142] delivery The CRISPR nuclease or CRISPR composition described herein may comprise or be delivered as a protein, a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or a combination thereof. In some embodiments, the RNA molecule comprises a chemical modification. Non-limiting examples of suitable chemical modifications include 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS) or 2'-O-methyl, 3'-thioPACE (MSP), pseudouridine, and 1-methylpseudouridine. Each possibility is a separate aspect of this invention.

[0143] Nucleotide molecules such as the CRISPR nucleases described herein and / or polynucleotides encoding same, and optionally additional proteins (e.g., ZFPs, TALENs, transcription factors, restriction enzymes) and / or guide RNAs, may be delivered to a target cell by suitable means. A target cell may be any cell, such as a eukaryotic or prokaryotic cell, in any environment, whether isolated or not, in culture, in vitro, ex vivo, in vivo or in planta.

[0144] In some embodiments, the composition delivered comprises a nuclease mRNA and a guide molecule RNA. In some embodiments, the composition delivered comprises a nuclease mRNA, a guide RNA, and a donor template. In some embodiments, the composition delivered comprises a CRISPR nuclease and a guide RNA. In some embodiments, the composition delivered comprises a CRISPR nuclease, a guide RNA, and a donor template for gene editing, e.g., by homology-directed repair. In some embodiments, the composition delivered comprises a nuclease mRNA, a DNA targeting RNA, and a tracrRNA. In some embodiments, the composition delivered comprises a nuclease mRNA, a DNA targeting RNA, a tracrRNA, and a donor template. In some embodiments, the composition delivered comprises a CRISPR nuclease, a DNA targeting RNA, and a tracrRNA. In some embodiments, the composition delivered comprises a CRISPR nuclease, a DNA targeting RNA, and a tracrRNA. In some embodiments, the composition delivered comprises a CRISPR nuclease, a DNA targeting RNA, a tracrRNA, and a donor template for gene editing, e.g., by homology-directed repair.

[0145] The RNA composition can be delivered using a suitable viral vector system. Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acid and / or CRISPR nuclease into cells (e.g., mammalian cells, plant cells, etc.) and target tissues. Such methods can also be used to administer the in vitro encoded nucleic acid and / or CRISPR nuclease protein to cells. In some embodiments, the nucleic acid and / or CRISPR nuclease is administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include naked nucleic acid and nucleic acid complexed with a delivery vehicle such as liposomes or poloxamers. For reviews of gene therapy procedures, see Anderson, Science (1992); Nabel and Felgner, TIBTECH (1993); Mitani and Caskey, TIBTECH (1993); Dillon, TIBTECH (1993); Miller, Nature (1992); Van Brunt, Biotechnology (1988); Vigne et al., Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin (1995); Haddada et al., Current Topics in Microbiology and Immunology (1995), and Yu et al., Gene Therapy 1:13-26 (1994).

[0146] Non-viral methods of delivery of nucleic acids and / or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, artificial virions, and drug-enhanced nucleic acid uptake, or can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboium meliloti, Mesorhizobium loti, Tobacco Mosaic Virus, Potato Virus X, Cauliflower Mosaic Virus, Cassava Vein Mosaic Virus). See, e.g., Chung et al. Trends Plant Sci. (2006). Sonoporation, e.g., using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids. Cationic lipid-mediated delivery of proteins and / or nucleic acids is also contemplated as an in vivo or in vitro delivery method, see Zuris et al., Nat. Biotechnol. (2015); Coelho et al., N. Engl. J. Med. (2013); Judge et al., Mol. Ther. (2006) and Basha et al., Mol. Ther. (2011).

[0147] Non-viral vectors, such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon system or recombinant PiggyBac transposon system), may also be used to deliver to target cells and transpose the polynucleotide sequences of, or encoding, the molecules of the composition in the target cells.

[0148] Other representative nucleic acid delivery systems include those provided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofectin is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and Lipofectamine® RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO 91 / 17424 and WO 91 / 16024. Delivery to cells (ex vivo administration) or target tissues (in vivo administration) is possible.

[0149] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is widely known to those of skill in the art (see, e.g., Crystal, Science (1995); Blaese et al., Cancer Gene Ther. (1995); Behr et al., Bioconjugate Chem. (1994); Remy et al., Bioconjugate Chem. (1994); Gao and Huang, Gene Therapy (1995); Ahmad and Allen, Cancer Res., (1992); U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028 and 4,946,787).

[0150] Another delivery method involves using packaging of the nucleic acid to be delivered in an EnGeneIC delivery vehicle (EDV). The EDV is delivered specifically to the target tissue using a bispecific antibody, where one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody carries the EDV to the surface of the target cell, where it is then carried into the cell by endocytosis. Once inside the cell, the contents are released (see MacDiamid et al., Nature Biotechnology (2009)).

[0151] The use of RNA or DNA virus-based systems for delivering nucleic acids utilizes highly evolved methods to target viruses to specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to the patient (in vivo) or can be used to treat cells in vitro and the modified cells are administered to the patient (ex vivo). RNA or DNA virus-based systems for delivering nucleic acids include, but are not limited to, recombinant retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and herpes simplex virus vectors for gene transfer. However, RNA viruses are preferred for delivery of the RNA compositions described herein. Also, high transduction efficiency has been observed in various cells and target tissues. The nucleic acids of the present invention may be delivered by non-integrating lentiviruses. Optionally, lentivirus-based RNA delivery is utilized. In some cases, the lentivirus includes a nuclease mRNA, a guide RNA. In some cases, the lentivirus includes a nuclease mRNA, a guide RNA and a donor template. In some cases, the lentivirus comprises a nuclease protein, a guide RNA. In some cases, the lentivirus comprises a nuclease protein, a guide RNA, and / or a donor template for gene editing, for example, by homology-directed repair. In some cases, the lentivirus comprises a nuclease mRNA, a DNA targeting RNA, and a tracrRNA. In some cases, the lentivirus comprises a nuclease mRNA, a DNA targeting RNA, a tracrRNA, and a donor template. In some cases, the lentivirus comprises a nuclease protein, a DNA targeting RNA, and a tracrRNA. In some cases, the lentivirus comprises a nuclease protein, a DNA targeting RNA, and a tracrRNA. In some cases, the lentivirus comprises a nuclease protein, a DNA targeting RNA, a tracrRNA, and a donor template for gene editing, for example, by homology-directed repair.

[0152] As previously described, the compositions described herein can be delivered to target cells using non-integrating lentiviral particle methods (e.g., the LentiFlash® system). Such methods may be used to deliver mRNA or other RNA to target cells such that delivery of the RNA to the target cell results in assembly of the compositions described herein inside the target cell. See also WO 2013 / 014537, WO 2014 / 016690, WO 2016 / 185125, WO 2017 / 194902, WO 2017 / 194903.

[0153] Retroviral tropism can be altered by incorporating foreign envelope proteins, expanding the potential target cell targeting. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and usually produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeat sequences that can package up to 6-10 kb of foreign sequences. A minimal number of cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells and provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher Panganiban, J. Virol. (1992); Johann et al., J. Virol. (1992); Sommerfelt et al., Virol. (1990); Wilson et al., J. Virol. (1989); Miller et al., J. Virol. (1991); WO 94 / 26877).

[0154] At least six viral vector approaches are currently available for gene transfer in clinical trials, using methods involving complementation of a defective vector by a gene inserted into a helper cell line to generate the transducing agent.

[0155] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood (1995);Kohn et al., Nat. Med. (1995);Malech et al., PNAS (1997)). PA317 / pLASN was the first therapeutic vector used in gene therapy (Blaese et al., Science (1995)). Transduction efficiencies of over 50% have been observed with MFG-S packaged vectors (Ellem et al., Immunol Immunother. (1997);Dranoff et al., Hum. Gene Ther. (1997)).

[0156] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus AAV, and psi.2 or PA317 cells, which package retrovirus. Viral vectors for use in gene therapy are usually obtained by producer cell lines that package nucleic acid vectors into viral particles. The vectors usually contain the minimal viral sequences required for packaging and subsequent integration into the host (if applicable), other viral sequences being replaced by expression cassettes that code for the proteins to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors for use in gene therapy usually only possess the terminal inverted repeat (ITR) sequences of the AAV genome, which are required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid that codes for other AAV genes, namely rep and cap, but lacks the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus facilitates the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. The helper plasmid is not packaged in large quantities because it lacks ITR sequences. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Furthermore, AAV can be produced on a clinical scale using the baculovirus system (see U.S. Patent No. 7,479,554).

[0157] In many gene therapies, it is desirable for the gene therapy vector to be delivered with high specificity to a particular tissue. Thus, viral vectors can be modified to have specificity for cells of interest by expressing a ligand on the outer surface of the virus as a fusion protein with the viral coat protein. The ligand is selected to have affinity for a receptor known to be present on the cells of interest. For example, Han et al., Proc. Natl. Acad. Sci. USA (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell combinations where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for the cell surface receptor. For example, filamentous phage can be modified to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for virtually any cellular receptor. Although this discussion applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be modified to contain uptake sequences that facilitate uptake by specific target cells.

[0158] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial injection) or local application as described below. Alternatively, vectors can be delivered ex vivo to cells, such as transplanted cells (e.g., lymphocytes, bone marrow aspirates, biopsy tissue) from an individual patient or hematopoietic stem cells from a universal donor, which are then re-implanted into the patient, typically after selection of cells that have incorporated the vector. In some embodiments, in vivo and ex vivo delivery of mRNA, as well as delivery of RNPs, may be utilized.

[0159] Ex vivo cell transfection for diagnostics, research, or gene therapy (e.g., by re-injection of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from a subject organism, transfected with an RNA composition, and re-injected into the subject organism (e.g., a patient). A variety of cells suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney, "Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications" (6th edition, 2010) and references cited therein for a discussion of how to isolate and culture cells from a patient).

[0160] Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells and / or cell lines. Non-limiting examples of such cells or cell lines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T) and perC6 cells, any plant cell (differentiated or undifferentiated), as well as insect cells such as Spodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and chizosaccharomyces. In some embodiments, the cell line is a CHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may be isolated and treated with a nuclease (e.g., ZFN or TALEN) or nuclease system (e.g., CRISPR) and then used ex vivo for reintroduction into the subject. Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and blood cell subsets, such as, but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells and mesenchymal stem cells.

[0161] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage of using stem cells is that they can be differentiated in vitro into other cells or introduced into a mammal (such as a cell donor) where they engraft in the bone marrow. Methods are known for differentiating CD34+ cells in vitro into clinically important immune cells using cytokines such as GM-CSF, IFNγ, and TNFα (see, for non-limiting examples, Inaba et al., J. Exp. Med. (1992)).

[0162] Stem cells are isolated for transduction and differentiation by known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies that bind to unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (pan B cells), GR-1 (granulocytes), and Iad (differentiated antigen presenting cells) (see, for non-limiting examples, Inaba et al., J. Exp. Med. (1992)). In some embodiments, modified stem cells can also be used.

[0163] In particular, the compositions described herein may be suitable for genome editing of post-mitotic cells or cells that are not actively dividing (e.g., arrested cells). Examples of post-mitotic cells that may be edited using the CRISPR nucleases of the invention include, but are not limited to, muscle cells, cardiomyocytes, liver cells, bone cells, and neurons.

[0164] Vectors (e.g., retroviruses, liposomes, etc.) containing therapeutic RNA compositions can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked RNA or mRNA can be administered. Administration is by routes including, but not limited to, injection, infusion, topical application, and electroporation, which are commonly used to introduce molecules with eventual contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and known to those of skill in the art, and although multiple routes of administration of a particular composition can be used, certain routes often result in more rapid and effective responses than others.

[0165] Suitable vectors for introducing transgenes into immune cells (e.g., T cells) include non-integrating lentiviral vectors, see, e.g., U.S. Patent Application Publication No. 2009 / 0117617.

[0166] Pharmaceutically acceptable carriers are determined in part by the composition being administered, as well as by the method used to administer the composition. Thus, there is a wide variety of suitable formulations of pharmaceutical compositions available, e.g., as described in Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0167] DNA repair by homologous recombination The term "homologous recombination repair" or "HDR" refers to a mechanism that repairs DNA damage in cells, for example, during repair of double-stranded and single-stranded breaks in DNA. HDR requires nucleotide sequence homology and uses a "nucleic acid template" (nucleic acid template or donor template, used interchangeably herein) to repair a sequence (e.g., a DNA target sequence) where a double-stranded or single-stranded break has occurred. This results, for example, in the transfer of genetic information from the nucleic acid template to the DNA target sequence. If the nucleic acid template sequence differs from the DNA target sequence and some or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence, HDR can lead to alterations of the DNA target sequence (e.g., insertions, deletions, mutations). In some embodiments, all or a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template, is incorporated into the site of the DNA target sequence.

[0168] The terms "nucleic acid template" and "donor" refer to a nucleotide sequence to be inserted or copied into a genome. A nucleic acid template comprises a sequence of, e.g., one or more nucleotides that may be added to a target nucleic acid, template a change in a target nucleic acid, or be used to modify a target sequence. The length of the nucleic acid template sequence may be any length, e.g., 2-10,000 nucleotides (or any integer therebetween or greater), preferably about 100-1,000 nucleotides (or any integer therebetween), more preferably about 200-500 nucleotides. A nucleic acid template may be a single-stranded nucleic acid, a double-stranded nucleic acid. In some embodiments, a nucleic acid template comprises a sequence of, e.g., one or more nucleotides, corresponding to a wild-type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template comprises a sequence of, e.g., one or more ribonucleotides, corresponding to a wild-type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template comprises modified ribonucleotides.

[0169] Insertion of exogenous sequences (also referred to as "donor sequences", "donor templates" or "donors") can also be performed, for example, to correct a mutant gene or increase expression of a wild-type gene. It is readily apparent that a donor sequence is not usually identical to the genomic sequence in which it is placed. A donor sequence can include a non-homologous sequence flanked by two homologous regions to allow efficient HDR at a target location. Additionally, a donor sequence can include a vector molecule that includes a sequence that is not homologous to a target region in cellular chromatin. A donor molecule can include discontinuous regions that are homologous to cellular chromatin. For example, to target insertion of a sequence that is not normally present in a target region, the sequence can be present in a donor nucleic acid molecule and can be flanked by regions that are homologous to a sequence of the target region.

[0170] The donor polynucleotide may be single-stranded and / or double-stranded DNA or RNA, and may be introduced into cells in linear or circular form. See, for example, US Patent Application Publication Nos. 2010 / 0047805; 2011 / 0281361; 2011 / 0207221 and 2019 / 0330620. When introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang and Wilson, Proc. Natl. Acad. Sci. USA (1987); Nehls et al., Science (1996). Other methods of protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages (e.g., phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues).

[0171] Thus, the embodiment of the present invention using a donor template for repair may use DNA or RNA, which is a single-stranded and / or double-stranded donor template that can be introduced into cells in a linear or circular form. In an embodiment of the present invention, the gene editing composition comprises (1) an RNA molecule comprising a guide sequence that makes a double-stranded break in the gene before repair, and (2) a donor RNA template for repair, where the RNA molecule comprising the guide sequence is a first RNA molecule and the donor RNA template is a second RNA molecule. In some embodiments, the guide RNA molecule and the template RNA molecule are linked as part of one molecule.

[0172] The donor sequence may be an oligonucleotide and may be used for gene correction or targeted modification of an endogenous sequence. The oligonucleotide may be introduced into the cell by a vector, electroporated into the cell, or by other methods known in the art. The oligonucleotide may be used to "correct" a mutant sequence in an endogenous gene (e.g., the sickle mutation of beta globin) or may be used to insert a sequence at an endogenous locus for a desired purpose.

[0173] Polynucleotides can be introduced into cells as part of a vector molecule that contains additional sequences such as, for example, an origin of replication, a promoter, and genes encoding antibiotic resistance. Additionally, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome, poloxamer, or delivered by recombinant virus (e.g., adenovirus, AAV, herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

[0174] The donor is generally inserted such that its expression is driven by the endogenous promoter of the integration site, i.e., the promoter that drives expression of the endogenous gene into which the donor is inserted. However, it will be apparent that the donor may also include a promoter and / or enhancer, e.g., a constitutive promoter or an inducible or tissue-specific promoter.

[0175] The donor molecule may be inserted into an endogenous gene such that all or a portion of the endogenous gene is expressed, or none of it is expressed. For example, a transgene as described herein may be inserted into an endogenous locus such that a portion of the endogenous sequence (e.g., the N-terminus and / or C-terminus of the transgene) is expressed, or none of it is expressed, e.g., as a fusion with the transgene. In other embodiments, the transgene (with or without additional coding sequences, e.g., an endogenous gene) is integrated into any endogenous locus, such as a safe harbor locus (e.g., the CCR5 gene, the CXCR4 gene, the PPP1R12c (also known as AAVS1) gene, the albumin gene, or the Rosa gene). See, e.g., U.S. Patent Nos. 7,951,925 and 8,110,379; U.S. Patent Application Publication Nos. 2008 / 0159996; 20100 / 0218264; 2010 / 0291048; 2012 / 0017290; 2011 / 0265198; 2013 / 0137104; 2013 / 0122591; 2013 / 0177983 and 2013 / 0177960, and U.S. Provisional Application No. 61 / 823,689).

[0176] When an endogenous sequence (endogenous or part of a transgene) is expressed along with a transgene, the endogenous sequence may be a full-length sequence (wild-type or mutant) or a partial sequence. Preferably, the endogenous sequence is functional. Examples of functions of these full-length or partial sequences include, but are not limited to, increasing the half-life of a polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0177] In addition, although not essential for expression, the exogenous sequence may also include transcriptional or translational regulatory sequences, such as a promoter, an enhancer, an insulator, an internal ribosome entry site, a sequence encoding a 2A peptide, and / or a polyadenylation signal.

[0178] In one embodiment, the donor molecule comprises a sequence selected from the group consisting of a gene encoding a protein (e.g., a coding sequence encoding a protein that is missing in a cell or individual, or an alternative version of a gene encoding a protein), a regulatory sequence, and / or a sequence encoding a structural nucleic acid such as a microRNA or siRNA.

[0179] It is intended that the embodiments described above are applicable to each other, for example, it is understood that an RNA molecule or composition of the invention may be utilized in a method of the invention.

[0180] In this specification, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of each section is equally applicable to all sections.

[0181] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various aspects and aspects of the present invention as described above and as claimed in the appended claims is experimentally supported in the following examples.

[0182] It will be understood that features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately, in any suitable subcombination, or in any other embodiment of the invention, as appropriate. Certain features described in the context of various embodiments should not be construed as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0183] Generally, the nomenclature used herein and the laboratory procedures utilized in this invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully described in the literature. See, e.g., Sambrook et al., "Molecular Cloning: A laboratory Manual" (1989); Ausubel, RM (Ed.), "Current Protocols in Molecular Biology" Volumes I-III (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.), "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998); the methods set forth in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; Cellis, JE (Ed.), "Cell Biology: A Laboratory Handbook", Volumes I-III (1994); Freshney, "Culture of Animal Cells - A Manual of Basic Technique" Third Edition, Wiley-Liss, NY (1994); Coligan JE (Ed.), "Current Protocols in Immunology" Volumes I-III (1994); Stites et al. (Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994);Mishell and Shiigi (Eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996);Clokie and Kropinski (Eds.), "Bacteriophage Methods and Protocols", Volume 1: Isolation, Characterization, and Interactions (2009), all of which are incorporated by reference. Other general references are provided throughout this specification.

[0184] In order to facilitate a more complete understanding of the present invention, the following examples are provided to illustrate representative modes of making and practicing the present invention. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are intended for illustrative purposes only. EXAMPLES

[0185] Experimental details In order to facilitate a more complete understanding of the invention, the following examples are provided. The following examples set forth representative modes of making and practicing the invention. However, the scope of the invention is not limited to the specific embodiments disclosed in the examples, which are intended for illustrative purposes only.

[0186] method The OMNI-103 sequences of CRISPR repeats (crRNA), transactivating crRNA (tracrRNA) and nuclease polypeptides were predicted from metagenomic sequence databases of environmental samples. The spacer-optimized full-length guide scaffold, NNRRHY PAM, and activity in mammals are discussed in WO 2022 / 170199 (published August 11, 2022), and the main elements are shown in Table 1.

[0187] Expression of OMNI-103 protein Briefly, the nuclease open reading frame was codon-optimized for human (Table 1) and cloned into a modified pET9a plasmid (sequence listed in Table 2) with SV40 NLS-OMNI-103 ORF (from the second amino acid, human optimized)-HA tag-SV40 NLS-8 His tag. The OMNI-103 construct was expressed in KRX cells (PROMEGA). Cells were grown in TB+0.4% glycerol supplemented with 6.66 mM rhamnose (26.4 ml from 0.5 M stock) and 0.05% glucose (2 ml from 0.5 M) and expressed in mid-log phase 4 hours after reducing the temperature to 20° C. Cells were lysed by chemical lysis and the clarified lysate was purified on Ni-NTA resin. The Ni-NTA eluted fractions were purified on CEX (SO3 fractogel) resin, followed by SEC purification on Superdex® 200 Increase 10 / 300 GL, AKTA Pure (GE Healthcare Life Sciences). Fractions containing OMNI-103 protein were pooled, concentrated to a 30 mg / ml stock, flash frozen in liquid nitrogen, and stored at -80°C.

[0188] Synthetic sgRNA used All synthetic sgRNAs for OMNI-103 were synthesized with three 2'-O-methyl 3'-phosphorothioates at the 3' and 5' ends (Agilent or Synthego).

[0189] Activity in mammalian cell lines The ability of OMNI-103 to promote editing with short sgRNA versions was tested at specific genomic locations in human cells (Table 4). For HeLa cells, the OMNI-103-P2A-mCherry expression vector (pmOMNI, Table 2) was transfected with sgRNA (pShuttle guide - Table 2, spacer sequence - Table 4).

[0190] For U2OS cells, RNPs were assembled by mixing 100uM nuclease with 120uM synthetic guide and 100uM Cas9 electroporation enhancer (IDT). After 10 minutes of incubation at room temperature, the RNP complex was mixed with 200,000 pre-washed U2OS cells and electroporated using the Lonza SE Cell Line 4D-Nucleofector®X Kit with DN100 program according to the manufacturer's protocol. At 72 hours, the cells were lysed and their genomic DNA content was used in a PCR reaction to amplify the corresponding putative genomic targets. The amplicons were subjected to NGS and the resulting sequences were then used to calculate the percentage of editing.

[0191] For T cells, RNPs were assembled by mixing 113 uM nuclease and 160 uM synthetic guide and incubating at room temperature for 10 minutes, and the RNP complexes were mixed with 200,000 primary activated T cells and electroporated with EH-115 pulse code using the P3 Primary Cell 4D-Nucleofector®X Kit. Cells were harvested after 3 and 8 days and CD3 and edited protein expression was measured by flow cytometry.

[0192] result Activity of short guides across genomic sites and cell types OMNI-103 nuclease activity was optimized for use with short sgRNA scaffolds. Five short sgRNA scaffolds were designed based on the "V2" double-stranded version containing up to four deletions around the tetraloop "GAAA" and terminator regions (Table 3, Figures 1A-1F). To test the activity level of OMNI-103 with the designed V2 scaffolds, sgRNAs with guide sequence portions of "TRAC-s91" or "PDCD-s40" were transfected into HeLa cells. Editing activity was calculated based on the NGS results (Figure 2). In all cases, the designed sgRNAs enabled editing. The next step was to test OMNI-103 activity as an RNP in U2OS and primary T cells. OMNI-103 was electroporated with sgRNAs with V2, V2.2, or V2.3 scaffolds and with guide sequence portions of "TRAC-s35" or "B2M-s12". Editing activity was calculated based on NGS results. It was shown that the level of OMNI-103 activity was not compromised when used with any of the scaffold variants (Figure 3). In primary T cells, activity was improved when the short scaffold variant was utilized.

[0193] Primary T cells were isolated from human PBMCs and activated with CD2, CD3 and CD28 according to the manufacturer's recommendations (#130-091-441, Miltenyi). After 3 days, most cells were activated (>85% CD25 positive cells, see Figure 5). 200,000 activated primary T cells were electroporated with OMNI-103 nuclease and guide RNA molecules with V2, V2.2 or V2.3 scaffolds and the spacer sequence (i.e., guide sequence portion) of TRAC-s35 or B2M-s12. Editing after 8 days of culture was measured by next generation sequencing (NGS) and the level of protein expression was measured by flow cytometry, showing a significant reduction in TCR or B2M with the V2 scaffold and an even greater reduction in protein expression with V2.2 or V2.3 (Figure 4).

[0194] [Table 1]

[0195] [Table 2]

[0196] [Table 3]

[0197] [Table 4]

[0198] [Table 5]

[0199] [Table 6]

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Claims

1. 1. A composition comprising a non-naturally occurring RNA molecule or a polynucleotide molecule encoding said RNA molecule, wherein said RNA molecule comprises a crRNA repeat sequence portion and a guide sequence portion, said RNA molecule forms a complex with an OMNI-103 nuclease in the presence of a tracrRNA sequence to target said OMNI-103 nuclease to a DNA target site, said tracrRNA sequence being encoded by the tracrRNA portion of said RNA molecule or the tracrRNA portion of a second RNA molecule, and optionally the crRNA repeat sequence portion is up to 17 nucleotides in length, preferably 14-17 nucleotides in length.

2. 2. The composition of claim 1, wherein the crRNA repeat sequence portion has at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence set forth in SEQ ID NO: 22 or 23, and / or the crRNA repeat sequence portion has at least 95% sequence identity to the sequence set forth in either SEQ ID NO: 22 or 23, and / or the crRNA repeat sequence is other than the sequence set forth in SEQ ID NO: 23, and / or the RNA molecule comprising the crRNA repeat sequence portion and the guide sequence portion further comprises the tracrRNA portion, optionally covalently linked to the tracrRNA portion via a polynucleotide linker portion.

3. 3. The composition of claim 1 or 2, wherein the composition comprises a second RNA molecule comprising the tracrRNA portion, and / or the OMNI-103 nuclease has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and / or the length of the guide sequence portion is 17 to 30 nucleotides, preferably 22 nucleotides.

4. 1. A composition comprising a non-naturally occurring RNA molecule, said RNA molecule comprising a tracrRNA portion, said RNA molecule forming a complex with and targeting an OMNI-103 nuclease to a DNA target site in the presence of a crRNA repeat sequence and a guide sequence portion, wherein the crRNA repeat sequence and the guide sequence portion are encoded by said RNA molecule or a second RNA molecule, and optionally, the tracrRNA portion is less than 85 nucleotides in length, preferably 84-80, 79-75, 74-70, 69-65, or 64-60 nucleotides in length.

5. The tracrRNA portion has at least 30-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the tracrRNA portion of the sequence set forth in any of SEQ ID NOs: 17-21, and / or the tracrRNA portion has at least 95% sequence identity to the tracrRNA portion of the sequence set forth in any of SEQ ID NOs: 17-21, and and / or the tracrRNA portion is other than the tracrRNA portion of the sequence set forth in SEQ ID NO: 4 or 5, and / or the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion of at most 19 nucleotides in length, preferably 16-19 nucleotides, and / or the tracrRNA portion is at least 60-70%, 71-80%, 81-90%, 90% or more of the sequence set forth in either SEQ ID NO: 24 or 25.

5. The composition of claim 4, wherein the RNA molecule comprises a tracrRNA anti-repeat sequence portion having 1-95% or 96-99% sequence identity, and / or the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having at least 95% sequence identity to the sequence set forth in either SEQ ID NO: 24 or 25, and / or the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having a sequence other than the sequence set forth in SEQ ID NO: 25, and / or the RNA molecule comprises a tracrRNA portion and further comprises a repeat sequence portion and a guide sequence portion, and / or the tracrRNA portion is covalently linked to the crRNA repeat sequence via a polynucleotide linker portion, optionally wherein the length of the polynucleotide linker portion is 4-10 nucleotides, and optionally wherein the sequence of the polynucleotide linker is GAAA.

6. The composition of claim 4 or 5, further comprising a second RNA molecule comprising a crRNA repeat sequence portion and a guide sequence portion, and / or the OMNI-103 nuclease has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and / or the length of the guide sequence portion is 17 to 30 nucleotides, preferably 22 nucleotides.

7. 1. A composition comprising a non-naturally occurring RNA molecule or a polynucleotide molecule encoding said RNA molecule, said RNA molecule comprising an RNA scaffold moiety, said RNA scaffold moiety having the structure: crRNA repeat sequence portion-tracrRNA portion wherein the RNA scaffold portion forms a complex with an OMNI-103 CRISPR nuclease and directs the OMNI-103 CRISPR nuclease to a DNA target site complementary to a guide sequence portion of the RNA molecule, and optionally the OMNI-103 nuclease has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

1.

8. The length of the RNA scaffold portion is 110-105, 104-100, 99-95, 94-90, 89-85, 84-80, 79-75 or 74-70 nucleotides, and / or the length of the RNA scaffold portion is 107, 101, 95, 85 or 79 nucleotides, and / or the RNA scaffold portion is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 100%, at least 15 ... and / or the length of the crRNA repeat sequence portion is at most 17 nucleotides, preferably 14-17 nucleotides; and / or the crRNA repeat sequence portion has at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence shown in SEQ ID NO: 22 or 23; and / or the crRNA repeat sequence portion has at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence shown in SEQ ID NO: 22 or 23. The tracrRNA portion has at least 95% sequence identity to the sequence set forth in any of SEQ ID NOs: 22 or 23, and / or the crRNA repeat sequence is other than the sequence set forth in SEQ ID NO: 23, and / or the length of the tracrRNA portion is less than 85 nucleotides, preferably 84-80, 79-75, 74-70, 69-65 or 64-60 nucleotides, and / or the tracrRNA portion has a tracrRNA sequence set forth in any of SEQ ID NOs: 17-21.

8. The composition of claim 7, wherein the tracrRNA portion has at least 30-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to a tracrRNA portion of a sequence set forth in any of SEQ ID NOs: 17-21, and / or the tracrRNA portion has at least 95% sequence identity to a tracrRNA portion of a sequence set forth in any of SEQ ID NOs: 17-21, and / or the tracrRNA portion is other than a tracrRNA portion of a sequence set forth in SEQ ID NO: 4 or 5.

9. The RNA scaffold portion has a structure in which the RNA scaffold is crRNA repeat sequence portion-linker portion-tracrRNA portion and / or the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion, wherein the crRNA repeat sequence and the tracrRNA anti-repeat sequence portion are covalently linked via the linker portion, optionally wherein the linker portion is a polynucleotide linker 4 to 10 nucleotides in length, and optionally wherein the sequence of the polynucleotide linker is GAAA.

10. The tracrRNA portion comprises a tracrRNA anti-repeat sequence portion up to 19 nucleotides in length, preferably 16-19 nucleotides, and / or the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having at least 60-70%, 71-80%, 81-90%, 91-95%, or 96-99% sequence identity to the sequence set forth in either SEQ ID NO: 24 or 25, and / or the tracrRNA portion comprises a tracrRNA anti-repeat sequence portion having at least 95% sequence identity to the sequence set forth in either SEQ ID NO: 24 or 25, and / or the tracrRNA anti-repeat sequence is other than the sequence set forth in SEQ ID NO: 25, and / or the tracrRNA 9. The composition of claim 7 or 8, wherein the RNA portion comprises a first section of nucleotides attached to the tracrRNA anti-repeat portion, wherein the first section of nucleotides has at least 95% sequence identity to a sequence set forth in any of SEQ ID NOs: 26-28; and / or the tracrRNA portion comprises a second section of nucleotides attached to the first section of nucleotides, wherein the second section of nucleotides has at least 95% sequence identity to a sequence set forth in any of SEQ ID NOs: 29-32; and / or the RNA scaffold portion has at least 95% identity to a nucleotide sequence set forth in any of SEQ ID NOs: 17-21; and / or the RNA scaffold portion has the predicted structure of any of V2, V2.1, V2.2, V2.3, V2.4 or V2.5 RNA scaffolds; and / or the RNA scaffold portion has a sequence other than the sequence set forth in SEQ ID NO: 4 or 5.

11. The guide sequence portion is covalently linked to the crRNA repeat sequence portion of the RNA molecule and has the structure: Guide sequence portion - crRNA repeat sequence portion - tracrRNA portion and / or the length of the guide sequence portion is 17 to 30 nucleotides, more preferably 20 to 23 nucleotides, more preferably 22 nucleotides.

12. 9. The composition of any one of claims 1, 2, 4, 5, 7 or 8, further comprising an OMNI-103 CRISPR nuclease, wherein the OMNI-103 CRISPR nuclease has at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 1; and / or the RNA molecule is formed by in vitro transcription (IVT) or solid-phase artificial oligonucleotide synthesis, optionally wherein the RNA molecule comprises modified nucleotides; and / or the RNA molecule comprises a sequence set forth in any one of SEQ ID NOs: 17-21, optionally wherein the RNA molecule comprises the sequence set forth in SEQ ID NO: 18, or wherein the RNA molecule comprises the sequence set forth in SEQ ID NO: 19; and / or wherein the RNA molecule consists of a guide sequence portion and a sequence set forth in any one of SEQ ID NOs: 17-21.

13. A polynucleotide molecule encoding an RNA molecule as defined in any one of claims 1, 2, 4, 5, 7 or 8.

14. 10. A method for modifying a nucleotide sequence at a DNA target site in the genome of a cell-free system or cell, comprising introducing into the system or cell a composition of any one of claims 1, 2, 4, 5, 7 or 8 and a CRISPR nuclease having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, wherein optionally the cell is a eukaryotic or prokaryotic cell, and optionally the eukaryotic cell is a human cell or a plant cell.

15. A kit for modifying a nucleotide sequence at a DNA target site in the genome of a cell-free system or cell, comprising: introducing into the system or cell a composition described in any one of claims 1, 2, 4, 5, 7 or 8; a CRISPR nuclease having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and instructions for delivering the RNA molecule and the CRISPR nuclease to the cell.

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