Genetically engineered high-fidelity OMNI-50 nuclease variants

Abstract

The present invention is directed, inter alia, to compositions and methods for genome editing, specifically non-naturally occurring OMNI-50 nuclease variants having a wild-type OMNI-50 protein sequence (SEQ ID NO:1) that contain amino acid substitutions at at least one of the following positions: R61, ¥437, R478, A493, ¥545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, V981, K965, and K1036.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 333,037, filed April 20, 2022, No. 63 / 332,214, filed April 18, 2022, and No. 63 / 232,571, filed August 12, 2021, the contents of each of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those referenced in parentheses. All publications mentioned in this application are incorporated by reference in their entirety into this specification to add context to the art to which this invention pertains and to which the invention may be utilized.

[0003] Reference to sequence listing This application incorporates by reference as part of this application the nucleotide sequence in the file "220812_91722-A-PCT_Sequence_Listing_AWG.xml", created on August 12, 2022 in IBM-PC format with operating system compatibility with MS-Windows®, contained in an XML file submitted on August 12, 2022, and having a size of 288 kilobytes. [Background technology]

[0004] Targeted genome modification is a powerful tool that can be used to reverse the effects of pathogenic genetic alterations and thus may provide novel therapies for human genetic diseases. Current genome engineering tools, including genetically modified zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently RNA-guided DNA endonucleases such as CRISPR / Cas, generate sequence-specific DNA breaks in the genome. The modification of the genome sequence occurs in the following steps and is the result of the activity of the cell's DNA repair mechanisms in response to the newly formed DNA breaks. These mechanisms may include, for example, (1) classical non-homologous end joining (NHEJ), in which the two ends of the break are ligated together in a rapid but imprecise manner (i.e., frequently resulting in DNA mutations at the break site in the form of small insertions or deletions), or (2) homology-directed repair (HDR), in which an intact homologous DNA donor is used to replace the DNA around the break site in a precise manner. Minimal off-target activity of the initial DNA damage inducer is required for efficient and safe genome editing. Summary of the Invention

[0005] Disclosed herein are genetically engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated OMNI-50 nucleases with altered and improved target specificity and their uses in genomic engineering, extragenomic engineering, genome targeting, genome editing and in vitro diagnostics.

[0006] In some embodiments, the OMNI-50 nuclease variants and improved variants are used to increase specificity compared to wild-type OMNI-50 nuclease.Advantageously, when the OMNI-50 nuclease of the genetically modified variant is active in the CRISPR endonuclease system, the CRISPR endonuclease system has reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR endonuclease system in which the wild-type OMNI-50 nuclease is active.For example, the OMNI-50 nuclease of the genetically modified variant may show improved allele-specific discrimination, for example, specific binding and activity in the target region containing heterozygous SNPs that are present only in the target allele and not in the non-target allele.

[0007] In some embodiments, OMNI-50 nickase variants are provided that have increased specificity compared to wild-type OMNI-50 nickase. In some embodiments, OMNI-50 catalytically inactive nuclease variants are provided that have increased specificity compared to wild-type OMNI-50 catalytically inactive nuclease. For example, the catalytic site of any one of the OMNI-50 nuclease variants of the present application may be modified so that the variant has nickase activity, thereby enabling it to perform single-stranded DNA cleavage. Alternatively, the catalytic site of any one of the OMNI-50 nuclease variants of the present application may be modified so that the variant does not have nuclease activity, i.e., is a catalytically inactive nuclease.

[0008] Some embodiments of the invention provide variants of OMNI-50 nuclease proteins that comprise a sequence that is at least 80% identical to the amino acid sequence of the wild-type OMNI-50 nuclease protein (SEQ ID NO:1).

[0009] In some aspects of the invention, non-naturally occurring OMNI-50 nuclease variants are provided having a wild-type OMNI-50 protein sequence (SEQ ID NO:1) that includes amino acid substitutions at at least one of the following positions: R61, Y437, R478, A493, Y545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, K965, V981, and K1036.

[0010] Some aspects of the invention provide a CRISPR system comprising any one of the OMNI-50 nuclease variants disclosed herein complexed with a guide RNA molecule targeted to a DNA target site, the CRISPR system having reduced off-target editing activity compared to a wild-type CRISPR system comprising a wild-type OMNI-50 nuclease protein and a guide RNA molecule.

[0011] In some embodiments, the OMNI-50 nuclease variant has increased specificity for a target site when complexed with a guide RNA that directs the OMNI-50 variant to the target site compared to wild-type OMNI-50 nuclease (SEQ ID NO:1).

[0012] In some embodiments, the OMNI-50 nuclease variant is a nickase having an inactivated RuvC domain made by amino acid substitutions at the positions provided for the CRISPR nuclease in column 1 of the table below. In some embodiments, the nickase further comprises an amino acid substitution at at least one of the following positions: R61, Y437, R478, A493, Y545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, K965, V981, and K1036.

[0013] In some embodiments, the OMNI-50 nuclease variant is a nickase with an inactivated HNH domain made by amino acid substitutions at the positions provided for the CRISPR nuclease in column 2 of the table below. In some embodiments, the nickase further comprises an amino acid substitution at at least one of the following positions: R61, Y437, R478, A493, Y545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, K965, V981, and K1036.

[0014] In some embodiments, the OMNI-50 nuclease variant is a catalytically inactive nuclease having an inactivated RuvC domain and an inactivated HNH domain made by substitutions at the positions provided for the CRISPR nuclease in column 3 of the table below. In some embodiments, the catalytically inactive nuclease comprises an amino acid substitution at at least one of the following positions: R61, Y437, R478, A493, Y545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, K965, V981, and K1036.

[0015] [Table 1]

[0016] This table lists alternative positions that may be substituted to generate a nickase with an inactivated RUVC domain, alternative positions that may be substituted to generate a nickase with an inactivated HNH domain, and alternative positions that may be substituted to generate a catalytically inactive nuclease with inactivated RUVC and HNH domains. Any other amino acid substitution is permissible for each of the amino acid positions shown in columns 1-3, except where followed by an asterisk indicating that any substitution other than aspartic acid (D) to glutamic acid (E) or glutamic acid (E) to aspartic acid (D) results in inactivation.

[0017] However, throughout the text, OMNI-50 nuclease variants means that any of these variants may be modified to have either nickase activity (i.e., a nuclease that generates single-stranded DNA breaks as opposed to double-stranded breaks) or no nuclease activity (i.e., a catalytically inactive nuclease).

[0018] Thus, point mutations can be introduced into the variants described herein to modify or abolish their nuclease activity while still retaining their ability to specifically bind DNA in a manner programmed by the sgRNA. Any one of these variants can specifically target a desired DNA target sequence via a guide RNA molecule. The variant-guide complex also delivers the molecule bound to the complex to the target site. Thus, this disclosure also contemplates the use of fusion proteins that include such variants and DNA-modifying domains (e.g., deaminase, nuclease, nickase, recombinase, methyltransferase, methylase, acetylase, acetyltransferase, transcriptional activator, or transcriptional repressor domains), as well as fusion proteins that correct mutations in a genome (e.g., the genome of a human subject) associated with a disease or create mutations in a genome (e.g., the human genome) to reduce or prevent expression of a gene.

[0019] In some embodiments, the variants of the present application may be fused to a protein having an enzymatic activity. In some embodiments, the enzymatic activity modifies 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 creates a double-stranded break in the target DNA. In some embodiments, the enzymatic activity modifies a target polypeptide associated with the target DNA. In some cases, 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. In some cases, 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.

[0020] Thus, any one of the OMNI-50 nucleases, nickases, or catalytically inactive nuclease variants may be fused (e.g., directly or via a linker) to another DNA regulatory or modifying enzyme, including, but not limited to, a deaminase, a reverse transcriptase (e.g., see Anzaolone et al. (2019) for use in prime editing), an enzyme that alters the methylation state of DNA (e.g., a methyltransferase), or a base editor such as a modifier of histones (e.g., a histone acetyltransferase). Indeed, the OMNI-50 nucleases, nickases, and inactive variants described herein may be fused to a DNA regulatory 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, any of the OMNI-50 variants of the present application are fused to a protein with enzymatic activity. In some embodiments, the enzymatic activity modifies a target DNA molecule. The OMNI-50 variants 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 expression of a gene.

[0021] In some embodiments of the invention, there is provided a method of gene editing with reduced off-target editing activity comprising contacting a DNA target site with an active CRISPR system comprising any one of the variants of OMNI-50 nuclease protein described herein.

[0022] In some embodiments, contacting the target site locus with an active CRISPR system comprising a variant of an OMNI-50 nuclease protein, any one of the variants described herein, wherein the active CRISPR system has reduced off-target editing activity and maintained on-target editing activity compared to a wild-type CRISPR system having a wild-type OMNI-50 nuclease protein. The present invention provides a gene editing method comprising:

[0023] Further aspects and the full scope of applicability of the present invention will become apparent from the detailed description given herein below. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0024] [Figure 1] Figure 1: A schematic diagram illustrating the nuclease optimization platform is shown. Libraries of nuclease variants are generated using a combination of rational design and random mutagenesis, followed by selection for activity, specificity, multiplicity (and any other desired traits) using specialized selection assays. [Diagram 2] Figure 2: Schematic diagram of the ELANE gene and target sites used to test the activity and fidelity of wild-type OMNI-50 nuclease and its variants. "alt" and "ref" indicate guide RNA molecules that direct the nuclease to the SNP position in alleles that either carry the "alt" version of the SNP or the "ref" version of the SNP, respectively. [Figure 3A]Figures 3A-3B: Allele specificity and off-target effects of wild-type OMNI-50 nuclease demonstrating that OMNI-50 nuclease could be improved by optimization. Figure 3A: HSCs edited with wild-type OMNI-50alt composition removed mutant alleles while maintaining prototype alleles intact. However, editing with wild-type OMNI-50ref composition resulted in reduction in prototype alleles and only partial preservation of mutant alleles, providing differential but not complete allele-specific editing. Statistical significance is indicated as **P<.01, ****P<.0001. [Figure 3B] FIG. 3B: rhAmpseq analysis in healthy (HD) and patient (ESCN-2)-edited HSCs (RNP(ref) or RNP(alt)) for off-targets identified for constant guide (Sg(constant)), prototype guide (Sg(ref)) and mutant guide (Sg(alt)). Results show that wild-type OMNI-50 has one off-target for each guide that should be removed to ensure target-specific editing. Editing rates in RNPref-treated HSCs are shown in the upper panel; editing rates in RNPalt-treated HSCs are shown in the lower panel. [Figure 4A]Figures 4A-4C: Optimized variants show improved allele specificity and comparable activity compared to wild-type OMNI-50 nuclease. Figure 4A: Allele specificity was determined in healthy HSCs edited with wild-type OMNI-50 or optimized variants. The variants show superior allele discrimination compared to wild-type OMNI-50. Specifically, graphs representative of allele-specific editing with sgRNA-564DS-ref or sgRNA-564DS-alt as measured by ddPCR are shown. Editing specificity is determined by two competitive probes, a FAM probe that binds to the mutant allele and a HEX probe that binds to the prototype allele, defining a reduced signal from the edited allele. The ratio between the concentrations of the prototype and mutant alleles in heterozygous untreated cells is 1. The graphs represent the average concentrations of the prototype allele (HEX), mutant allele (FAM) normalized to the endogenous gene controls RPP30 and STAT1 for each gDNA sample. [Figure 4B] Figure 4B: Excision rates were measured in healthy HSCs edited with wild-type OMNI-50 (WT OMNI-50) or the optimized variant. The 3795 variant shows comparable activity compared to WT OMNI-50. The 3795 variant showed the best allele specificity without compromising activity. [Figure 4C] Figure 4C: Variant fidelity was measured by next-generation sequencing (NGS) analysis specific for known OMNI-50 nuclease off-targets. Note: All variants showed high fidelity. [Figure 5A] Figures 5A-5B: rhAmpSeq analysis of healthy HSCs edited with either WT OMNI-50 or 3795 nuclease variants for off-targets identified for constant guide (Sg(constant)), prototype guide (Sg(ref)) (RNP(ref), Figure 5A), and mutant guide (Sg(alt)) molecules (RNP(alt)), Figure 5B). [Figure 5B]Figures 5A-5B: rhAmpSeq analysis of healthy HSCs edited with either WT OMNI-50 or 3795 nuclease variants for off-targets identified for constant guide (Sg(constant)), prototype guide (Sg(ref)) (RNP(ref), Figure 5A), and mutant guide (Sg(alt)) molecules (RNP(alt)), Figure 5B). [Figure 5C] Figure 5C shows additional results validating the fidelity of the 3795 nuclease variants are demonstrated by examining off-targets when using three different guide molecules (g35, g62Ref, or g62Alt) on HSC samples with either WT OMNI-50 or the 3795 nuclease variants (Figure 5C). These results show that there were strong validated off-target effects for each guide when using WT OMNI-50 nuclease, but no off-targets were validated with the 3795 nuclease variants, indicating that the 3795 nuclease variants remove off-targets compared to WT OMNI-50 nuclease. [Figure 6A] Figures 6A-6C: Single allele ablation in ELANE restores neutrophil differentiation. Figure 6A: Schematic of gene editing therapy to restore neutrophil differentiation. [Figure 6B] FIG. 6B: Flow cytometry analysis showing healthy HSCs (HD-2) and patient-derived HSCs (ESCN-2) edited ex-vivo only for the targeted mutant allele and differentiated into mature neutrophils. [Figure 6C] FIG. 6C: Quantification of the data shown in FIG. 6B. [Figure 7A]Figure 7A: Linear representation of the five exons and four introns of ELANE showing the location of representative heterozygous mutations associated with SCN depicted as black inverted triangles. Based on Makaryan et al. (Figure 7B, I-III). Schematic diagram of the three identified SNPs (white inverted triangles) and common cut sites (grey inverted triangles) associated with the majority of ELANE mutations, based on which three allele-specific sgRNA guides and constant guides were designed, representing three single-allele excision strategies. [Figure 7B] Figure 7A: Linear representation of the five exons and four introns of ELANE showing the location of representative heterozygous mutations associated with SCN depicted as black inverted triangles. Based on Makaryan et al. (Figure 7B, I-III). Schematic diagram of the three identified SNPs (white inverted triangles) and common cut sites (grey inverted triangles) associated with the majority of ELANE mutations, based on which three allele-specific sgRNA guides and constant guides were designed, representing three single-allele excision strategies. [Figure 8A] Figures 8A-8G: Allele specificity and excision efficiency of OMNI variant 3795 nuclease compositions. Figure 8A: Schematic depicting the experimental workflow. HSCs derived from healthy individuals and SCN patients were electroporated with RNP or left untreated, followed by recovery for 3 days in CD34+ growth medium. Cells were then subjected to differentiation by culturing for 7 days with IL-3, SCF, GMCSF and GCSF for proliferation and myeloid progenitor differentiation, followed by culturing for 7 days in GCSF for neutrophil differentiation. [Figure 8B]Figure 8B: Bar graphs showing the percentage of unedited prototype alleles (black) and mutant alleles (grey) in HSCs from healthy subjects (HD-V3) or SCN patients (SCN-P41) treated or not (NT) with RNP(ref) composition at day 6 of differentiation as measured by ddPCR. The mean of each allele concentration was normalized to the endogenous gene controls RPP30 and STAT1 and presented relative to untreated cells. (n=3 groups of cells from HD-V3 healthy / SCN-P41 patient donors). Statistical significance is shown as ****P<.0001, ns=not statistically significant. [Figure 8C] Figure 8C: Bar graphs representing the percentage of ablation at days 6 and 14 of differentiation in HSCs obtained from either healthy subjects (HD-V3): untreated (NT, black) or RNP(ref)-treated (gray) or SCN patients (SCN-P41): untreated (NT, white) or RNP(ref)-treated (dark diagonal lines) as measured by ddPCR. (n=3 groups of cells from HD-V3 healthy / SCN-P41 patient donors). Statistical significance is indicated as **P<.01, ****P<.0001. [Figure 8D] Figure 8D: Bar graph showing the percentage of wild type (black) and mutant (gray) alleles in cDNA derived from HSCs of SCN-P41 patients treated with RNP(ref) composition or not (NT) as measured by next generation sequencing (NGS) targeting the mutation site (n=3 groups of cells from SCN-P41 patients). Statistical significance is indicated as ***P<.001. [Figure 8E] FIG8E: Bar graphs showing the percentage of unedited prototype alleles (black) and mutant alleles (grey) in HSCs from SCN patients (SCN-P55) treated with RNP(alt) compositions or not (NT) at day 6 of differentiation as measured by ddPCR. The mean of each allele concentration was normalized to endogenous gene controls RPP30 and STAT1 and presented relative to untreated cells. (n=3 groups of cells from SCN-P55 patient donors). Statistical significance is shown as ****P<.0001, ns=not statistically significant. [Figure 8F]Figure 8F: Bar graphs representing the percentage of ablation at days 6 and 14 of differentiation in HSCs obtained from SCN patients (SCN-P55): untreated (NT, white) or RNP(alt)-treated (dark diagonal lines) as measured by ddPCR. (n=3 groups of cells from SCN-P55 patient donors). Statistical significance is indicated as ***P<.001, ****P<.0001. [Figure 8G] Figure 8G: Bar graph showing the percentage of wild type alleles (black) and mutant alleles (gray) in cDNA from HSCs of SCN-P55 patients treated with RNP(alt) composition or not (NT) as measured by NGS targeting the mutation site. (n=3 groups of cells from SCN-P55 patients). Statistical significance is indicated as ***P<.001. [Figure 8H] Figure 8H: Bar graphs representing ELANE mRNA levels in day 6 differentiated HSCs of SCN-P41 and SCN-P55 patients either untreated (NT, black) or RNP(ref) / RNP(alt) treated (truncated, grey), respectively. Data are presented relative to the NT group. (n=3 groups of cells from SCN-P41 and SCN-P55 patients). Statistical significance is indicated as *P<.05. Bars represent mean values ​​with standard deviation. [Figure 9A] Figures 9A-9K: OMNI variant 3795-promoted editing enhances neutrophil differentiation and maturation in vitro. Figure 9A: Representative FACS plots of untreated (NT, left panel) and RNP(ref)-treated (right panel) healthy (HD-V3, upper panel) and SCN-P41 patient (lower panel) differentiated HSCs analyzed for neutrophil (CD66b+) and monocyte (CD14+ / CD66b-) subsets. [Figure 9B]Figure 9B: Quantitative analysis of individual FACS data for the percentage of neutrophils (CD66b+ cells) in healthy (HD-V3) and SCN patient (SCN-P41) differentiated HSCs untreated (NT, black) or treated with RNP (ref) (gray). (n=3 groups of cells from HD-V3 healthy / SCN-P41 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. [Figure 9C] Figure 9C: Quantitative analysis of individual FACS data for the percentage of monocytes (CD14+ / CD66b- cells) in healthy (HD-V3) and SCN patient (SCN-P41) differentiated HSCs untreated (NT, black) or treated with RNP (ref) (gray). (n=3 groups of cells from HD-V3 healthy / SCN-P41 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. [Figure 9D] (D) Quantification of the percentage of Zymosan Green uptake by healthy (HD-V3) and SCN patient (SCN-P41) differentiated HSCs untreated (NT, black) or treated with RNP (ref) (gray). ns = not statistically significant. [Figure 9E] Figure 9E: The graph depicts the real-time change in luminescence, relative light units (RLU), from 200,000 luciferase-expressing bacterial cells incubated with healthy untreated (HD-V3, NT; squares) or patient RNP(ref)-treated (SCN-P41, RNP(ref); triangles) HSC-derived differentiated neutrophils compared to control-only bacterial cells (E. coli, circles). Statistical significance for each one of the groups versus E. coli control at the final presented time point where RLU levels reached a plateau is indicated as *P<.05, **P<.01. [Figure 9F] FIG. 9F: Representative FACS plots of untreated healthy (HD-V4 NT, left panel), untreated SCN patient (SCN-P55 NT, center panel) and RNP(alt)-treated SCN patient (right panel) differentiated HSCs analyzed for neutrophil (CD66b+) and monocyte (CD14+ / CD66b-) subsets. [Figure 9G] FIG9G: Quantitative analysis of individual FACS data for the percentage of neutrophils (CD66b+ cells) in differentiated HSCs from untreated healthy (HD-V4, NT; black) and SCN patients (SCN-P55) either untreated (NT, black) or treated with RNP (alt) (gray). (n=cells from 3 groups of HD-V4 healthy / SCN-P55 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. [Figure 9H] Figure 9H: Quantitative analysis of individual FACS data for the percentage of monocytes (CD14+ / CD66b- cells) in differentiated HSCs from untreated healthy (HD-V4, NT; black) and SCN patients (SCN-P55) either untreated (NT, black) or treated with RNP (alt) (gray). (n=cells from 3 groups of HD-V4 healthy / SCN-P55 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. [Figure 9I] Figure 9I: Diff-Quik staining of differentiated HSCs derived from a P55 SCN patient treated with RNP(alt) or electroporated without nuclease composition (SCN-P55 Mock). Photomicrographs were obtained on a LEITZ LABORLUX S polarizing microscope at 400x magnification using a Nikon DSLR digital camera. [Figure 9J] FIG 9J: Quantification of the percentage of Zymosan Green uptake by differentiated HSCs from untreated healthy (HD-V4, NT; black) and SCN patients (SCN-P55) either untreated (NT, black) or treated with RNP (alt) (gray). (n=3 groups of cells from HD-V4 healthy / SCN-P55 patient donors). Statistical significance is indicated as *P<.05. [Figure 9K]FIG. 9K: The graph depicts the real-time change in luminescence, relative light units (RLU), from 200,000 luciferase-expressing bacterial cells incubated with differentiated neutrophils derived from healthy untreated (HD-V4, NT; squares) HSCs and patient RNP(alt)-treated (SCN-P55, RNP(alt); triangles) HSCs compared to control-only bacterial cells (E. coli, circles). Statistical significance for each one of the groups versus E. coli control at the final time point presented, when RLU levels reached a plateau, is indicated as *P<.05, ***P<.001. Bars represent mean values ​​with standard deviations. [Figure 10A] Figures 10A-10B: Heterozygosity frequency and coverage in patient and healthy populations by three SNPs. Figure 10A: Heterozygosity frequency for each of the three selected SNPs in the healthy (left) or patient (right) populations. Heterozygosity frequencies were similar between healthy and patient populations for each of the three SNPs. rs10414837, p-value=0.126, odds ratio=0.653; rs3761005, p-value=0.9615, odds ratio=1.014; rs1683564, p-value=0.9475, odds ratio=1.019, all analyzed by χ2. [Figure 10B] Figure 10B: Pie charts representing the proportion of the population heterozygous for at least one of the three selected SNPs (grey) in the healthy (left) or patient (right) populations. Similar coverage of the healthy and patient populations by the three SNPs (>75%, p-value=0.1285, odds ratio=0.56, χ2). [Figure 11] Figure 11: Mutation-SNP linkage determination in SCN-P41 and SCN-P55 patients. Electropherogram of the mutation site and sequencing analysis of the rs1683564 SNP. SCN-P41 patients carry the mutation on the same allele as the prototype form of the SNP (C, cytosine), whereas SCN-P55 patients carry the mutation on the same allele as the variant form of the SNP (A, adenosine). [Figure 12]Figure 12: Same editing results using RNP(ref) and RNP(alt) compositions. The ELANE gene is cut at two locations: 1) intron 4, a biallelic site guided by sgRNA(constant) guide, and 2) a heterozygous SNP site, rs1683564, a monoallelic site guided by either sgRNA(ref) or sgRNA(alt) depending on the linkage to the mutation site. If the mutation is located in an allele carrying the basic form of the SNP (C, cytosine), an RNP(ref) composition comprising nuclease, sgRNA(ref) and sgRNA(constant) is selected and the section containing the mutation is cut from the basic allele (Patient A, upper panel). If the mutation is located in an allele carrying the mutant form of the SNP (A, adenosine), an RNP(alt) composition comprising nuclease, sgRNA(alt) and sgRNA(constant) is selected and the section containing the mutation is cut from the mutant allele (Patient B, lower panel). Figure created by BioRender.com. [Figure 13A] Figures 13A-13C: Inversion events after excision. Figure 13A: Schematic of detection of inversion events: Specific primers were designed to amplify the inversion variations of the excision fragment. EvaGreen dye, a fluorescent DNA-binding dye that binds to dsDNA, was used in a ddPCR assay to measure all inversion events. Figure created by BioRender.com. [Figure 13B] (B) Quantification of total reversal events measured by EvaGreen-based ddPCR assay in differentiated HSCs derived from unedited (black) and RNP(ref)-treated (grey), HD-V3 healthy subjects and SCN-P41 patients. Statistical significance is indicated as ****P<.0001. [Figure 13C]Figure 13C: Quantification of total reversal events measured by EvaGreen-based ddPCR assay in unedited HD-V4 healthy and SCN-P55 patient-derived differentiated HSCs (black) and RNP(alt)-treated (grey) SCN-P55 patient-derived differentiated HSCs. Statistical significance is indicated as ****P<.0001. Bars represent mean values ​​with standard deviation. (n=3-4 groups of cells from HD-V3 or HD-V4 healthy / SCN-P41 or SCN-P55 patient donors). [Figure 14A] Figures 14A-14B: Excision levels and allele specificity in additional healthy individuals. Figure 14A: Bar graphs depicting the percentage of excisions in HSCs obtained from healthy individuals that were either untreated (NT, black), RNP(ref)-treated (gray), or RNP(alt)-treated (white) as measured by ddPCR. (n=4 cells from 2 healthy individuals in each group). Statistical significance is indicated as ****P<.0001. [Figure 14B] Figure 14B: Bar graphs representing the percentage of unedited prototype alleles (black) and mutant alleles (grey) in HSCs from healthy individuals treated with untreated (NT), RNP(ref) or RNP(alt) as measured by ddPCR. The mean of each allele concentration was normalized to the endogenous gene controls RPP30 and STAT1 and presented relative to untreated cells. (n=4 cells from 2 healthy individuals in each group). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. Bars represent mean values ​​with standard deviation. [Figure 15A] Figures 15A-15C: Excision levels in long-term HSC populations. Figure 15A: Representative FACS plots of CD34+ HSCs from healthy subjects before sorting (total, left panel) and after sorting into CD90- (middle panel) and CD90+ (right panel) populations. [Figure 15B]Figures 15B and 15C: Bar graphs representing the percentage of excision from HSCs obtained from two healthy individuals (MLP1; Fig. 15B - heterozygous for SNP variants and MLP2, Fig. 15C - homozygous for SNP variants) before sorting (total, black) and after sorting into CD90+ (light grey) and CD90- (dark grey) populations, as measured by ddPCR. Naïve HSCs before sorting served as control (NT) (n=2 cells from each healthy individual in each group). Bars represent mean values ​​with standard deviations. [Figure 15C] Figures 15B and 15C: Bar graphs representing the percentage of excision from HSCs obtained from two healthy individuals (MLP1; Fig. 15B - heterozygous for SNP variants and MLP2, Fig. 15C - homozygous for SNP variants) before sorting (total, black) and after sorting into CD90+ (light grey) and CD90- (dark grey) populations, as measured by ddPCR. Naïve HSCs before sorting served as control (NT) (n=2 cells from each healthy individual in each group). Bars represent mean values ​​with standard deviations. [Figure 16A] Figures 16A-16D: Differentiation into CD11b+ / CD15+ neutrophils. Figure 16A: Representative FACS plots of untreated (NT, left panel) and RNP(ref)-treated (right panel) healthy (HD-V3, upper panel) and SCN-P41 patient (lower panel) differentiated HSCs analyzed for the neutrophil (CD11b+ / CD15+) subset. [Figure 16B] Figure 16B: Quantitative analysis of individual FACS data for the percentage of neutrophils (CD11b+ / CD15+ cells) in healthy (HD-V3) and SCN patient (SCN-P41) differentiated HSCs untreated (NT, black) or treated with RNP (ref) (gray). (n=3 groups of cells from HD-V3 healthy / SCN-P41 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. [Figure 16C]FIG. 16C: Representative FACS plots of untreated healthy (HD-V4 NT, left panel), untreated SCN patient (SCN-P55 NT, center panel) and RNP(alt)-treated SCN patient (right panel) differentiated HSCs analyzed for neutrophil (CD11b+ / CD15+) subsets. [Figure 16D] FIG 16D: Quantitative analysis of individual FACS data for the percentage of neutrophils (CD11b+ / CD15+ cells) in differentiated HSCs from untreated healthy (HD-V4 NT; black) and SCN patients (SCN-P55) that were either untreated (NT, black) or treated with RNP (alt) (gray). (n=3 groups of cells from HD-V4 healthy / SCN-P55 patient donors). Statistical significance is indicated as ****P<.0001. Bars represent mean values ​​with standard deviation. [Figure 17A] 17A-17B: OMNI-50 variant editing activity of the ref and alt alleles of g62 in LCL cells. Editing activity was determined by NGS analysis. The average and standard deviation of three replicates are shown. [Figure 17B] 17A-17B: OMNI-50 variant editing activity of the ref and alt alleles of g62 in LCL cells. Editing activity was determined by NGS analysis. The average and standard deviation of three replicates are shown. [Figure 18A] Figures 18A-18B: OMNI-50 variant editing activity of g62 off-targets. Two different off-targets were tested: g62 OT1 and g62 OT2. The mean and standard deviation of three replicates are shown. [Figure 18B] Figures 18A-18B: OMNI-50 variant editing activity of g62 off-targets. Two different off-targets were tested: g62 OT1 and g62 OT2. The mean and standard deviation of three replicates are shown. [Figure 19A]Figures 19A-19B: Specificity of probes and guides. Figure 19A: Binding of each probe (FAM, black; HEX, grey) to DNA extracted from healthy donor (HD) cells homozygous for either the prototype or variant form of the SNP was measured by ddPCR. Bar graphs showing the concentration of positive events. (n=3 groups of cells). Statistical significance is indicated as ****P<.0001. [Figure 19B] Figure 19B: Bar graph showing the percentage of excision in healthy HSCs homozygous for the prototype of rs1683564 SNP, untreated (NT) or treated with RNP(alt) or RNP(ref), as measured by ddPCR (n=3 groups of cells). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. Bars represent mean values ​​with standard deviation. [Figure 20]Figures 20A-20B: No off-targets detected after editing with OMNI variant 3795 nuclease and sgRNA, respectively. (Figures 20A-C) Unbiased survey of genome-wide off-target cleavage (GUIDE-seq) with OMNI variant 3795 nuclease and each of the constant (Figure 20A, SgRNA(constant)), archetype (Figure 20B, SgRNA(ref)) and mutant (Figure 20C, SgRNA(alt)) guides showing that all reads are to the target sequence and no off-targets are detected (4 mismatches). Note: Analysis was performed in U2OS cells homozygous for the archetype of rs1683564 SNP. As OMNI variant 3795 nuclease is highly discriminatory of alleles, there is very little on-target editing of the archetype allele when using sgRNA(alt) (13 reads of the reference cytosine genotype) and no detectable off-targets. (FIG. 20D) A table summarizing the results of in silico off-target analysis for constant, mutant, and prototype guides depicting several possible off-targets. None of these off-targets were validated by rhAmpSeq analysis performed on HSCs from SCN-P41 and SCN-P55 patients edited with RNP(ref) and RNP(alt), respectively, see the two right columns. The rhAmpSeq validation threshold was set at 0.2% or higher edits. [Figure 21]Figures 21A-21J. Excision with RNP(ref) in SCN-P42 and HD-V5. (Figure 21A) Bar graphs representing the percentage of unedited prototype alleles (black) and mutant alleles (grey) at day 6 of differentiation in HSCs from healthy (HD-V5) or SCN patients (SCN-P42) treated with RNP(ref) composition or electroporated without nuclease composition (mock), as measured by ddPCR. (n=3 groups of cells from HD-V5 healthy / SCN-P42 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. (FIG. 21B) Bar graphs representing the percentage of ablation at day 6 and day 14 of differentiation of HSCs from either healthy subjects (HD-V5): mock-treated (mock, black) or RNP(ref)-treated (gray), or SCN patients (SCN-P42): mock-treated (mock, white) or RNP(ref)-treated (dark diagonal lines), as measured by ddPCR. (n=3 groups of cells from HD-V5 healthy / SCN-P42 patient donors). Statistical significance is indicated as ****P<.0001. (FIG. 21C) Bar graphs representing ELANE mRNA levels at day 6 in differentiated HSCs from SCN-P42 patients that were either mock-treated (black) or RNP(ref)-treated (gray). Data are presented relative to the mock group. (n=3 groups of cells from SCN-P42 patients). Statistical significance is indicated as **P<.01. (FIG. 21D) Bar graphs representing the percentage of wild-type (black) and mutant (gray) alleles in cDNA from SCN-P42 patient HSCs that were either RNP(ref)-treated or mock-treated (Mock) as measured by NGS targeting the mutation site. (n=3 groups of cells from SCN-P42 patients). Statistical significance is indicated as *P<.05. (FIG. 21E) Representative FACS plots of mock-treated (Mock, left panel) and RNP(ref)-treated (right panel) healthy (HD-V5, upper panel) and SCN-P42 patient (lower panel) differentiated HSCs analyzed for neutrophil (CD66b+) and monocyte (CD14+ / CD66b-) subsets.(FIG. 21F) Quantitative analysis of individual FACS data for the percentage of neutrophils (CD66b+ cells) in healthy (HD-V5) and SCN patient (SCN-P42) differentiated HSCs mock-treated (mock, black) or treated with RNP (ref) (gray). (n=3 groups of cells from HD-V5 healthy / SCN-P42 patient donors). Statistical significance is shown as ****P<.0001, ns=not statistically significant. (FIG. 21G) Quantitative analysis of individual FACS data for the percentage of monocytes (CD14+ / CD66b- cells) in healthy (HD-V5) and SCN patient (SCN-P42) differentiated HSCs mock-treated (mock, black) or treated with RNP (ref) (gray). (n=3 groups of cells from HD-V5 healthy / SCN-P42 patient donors). Statistical significance is indicated as ****P<.0001, ns=statistically not significant. (FIG. 21H) Diff-Quik staining of differentiated HSCs derived from P42 SCN patient treated with RNP(ref) or electroporated without nuclease composition (SCN-P42Mock). Photomicrographs were obtained on a LEITZ LABORLUX S polarizing microscope at 400x magnification with a Nikon DSLR digital camera. (FIG. 21I) Quantification of the percentage of Zymosan Green uptake by healthy (HD-V5) and SCN patient (SCN-P42) differentiated HSCs mock-treated (mock, black) or treated with RNP(ref) (gray). Statistical significance is indicated as ****P<.0001, ns=statistically not significant. (FIG. 21J) The graph depicts the real-time change in luminescence, relative light units (RLU), from 200,000 luciferase-expressing bacterial cells incubated with healthy mock-treated (HD-V5, mock; open squares), patient mock-treated (SCN-42 mock; cross circle) or patient RNP(ref)-treated (SCN-P42 RNP(ref); triangles) HSC-derived differentiated neutrophils compared to control-only bacterial cells (E. coli, circles). Statistical significance for each one of the groups versus E. coli control at the final presented time point where RLU levels reached a plateau is shown as ****P<.0001. Bars represent mean values ​​with standard deviations. [Figure 22]Figures 22A-22I: Excision with RNP(alt) in SCN-P12 and HD-V1. (Figure 22A) Bar graphs representing the percentage of unedited prototype alleles (black) and mutant alleles (grey) at day 6 of differentiation in HSCs from healthy (HD-V1) or SCN patients (SCN-P12) treated with RNP(alt) composition or electroporated without nuclease composition (mock), as measured by ddPCR. (n=3 groups of cells from HD-V1 healthy / SCN-P12 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. (FIG. 22B) Bar graphs representing the percentage of ablation at day 6 and day 14 of differentiation of HSCs from either healthy subjects (HD-V1): mock-treated (mock, black) or RNP(alt)-treated (gray), or SCN patients (SCN-P12): mock-treated (mock, white) or RNP(alt)-treated (dark diagonal lines), as measured by ddPCR. (n=3 groups of cells from HD-V1 healthy / SCN-P12 patient donors). Statistical significance is indicated as ***P<.001, ****P<.0001. (FIG. 22C) Bar graphs representing ELANE mRNA levels at day 6 in differentiated HSCs from SCN-P12 patients that were either mock-treated (black) or RNP(alt)-treated (gray). Data are presented relative to the mock group. (n=3 groups of cells from SCN-P12 patients). Statistical significance is indicated as *P<.05. (FIG. 22D) Bar graphs depicting the percentage of wild-type (black) and mutant (gray) alleles in cDNA from SCN-P12 patient HSCs that were either RNP(alt)-treated or mock-treated (Mock) as measured by NGS targeting the mutation site. (n=3 groups of cells from SCN-P12 patients). Statistical significance is indicated as ***P<.001. (FIG. 22E) Representative FACS plots of mock-treated (Mock, left panel) and RNP(alt)-treated (right panel) healthy (HD-V1, upper panel) and SCN-P12 patient (lower panel) differentiated HSCs analyzed for neutrophil (CD66b+) and monocyte (CD14+ / CD66b-) subsets.(FIG. 22F) Quantitative analysis of individual FACS data for the percentage of neutrophils (CD66b+ cells) in healthy (HD-V1) and SCN patient (SCN-P12) differentiated HSCs mock-treated (mock, black) or treated with RNP (alt) (gray). (Cells from HD-V1 healthy / SCN-P12 patient donors, n=3 groups). Statistical significance is indicated as **P<.01, ***P<.001. (FIG. 22G) Quantitative analysis of individual FACS data for the percentage of monocytes (CD14+ / CD66b- cells) in healthy (HD-V1) and SCN patient (SCN-P12) differentiated HSCs mock-treated (mock, black) or treated with RNP (alt) (gray). (Cells from HD-V1 healthy / SCN-P12 patient donors, n=3 groups). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. (FIG. 22H) Quantification of the percentage of Zymosan Green uptake by healthy (HD-V1) and SCN patient (SCN-P12) differentiated HSCs mock-treated (mock, black) or treated with RNP (alt) (gray). Statistical significance is indicated as ***P<.001, ns=not statistically significant. (FIG. 22I) The graph depicts the real-time change in luminescence, relative light units (RLU), from 200,000 luciferase-expressing bacterial cells incubated with healthy mock-treated (HD-V1, mock; open squares), patient mock-treated (SCN-12 mock; cross circles) or patient RNP(alt)-treated (SCN-P12 RNP(alt); triangles) HSC-derived differentiated neutrophils compared to control-only bacterial cells (E. coli, circles). Statistical significance for each one of the groups versus E. coli control at the final time point presented, when RLU levels reached a plateau, is indicated as *P<.05, **P<.01, ***P<.001. Bars represent mean values ​​with standard deviations. [Diagram 23]Figures 23A-23I: Excision with RNP(alt) in SCN-P56 and HD-V3. (Figure 23A) Bar graphs depicting the percentage of unedited prototype alleles (black) and mutant alleles (grey) at day 6 of differentiation in HSCs from healthy subjects (HD-V3) or SCN patients (SCN-P56) treated with RNP(alt) composition or electroporated without nuclease composition (mock), as measured by ddPCR. (n=3 groups of cells from HD-V3 healthy / SCN-P56 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. (FIG. 23B) Bar graphs representing the percentage of excision at day 6 of differentiation of HSCs from either healthy individuals (HD-V3): mock-treated (mock, black) or RNP(alt)-treated (gray), or SCN patients (SCN-P56): mock-treated (mock, white) or RNP(alt)-treated (dark diagonal lines), as measured by ddPCR. (n=3 groups of cells from HD-V3 healthy / SCN-P56 patient donors). Statistical significance is indicated as ***P<.001. (FIG. 23C) Bar graphs representing the percentage of wild-type (black) and mutant (gray) alleles in cDNA from SCN-P56 patient HSCs that were either RNP(alt)-treated or mock-treated (mock), as measured by NGS targeting the mutation site. (n=3 groups of cells from SCN-P56 patient). Statistical significance is indicated as ****P<.0001. (FIG. 23D) Representative FACS plots of mock-treated (mock, left panel) and RNP(alt)-treated (right panel) healthy (HD-V3, upper panel) and SCN-P56 patient (lower panel) differentiated HSCs analyzed for neutrophil (CD66b+) and monocyte (CD14+ / CD66b-) subsets. (FIG. 23E) Quantitative analysis of individual FACS data for the percentage of neutrophils (CD66b+ cells) in healthy (HD-V3) and SCN patient (SCN-P56) differentiated HSCs mock-treated (mock, black) or treated with RNP(alt) (grey). (n=cells from 3 groups of HD-V3 healthy / SCN-P56 patient donors). Statistical significance is indicated as ****P<.0001, ns=not statistically significant.(FIG. 23F) Quantitative analysis of individual FACS data for the percentage of monocytes (CD14+ / CD66b- cells) in healthy (HD-V3) and SCN patient (SCN-P56) differentiated HSCs mock treated (mock, black) or treated with RNP(alt) (gray). (n=3 groups of cells from HD-V3 healthy / SCN-P56 patient donors). Statistical significance is indicated as P=0.04, ****P<.0001. (FIG. 23G) Diff-Quik staining of differentiated HSCs derived from P56 SCN patient treated with RNP(alt) or electroporated without nuclease composition (SCN-P56 mock). Photomicrographs were obtained on a LEITZ LABORLUX S polarizing microscope at 400x magnification using a Nikon DSLR digital camera. (FIG. 23H) Quantification of the percentage of Zymosan Green uptake by healthy (HD-V3) and SCN patient (SCN-P56) differentiated HSCs mock-treated (Mock, black) or treated with RNP(alt) (gray). Statistical significance is indicated as ****P<.0001, ns=not statistically significant. (FIG. 23I) Graph depicts real-time change in luminescence, relative light units (RLU), from 200,000 luciferase-expressing bacterial cells incubated with differentiated neutrophils derived from healthy mock-treated (HD-V3, Mock; white squares), patient mock-treated (SCN-56 Mock; cross circle) or patient RNP(alt)-treated (SCN-P56 RNP(alt); triangles) HSCs compared to control-only bacterial cells (E. coli, circles). Statistical significance for each one of the groups versus the E. coli control at the final time point presented when RLU levels reached a plateau is indicated as **P<.01. Bars represent the mean with standard deviation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description The present disclosure provides a genetically modified OMNI-50 nuclease with increased specificity for a target site compared to wild-type OMNI-50 nuclease (SEQ ID NO: 1). Wild-type OMNI-50 nuclease is disclosed in WO 2020 / 030782 (incorporated by reference herein). When the genetically modified OMNI-50 nuclease variant is active in a CRISPR endonuclease system, the CRISPR endonuclease system has reduced off-target editing activity and maintained on-target editing activity compared to a CRISPR endonuclease system that includes wild-type OMNI-50 nuclease. In some embodiments, the genetically modified OMNI-50 nuclease is an OMNI-50 nuclease variant that includes at least one amino acid substitution compared to wild-type OMNI-50 nuclease. In some embodiments, the genetically modified OMNI-50 nuclease includes multiple amino acid substitutions compared to wild-type OMNI-50 nuclease.

[0026] In some embodiments, an OMNI-50 nuclease variant is at least 80%, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1. By way of non-limiting example, an OMNI-50 nuclease variant may have amino acid differences at up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 20% of its residues compared to SEQ ID NO: 1. Such sequence differences may be revealed by sequence alignment. An OMNI-50 nuclease variant may be generated by replacing at least one amino acid residue of an OMNI-50 wild-type nuclease with another amino acid residue, e.g., by a conservative or non-conservative amino acid substitution, and / or by inserting or deleting an amino acid residue of an OMNI-50 wild-type nuclease. Any such mutation, including but not limited to substitutions, insertions, or deletions, in addition to or in addition to any other mutations described herein, may be used to generate an OMNI-50 nuclease variant from an OMNI-50 wild-type nuclease. In some embodiments, an OMNI-50 nuclease variant retains a desired activity of the parent wild-type OMNI-50 nuclease, e.g., the ability to interact with guide RNA and target DNA and / or nuclease activity (e.g., the ability to cause double-stranded DNA breaks, single-stranded DNA breaks, or the lack of any nuclease or nickase activity). In some embodiments, the variants retain the desired activity of the parent, e.g., nuclease activity, at a level that exceeds or is equal to the level of activity of the parent. In some embodiments, the variants retain the desired activity of the parent at a level that is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of activity of the parent. In some embodiments, the OMNI-50 nuclease variants have reduced off-target effects compared to OMNI-50 wild-type nuclease.

[0027] In some embodiments, variants of OMNI-50 nuclease proteins are provided that comprise a sequence at least 80% identical to the amino acid sequence of wild-type OMNI-50 (SEQ ID NO:1) and have at least one amino acid substitution. In some embodiments, the amino acid substitution comprises the replacement of an amino acid residue with a positive, negative, uncharged, hydrophilic, hydrophobic, polar, or non-polar amino acid. In some embodiments, the amino acid substitution is selected from the replacement of an amino acid with any one of a different amino acid selected from the group consisting of R, K, H, D, E, S, T, N, Q, C, U, G, P, A, I, L, M, F, W, Y, and V.

[0028] Positive amino acids include any amino acid with a positively charged R group, such as lysine (K), arginine (R), or histidine (H). Negative amino acids include any amino acid with a negatively charged R group, such as aspartic acid (D) or glutamic acid (E). Uncharged or neutral amino acids include amino acids whose R group is normally uncharged. Polar amino acids include any amino acid with a polar R group, such as serine (S), threonine (T), tyrosine (Y), asparagine (N), or glutamine (Q). Nonpolar amino acids include any amino acid with a nonpolar R group, such as glycine (G), alanine (A), valine (V), cysteine ​​(C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), or phenylalanine (F).

[0029] The properties of an original variant protein with an original amino acid substitution at a given position may extend to different variants with different amino acid substitutions at the same position, provided that the different amino acid substitutions have R groups with similar properties as the original amino acid substitution. For example, if a variant protein is shown to have higher specificity compared to the wild-type protein by substituting a lysine (K) residue with a glutamic acid (E) residue, it is reasonable to assume that a similar variant substituting an arginine (R) residue with a glutamic acid (E) residue would also exhibit higher specificity, since both lysine (K) and arginine (R) share similar properties (e.g., they both contain positively charged R groups). Conversely, a variant with a glutamic acid (E) residue with an aspartic acid (D) substitution is unlikely to exhibit higher specificity properties, since both glutamic acid (E) and aspartic acid (D) share similar properties, both containing negatively charged R groups.

[0030] In some embodiments, variants of OMNI-50 nuclease proteins contain amino acid substitutions at at least one of the following positions in the wild-type OMNI-50 protein sequence (SEQ ID NO:1): R61, Y437, R478, A493, Y545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, K965, V981, and K1036. Each possibility is a separate embodiment. In some aspects, variants of OMNI-50 nuclease proteins are provided that comprise a sequence that is at least 80% identical to the amino acid sequence of wild-type OMNI-50 nuclease (SEQ ID NO:1) and have at least one amino acid substitution at at least one of the following positions in the wild-type OMNI-50 protein sequence: R61, Y437, R478, A493, Y545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, K965, V981, and K1036. In some embodiments, the variant of the OMNI-50 nuclease protein comprises at least one of the following amino acid substitutions at the following positions in the wild-type OMNI-50 protein sequence: R478I, Y545H, Q803V, L805I, R61A, Y437W, R788K, L844N, V981M, G606P, L690V, E695Q, R478T, A493G, K688E, L718C, K965V and K1036V. Each possibility is a separate embodiment. In some embodiments, the substitution corresponds to the mutations listed in Table 1.

[0031] In some embodiments, the OMNI-50 nuclease protein variant comprises at least one amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: R478, Y545, Q803, and L805. In some embodiments, the OMNI-50 nuclease protein variant comprises an amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: R478, Y545, Q803, and L805. In some embodiments, the OMNI-50 nuclease protein variant comprises the following amino acid substitutions from the wild-type OMNI-50 protein sequence: R478I, Y545H, Q803V, and L805I.

[0032] In some embodiments, the OMNI-50 nuclease protein variant comprises at least one amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: R61, Y437, R788, L844, and V981. In some embodiments, the OMNI-50 nuclease protein variant comprises an amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: R61, Y437, R788, L844, and V981. In some embodiments, the OMNI-50 nuclease protein variant comprises the following amino acid substitutions from the wild-type OMNI-50 protein sequence: R61A, Y437W, R788K, L844N, and V981M.

[0033] In some embodiments, the OMNI-50 nuclease protein variant comprises at least one amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: G606, L690, and E695. In some embodiments, the OMNI-50 nuclease protein variant comprises an amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: G606, L690, and E695. In some embodiments, the OMNI-50 nuclease protein variant comprises the following amino acid substitutions from the wild-type OMNI-50 protein sequence: G606P, L690V, and E695Q.

[0034] In some embodiments, the OMNI-50 nuclease protein variant comprises at least one amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: R478, A493, K688, L718, K965, and K1036. In some embodiments, the OMNI-50 nuclease protein variant comprises an amino acid substitution at the following positions in the wild-type OMNI-50 protein sequence: R478, A493, K688, L718, K965V, and K1036. In some embodiments, the OMNI-50 nuclease protein variant comprises the following amino acid substitutions from the wild-type OMNI-50 protein sequence: R478T, A493G, K688E, L718C, K965V, and K1036V.

[0035] In some embodiments, the OMNI-50 nuclease variant further comprises one or more of a nuclear localization sequence (NLS), a cell membrane-permeable peptide sequence, and / or an affinity tag. In some embodiments, the OMNI-50 nuclease variant comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of a CRISPR complex comprising a CRISPR nuclease in detectable amounts in the nucleus of a eukaryotic cell.

[0036] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution selected from those corresponding to the substitutions displayed in Table 1 compared to wild-type OMNI-50.

[0037] In some aspects, there is provided an isolated variant of an OMNI-50 nuclease protein that comprises one or more substitutions or mutations compared to a wild-type OMNI-50 nuclease sequence, wherein said isolated OMNI-50 nuclease variant is active in a CRISPR system, wherein said CRISPR system has reduced off-target editing activity and maintained on-target editing activity compared to a wild-type CRISPR system.

[0038] In some embodiments, additional mutations to the OMNI-50 nuclease variants described herein may be implemented. Examples include, but are not limited to, mutations that change the PAM recognition sequence or alter the nuclease activity of the enzyme, and truncation or removal of a portion of the nuclease. In some embodiments, the OMNI-50 nuclease variant may be encoded by any nucleic acid sequence that produces the desired amino acid sequence of the variant. For example, the nucleic acid sequence may be codon-optimized for a cell, such as a bacterial cell, a plant cell, or a mammalian cell.

[0039] In an embodiment of the present invention, the CRISPR nuclease and the targeting molecule form a CRISPR complex that binds to a target DNA sequence to effect cleavage of the target DNA sequence. The CRISPR nuclease may form a CRISPR complex that includes the CRISPR nuclease and a single guide RNA (sgRNA) molecule. Alternatively, the CRISPR nuclease may form a CRISPR complex that includes the CRISPR nuclease, a crRNA molecule, and a tracrRNA molecule.

[0040] In some aspects of the invention, methods of gene editing with reduced off-target editing activity and / or increased on-target editing activity are provided, comprising contacting a target site locus with an active CRISPR endonuclease system having an OMNI-50 protein variant complexed with a suitable guide RNA or guide RNA complex, wherein the active CRISPR endonuclease system has reduced off-target editing activity and maintained on-target editing activity compared to a wild-type OMNI-50 CRISPR system.

[0041] In some embodiments, non-naturally occurring OMNI-50 nuclease variants are provided having a wild-type OMNI-50 protein sequence (SEQ ID NO:1) that includes amino acid substitutions at at least one of the following positions: R61, Y437, R478, A493, Y545, G606, K688, L690, E695, L718, R788, Q803, L805, L844, V981, K965, and K1036.

[0042] In some embodiments, an amino acid substitution at position R478 and / or Y545, preferably at both positions R478 and Y545.

[0043] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions R478 and Y545.

[0044] In some embodiments, the amino acid substitution at position R478 is any one of the following substitutions: R478D, R478E, R478S, R478T, R478N, R478Q, R478G, R478P, R478C, R478A, R478V, R478I, R478L, R478M, R478F, R478Y, or R478W, preferably R478V, R478H, R478L, R478M, R478P, R478F, R478W, R478Y, R478S, R478C, R478T, R478N, or R478Q.

[0045] In some embodiments, the amino acid substitution is at position R478, and the amino acid substituting the arginine is an amino acid having a negatively charged or uncharged R group.

[0046] In some embodiments, the amino acid substitution is at position R478 and the amino acid substituting the arginine is a polar amino acid or a non-polar amino acid.

[0047] In some embodiments, the amino acid substitution is at position R478 and the amino acid substituting the arginine is a non-polar amino acid.

[0048] In some embodiments, the amino acid substitution at position Y545 is any one of the following substitutions: Y545D, Y545E, Y545S, Y545T, Y545N, Y545Q, Y545G, Y545P, Y545C, Y545A, Y545V, Y545I, Y545L, Y545M, Y545F, Y545R, Y545K, Y545H, or Y545W, preferably Y545W, Y545F, Y545H, Y545V, or Y545G.

[0049] In some embodiments, the amino acid substitution is at position Y545, and the amino acid substituting the tyrosine is an amino acid having a negatively charged R group or a positively charged R group.

[0050] In some embodiments, the amino acid substitution is at position Y545 and the amino acid substituting the tyrosine is a non-polar amino acid.

[0051] In some embodiments, the amino acid substitution is at position Y545, and the amino acid substituting the tyrosine lacks a phenyl ring or is phenylalanine.

[0052] In some aspects, the amino acid substitutions are R478I and Y545H.

[0053] In some aspects, the amino acid substitution is any one of the following substitutions: R478I, Y545H, Q803V, L805I, R61A, Y437W, R788K, L844N, V981M, G606P, L690V, E695Q, R478T, A493G, K688E, L718C, K965V and K1036V.

[0054] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions R478, Y545, Q803, and L805.

[0055] In some aspects, the amino acid substitutions are R478I, Y545H, Q803V, and L805I.

[0056] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of the following positions: R61, Y437, R788, L844, and V981.

[0057] In some aspects, the amino acid substitutions are R61A, Y437W, R788K, L844N, and V981M.

[0058] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions G606, L690, and E695.

[0059] In some aspects, the amino acid substitutions are G606P, L690V, and E695Q.

[0060] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions R478, A493, K688, L718, K965, and K1036.

[0061] In some aspects, the amino acid substitutions are R478T, A493G, K688E, L718C, K965V and K1036V.

[0062] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at position Q803.

[0063] In some embodiments, the amino acid substitution is Q803V.

[0064] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at position Y545.

[0065] In some embodiments, the amino acid substitution is Y545H.

[0066] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at position L805.

[0067] In some embodiments, the amino acid substitution is L805I.

[0068] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions R478, Y545, and Q803.

[0069] In some aspects, the amino acid substitutions are R478I, Y545H, and Q803V.

[0070] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions R478, Y545, and L805.

[0071] In some aspects, the amino acid substitutions are R478I, Y545H, and L805I.

[0072] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions R478, Q803, and L805.

[0073] In some aspects, the amino acid substitutions are R478I, Q803V, and L805I.

[0074] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions Y545, Q803, and L805.

[0075] In some aspects, the amino acid substitutions are R478I, Y545H, and L805I.

[0076] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at position R478 and / or Y545.

[0077] In some embodiments, the amino acid substitution at position R478 is any one of the following substitutions: R478D, R478E, R478S, R478T, R478N, R478Q, R478G, R478P, R478C, R478A, R478V, R478I, R478L, R478M, R478F, R478Y, or R478W.

[0078] In some embodiments, the amino acid substitution is at position R478, and the amino acid substituting the arginine is an amino acid having a negatively charged or uncharged R group.

[0079] In some embodiments, the amino acid substitution is at position R478 and the amino acid substituting the arginine is a polar amino acid or a non-polar amino acid.

[0080] In some embodiments, the amino acid substitution is at position R478 and the amino acid substituting the arginine is a non-polar amino acid.

[0081] In some embodiments, the amino acid substitution is at position R478, and the amino acid substituting the arginine is selected from large hydrophobic amino acids (e.g., leucine, methionine, proline, valine), aromatic amino acids (e.g., histidine, phenylalanine, tryptophan and tyrosine), and polar uncharged amino acids (e.g., serine, cysteine, threonine, asparagine, and glutamine).

[0082] In some aspects, the amino acid substitution at position Y545 is any one of the following substitutions: Y545D, Y545E, Y545S, Y545T, Y545N, Y545Q, Y545G, Y545P, Y545C, Y545A, Y545V, Y545I, Y545L, Y545M, Y545F, Y545R, Y545K, Y545H, or Y545W.

[0083] In some embodiments, the amino acid substitution is at position Y545, and the amino acid substituting the tyrosine is an amino acid having a negatively charged R group or a positively charged R group.

[0084] In some embodiments, the amino acid substitution is at position Y545, and the amino acid substituting the tyrosine is an amino acid having a positively charged R group. Non-limiting examples of positively charged amino acids include histidine, lysine and arginine.

[0085] In some embodiments, the amino acid substitution is at position Y545 and the amino acid substituting the tyrosine is a non-polar amino acid. Non-limiting examples of non-polar amino acids include glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan, and phenylalanine.

[0086] In some embodiments, the amino acid substitution is at position Y545, and the amino acid substituting the tyrosine lacks a phenyl ring or is phenylalanine.

[0087] In some embodiments, the amino acid substitution is at position Y545 and the amino acid substituting the tyrosine comprises a phenyl ring (eg, phenylalanine).

[0088] In some embodiments, the OMNI-50 nuclease variant comprises an amino acid substitution at each of positions R478 and Y545.

[0089] In some embodiments, the amino acid substitutions are R478I and Y545H. However, other amino acid substitutions at positions R478 and Y545 are also contemplated. As a non-limiting example, amino acids with R groups that have non-polar properties similar to isoleucine, such as alanine (A), valine (V), and leucine (L), are substitutions considered at position R478. In another non-limiting example, amino acids with R groups that have non-polar properties similar to isoleucine, such as methionine (M), phenylalanine (F), tyrosine (Y), and tryptophan (W), are substitutions considered at position R478.

[0090] In some embodiments, the OMNI-50 nuclease variant has an amino acid sequence set forth in a SEQ ID NO: selected from the group consisting of SEQ ID NOs: 2-5, 63-70, and 89-97.

[0091] In some embodiments, the OMNI-50 nuclease variant has at least 80% sequence identity to the wild-type OMNI-50 protein sequence (SEQ ID NO:1).

[0092] In some embodiments, the OMNI-50 nuclease variant further comprises a nuclear localization sequence (NLS).

[0093] In some embodiments, the OMNI-50 nuclease variant has increased specificity for a DNA target site when complexed with a guide RNA molecule that directs the variant to the DNA target site compared to a wild-type OMNI-50 nuclease complexed with a guide RNA molecule.

[0094] Some aspects of the invention provide a CRISPR system comprising any one of the OMNI-50 nuclease variants described herein complexed with a guide RNA molecule targeted to a DNA target site, the CRISPR system having reduced off-target editing activity compared to a wild-type CRISPR system comprising a wild-type OMNI-50 nuclease protein and a guide RNA molecule.

[0095] In some embodiments of the invention, there is provided a method of gene editing with reduced off-target editing activity comprising contacting a DNA target site with an active CRISPR system comprising any one of the OMNI-50 nuclease variant proteins described herein.

[0096] In some embodiments, the active CRISPR system has reduced off-target editing activity compared to a wild-type CRISPR system that includes a wild-type OMNI-50 nuclease protein.

[0097] In some aspects, gene editing is performed in eukaryotic or prokaryotic cells.

[0098] In some aspects, the eukaryotic cell is a plant cell or a mammalian cell.

[0099] In some embodiments, the mammalian cell is a human cell.

[0100] In some embodiments, the DNA target site is located within or near a pathogenic allele of a gene.

[0101] In some embodiments, the DNA target site is located within a gene selected from the group consisting of ELANE, CXCR4, EMX, RyR2, KNCQ1, KCNH2, SCN5a, GBA1, GBA2, rhodopsin, GUCY2D, IMPDH1, FGA, BEST1, PRPH2, KRT5, KRT14, ApoA1, STAT3, STAT1, ADA2, RPS19, SBDS, GATA2, RPE65, LDLR, ANGPTL3, B2M, TRAC, TCF4, TGFBi, PAX6, C3, LRRK2, SARM1, SAMD9, SAMD9L, HAVCR2, CD3E, APLP2, CISH, TIGIT, TNNT2, TNN, MYH7, and HLA-E.

[0102] In some embodiments, the DNA target is repaired by an exogenous donor molecule.

[0103] In some embodiments, the off-target editing activity is at least 2-fold, 10-fold, 10-fold, 2 1 / 10 3 1 / 10 4 1 / 10 5 1 / 10 6 It will be reduced to one-fifth.

[0104] In some aspects of the invention, modified cells obtained by the methods described herein are provided.

[0105] In some aspects, the cells are capable of engraftment.

[0106] In some aspects, the cells are capable of giving rise to progeny cells after engraftment.

[0107] In some aspects, the cells are capable of giving rise to progeny cells following autoengraftment.

[0108] In some aspects, the cells are capable of giving rise to progeny cells for at least 12 months or at least 24 months after engraftment.

[0109] In some embodiments, the cells are selected from the group consisting of hematopoietic stem cells, progenitor cells, CD34+ hematopoietic stem cells, bone marrow cells, and peripheral mononuclear cells.

[0110] In some aspects of the invention, there is provided a composition comprising any one of the modified cells described herein and a pharma- ceutically acceptable carrier. In some aspects of the invention, there is provided an in vitro or ex vivo method of preparing the composition comprising mixing the cells with a pharma- ceutically acceptable carrier.

[0111] In some aspects of the invention, there are provided polynucleotide molecules encoding any one of the OMNI-50 variant proteins described herein.

[0112] delivery The OMNI-50 variant compositions described herein may be delivered as a protein, a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or any 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.

[0113] The OMNI-50 variant and / or a polynucleotide encoding the OMNI-50 variant described herein, and / or an additional molecule such as a single guide RNA molecule, a crRNA molecule, a tracrRNA molecule, or a nucleotide molecule encoding any one thereof, may be delivered to the target cell by any suitable means. The target cell may be any type of cell, e.g., a eukaryotic or prokaryotic cell, maintained in any environment, e.g., isolated or not, in culture, in vitro, ex vivo, in vivo or in planta. The target site in the target cell may be within the nucleus of the cell.

[0114] The compositions described herein may be introduced into cells as part of a vector molecule with additional sequences, such as, for example, an origin of replication, a promoter, and a gene encoding antibiotic resistance. Additionally, the compositions may be introduced into cells as naked nucleic acid or protein, as nucleic acid or protein complexed with or packaged within an agent, such as a liposome, an exosome, or a poloxamer, or delivered by a recombinant virus (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)) or virus-like particle. By way of non-limiting example, the compositions may be packaged into an adeno-associated virus (AAV) or into a lentivirus, such as a non-integrating lentivirus or a lentivirus lacking reverse transcription. Additional non-limiting examples include packaging the compositions into liposomes, extracellular vesicles, or exosomes, which may be pseudotyped with vesicular stomatitis glycoprotein (VSVG) or conjugated to a cell membrane-permeable peptide, an antibody, a targeting moiety, or any combination thereof.

[0115] In some embodiments, the composition to be delivered includes a nuclease mRNA and a guide RNA. In some embodiments, the composition to be delivered includes a nuclease mRNA, a guide RNA, and a donor template. In some embodiments, the composition to be delivered includes a CRISPR nuclease and a guide RNA. In some embodiments, the composition to be delivered includes a CRISPR nuclease, a guide RNA, and a donor template for gene editing, e.g., via homology directed repair. In some embodiments, a lentivirus includes a nuclease mRNA and a guide RNA molecule (e.g., a single guide RNA molecule or a crRNA molecule) and is used to guide the nuclease to a target site. In some embodiments, the composition to be delivered to a cell includes a nuclease mRNA, a guide RNA molecule, and a donor template molecule. In some embodiments, a lentivirus includes a nuclease protein variant and a guide RNA molecule. In some embodiments, the composition to be delivered to a cell includes a nuclease protein variant, a guide RNA molecule, and / or a donor template for homology directed repair. In some cases, the composition delivered to the cell includes the mRNA of the nuclease variant, the DNA targeting crRNA molecule and the tracrRNA molecule. The composition delivered to the cell includes the mRNA of the nuclease variant, the DNA targeting crRNA molecule and the tracrRNA molecule, and the donor template molecule. The composition delivered to the cell includes the nuclease protein variant, the DNA targeting crRNA molecule and the tracrRNA molecule. In some cases, the composition delivered to the cell includes the nuclease protein variant, the DNA targeting crRNA molecule and the tracrRNA molecule, and the DNA donor template molecule for homologous recombination repair.

[0116] Any suitable viral vector system may be used to deliver such compositions. Conventional viral or non-viral based gene transfer methods can be used to introduce nucleic acids and / or OMNI-50 variant proteins into cells (e.g., mammalian cells, plant cells, etc.) and target tissues. Such methods can also be used to administer nucleic acids encoding OMNI-50 variant proteins and / or OMNI-50 variant proteins to cells in vitro. In certain embodiments, nucleic acids and / or OMNI-50 variant proteins are administered for in vivo or ex vivo gene therapy applications. Non-viral vector delivery systems include naked nucleic acids and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992);Nabel & Felgner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988);Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and See Bohm (eds.) (1995) and Yu et al., Gene Therapy 1:13-26 (1994).

[0117] Non-viral methods of delivery of nucleic acids and / or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, virus-like particles, exosomes, 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, for example 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).

[0118] 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 transfer the polynucleotide sequences of, or polynucleotide sequences encoding, the molecules of the composition in the target cells.

[0119] 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.

[0120] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820). (1992); see U.S. Patent 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.

[0121] 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 (2009) Nature Biotechnology 27(7) p. 643).

[0122] The use of RNA or DNA virus-based systems for the delivery of nucleic acids utilizes highly developed 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 patients (in vivo) or used to treat cells in vitro, and the modified cells are administered to patients (ex vivo). Conventional virus-based systems for the delivery of nucleic acids include, but are not limited to, retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible using retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different cell types and target tissues. OMNI-50 variants or nucleic acids expressing variants, and any related nucleic acids, may be delivered by non-integrating lentiviruses. In some cases, RNA delivery using lentiviruses is utilized. In some cases, the lentivirus comprises a nuclease mRNA and a guide RNA molecule (e.g., a single guide RNA molecule or a crRNA molecule) that is used to target the nuclease to a target site. In some cases, the lentivirus comprises a nuclease mRNA, a guide RNA molecule, and a donor template molecule. In some cases, the lentivirus comprises a nuclease protein variant and a guide RNA molecule. In some cases, the lentivirus comprises a nuclease protein variant, a guide RNA molecule, and / or a donor template molecule for homologous recombination repair. In some cases, the lentivirus comprises a nuclease variant mRNA, a DNA targeting crRNA molecule, and a tracrRNA molecule. In some cases, the lentivirus comprises a nuclease variant mRNA, a DNA targeting crRNA molecule, and a tracrRNA molecule, and a donor template molecule. In some cases, the lentivirus comprises a nuclease protein variant, a DNA targeting crRNA molecule, and a tracrRNA molecule.In some cases, the lentivirus comprises a nuclease protein variant, a DNA targeting crRNA molecule and a tracrRNA molecule, and a donor template molecule for homology directed repair.

[0123] As previously described, the compositions described herein may be delivered to target cells using non-integrating lentiviral particle methods, such as the LentiFlash® system. Such methods may be used to deliver mRNA or other types of 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 and WO 2017 / 194903.

[0124] 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 repeats 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).

[0125] 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.

[0126] 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)).

[0127] 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).

[0128] In many gene therapies, it is desirable to deliver gene therapy vectors with high specificity to specific tissues. 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 92:9747-9751 (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 pairs, 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. This description applies primarily to viral vectors, but 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.

[0129] 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 transplanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, biopsy tissue) or to cells such as 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.

[0130] 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 the subject organism, transfected with an RNA composition, and re-injected into the subject organism (e.g., patient). A variety of cells suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994) and references cited therein for a discussion of methods for isolating and culturing cells from patients).

[0131] 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 one embodiment, the cell line is a CHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may be isolated and treated with a nuclease system (e.g., CRISPR / Cas) before being 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.

[0132] 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 cell types 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 cell types using cytokines such as GM-CSF, IFNγ, and TNFα (see, for non-limiting examples, Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).

[0133] 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, J. Exp. Med. 176:1693-1702 (1992)). In some embodiments, modified stem cells can also be used.

[0134] In particular, the OMNI-50 variants of the present application 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 OMNI-50 variants of the present invention include, but are not limited to, muscle cells, cardiomyocytes, hepatocytes, bone cells, and neurons.

[0135] 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.

[0136] 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.

[0137] 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, see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0138] DNA repair by homologous recombination In some aspects of the invention, OMNI-50 nuclease variants are utilized to cleave DNA at target sites to induce cellular repair mechanisms, such as, but not limited to, non-homologous end joining (NHEJ) or homology directed repair (HDR).

[0139] The term "homologous recombination repair" or "HDR" refers to a mechanism for repairing 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" (used interchangeably herein, nucleic acid template or donor template) to repair a sequence (e.g., a DNA target sequence) where a double-stranded or single break has occurred. This results, for example, in the transfer of genetic information from the nucleic acid template to the DNA target sequence. HDR may result in changes (e.g., insertions, deletions, mutations) in the DNA target sequence when the nucleic acid template sequence differs from the DNA target sequence and a portion or all of the nucleic acid template polynucleotide or oligonucleotide is integrated into the DNA target sequence. In some embodiments, the entire nucleic acid template polynucleotide, a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template is integrated at the site of the DNA target sequence.

[0140] The terms "nucleic acid template" and "donor" refer to a nucleotide sequence that is inserted or copied into a genome. A nucleic acid template includes a nucleotide sequence, e.g., one or more nucleotides, that may be added to a target nucleic acid or template a change in a target nucleic acid or used to change a target sequence. The nucleic acid template sequence may be any length, e.g., 2-10,000 nucleotides long (or any integer value therebetween or greater), preferably about 100-1,000 nucleotides long (or any integer value therebetween), more preferably about 200-500 nucleotides long. A nucleic acid template may be a single stranded nucleic acid, a double stranded nucleic acid. In some embodiments, a nucleic acid template includes a nucleotide sequence, e.g., one or more nucleotides, that corresponds to the wild type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template includes a ribonucleotide sequence, e.g., one or more ribonucleotides, that corresponds to the wild type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template includes modified ribonucleotides.

[0141] Insertion of exogenous sequences (also referred to as "donor sequences", "donor templates" or "donors") can also be performed, for example, for the correction of mutant genes or for increased expression of wild-type genes. It is immediately clear that donor sequences are usually not identical to the genomic sequence in which they are located. Donor sequences can contain non-homologous sequences flanked by two homologous regions to allow efficient HDR at the location of interest. In addition, donor sequences can contain vector molecules that contain sequences in the chromatin of a cell that are not homologous to the region of interest. Donor molecules can contain several discontinuous regions that are homologous to the chromatin of a cell. For example, for targeted insertion of a sequence that is not normally present in the region of interest, the sequence can be present in the donor nucleic acid molecule and can be flanked by regions that are homologous to sequences in the region of interest.

[0142] The donor polynucleotide can be DNA or RNA, single-stranded and / or double-stranded, and can 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. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolysis) 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). Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages, such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[0143] Thus, the embodiment of the present invention that uses a donor template for repair may use DNA or RNA, single-stranded and / or double-stranded donor template, which can be introduced into cells in linear or circular form.In an embodiment of the present invention, the gene editing composition comprises: (1) an RNA molecule that comprises a guide sequence that breaks the double strand in the gene before repair, and (2) a donor RNA template for repair, and the RNA molecule that comprises a guide sequence is the first RNA molecule and the donor RNA template is the second RNA molecule.In some embodiments, the guide RNA molecule and the template RNA molecule are linked as part of a single molecule.

[0144] The donor sequence may be an oligonucleotide and used for gene correction or targeted alteration of an endogenous sequence. The oligonucleotide may be introduced into the cell on a vector, electroporated into the cell, or introduced via 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 of a desired purpose into the endogenous locus.

[0145] Polynucleotides can be introduced into cells as part of vector molecules with additional sequences, such as origins of replication, promoters, and genes encoding antibiotic resistance. In addition, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with or packaged within agents such as liposomes, exosomes, or poloxamers, or delivered by recombinant viruses (e.g., adenoviruses, AAV, herpes viruses, retroviruses, lentiviruses, and integrase-deficient lentiviruses (IDLV)) or virus-like particles. Non-viral vectors, such as transposon-based systems, such as recombinant Sleeping Beauty transposon systems or recombinant PiggyBac transposon systems, can also be utilized for transposition of polynucleotide sequences in target cells.

[0146] 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 include a promoter and / or enhancer, e.g., a constitutive promoter or an inducible or tissue-specific promoter.

[0147] The donor molecule may be inserted into an endogenous gene such that all, part, or none of the endogenous gene is expressed. For example, a transgene as described herein may be inserted into an endogenous locus, e.g., as a fusion with the transgene, such that a portion of the endogenous sequence (N-terminal and / or C-terminal to the transgene) is expressed, or the endogenous sequence is not expressed. In other embodiments, the transgene (e.g., with or without additional coding sequences, such as to the endogenous gene) is integrated into any endogenous locus, e.g., 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; 2010 / 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.

[0148] When an endogenous sequence (endogenous or a portion 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. Non-limiting examples of functions of these full-length or partial sequences include increasing the serum half-life of a polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0149] In addition, although not required for expression, the exogenous sequence may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding the 2A peptide, and / or polyadenylation signals.

[0150] In certain embodiments, 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 the cell or an individual or 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.

[0151] DNA-targeting RNA molecules In an embodiment of the present invention, the DNA targeting RNA molecule comprises a guide sequence. The "guide sequence" of an RNA molecule refers to a nucleotide sequence that can hybridize with a specific target DNA sequence, for example, the guide sequence has a nucleotide sequence that is completely complementary to the targeted DNA sequence along the length of the guide sequence. In some embodiments, the length of the guide sequence 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, 27-25, 17-24, 18-22, 19-22, 18-20, 17-20 or 21-22 nucleotides. The entire length of the guide sequence is completely complementary to the targeted DNA sequence along the length of the guide sequence. Guide sequence can be a part of RNA molecule that can form a complex with CRISPR nuclease with guide sequence, which serves as the DNA targeting part of CRISPR complex.When RNA molecule with guide sequence is present at the same time with CRISPR molecule, RNA molecule can target CRISPR nuclease to specific target DNA sequence.Each possibility is a separate embodiment.RNA molecule can be specially designed to target desired sequence.

[0152] In some aspects of the invention, the disclosed methods include methods of modifying a nucleotide sequence in a cell-free system or at a target site in the genome of a cell, comprising introducing into a cell a composition as described herein.

[0153] In some embodiments, the cell is a eukaryotic cell, preferably a mammalian cell or a plant cell. In some embodiments, the modification of the genome occurs within the nucleus of the cell.

[0154] In some aspects of the invention, the disclosed methods include the use of the compositions of the present application for the treatment of a subject suffering from a disease associated with a genomic mutation, comprising modifying a nucleotide sequence at a target site in the subject's genome.

[0155] In some aspects of the invention, the disclosed methods include methods of treating a subject having a mutational disorder comprising targeting a composition of the present application to an allele associated with the mutational disorder.

[0156] 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, beta thalassemia, sickle cell anemia, cellular dysregulation, tumor-related diseases and disorders, inflammation and immune-related diseases and disorders, metabolic, liver, hypercholesterolemia, kidney and protein diseases and disorders, muscular and skeletal diseases and disorders, skin diseases and disorders, neurological diseases and disorders, pulmonary diseases and disorders, corneal diseases and disorders, retinal diseases and disorders, and ophthalmic diseases and disorders.

[0157] 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 novel nucleases disclosed herein may be specifically targeted to pathogenic mutant alleles of genes using specially designed guide RNA molecules 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-50 nuclease to a target site are designed to include a spacer sequence complementary to the DNA strand of the DNA double-stranded region adjacent to the OMNI-140 PAM sequence (e.g., "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.

[0158] 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, the non-homologous end joining (NHEJ) pathway is induced, resulting in silencing of the mutant allele by introducing a frameshift mutation. 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, the homology-directed repair (HDR) pathway is 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.

[0159] 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.

[0160] Mutations in ELANE gene are associated with neutropenia.Therefore, the embodiment of the present invention that targets ELANE may be used without limitation in the method of treating subjects suffering from neutropenia.Guide RNA molecules that target ELANE gene and are useful for treating neutropenia are disclosed in PCT International Patent Application PCT / US2020 / 059186, which is incorporated herein by reference.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] The present invention may be used to target disease-associated genes in the study, modification or treatment of diseases or disorders listed below in Table A or Table B. Indeed, disease-associated genes having a genetic locus may be studied, modified or treated by using the nucleases disclosed herein to target the appropriate disease-associated gene, such as those listed in US Patent Application Publication No. 2018 / 0282762 and EP Patent No. 3079726 (B1).

[0165] [Table 2]

[0166] [Table 3-1]

[0167] [Table 3-2]

[0168] [Table 3-3]

[0169] 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 herein can be used in the practice or testing of embodiments of this 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.

[0170] 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" and indicates at least one or any combination of the items it conjugates.

[0171] The term "a" or "an" as used herein should be understood to refer to "one or more" of the listed components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically noted otherwise. Thus, the terms "a" or "an" and "at least one" have the same meaning in this application.

[0172] 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 in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0173] In this specification and 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.

[0174] In this specification, the term "targeting sequence" or "targeting molecule" refers to a nucleotide sequence or a molecule that contains a nucleotide sequence that can hybridize to a specific target sequence, for example, a targeting sequence has a nucleotide sequence that is at least partially complementary to the sequence that is targeted along its length. A targeting sequence or a targeting molecule can be a part of an RNA molecule that can form a complex with CRISPR nuclease that has a targeting sequence that functions as the targeting part of the CRISPR complex. When a molecule that has a targeting sequence is present simultaneously with a CRISPR molecule, the RNA molecule can target CRISPR nuclease to a specific target sequence. Each possibility is a separate embodiment. An RNA molecule can be specifically designed to target a desired sequence.

[0175] In this specification, the term "target" refers to the preferential hybridization of targeting sequence or targeting molecule to the nucleic acid having the target nucleotide sequence.It is understood that the term "target" encompasses variable hybridization, such that there is preferential targeting of the nucleic acid having the target nucleotide sequence, but 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 the sequence due to nuclease activity.

[0176] In this specification, the term "wild type" refers to a typical form of an organism, strain, gene or characteristic that exists in nature, as distinguished from a variant or a variant, as understood by those skilled in the art. Thus, in this specification, when an amino acid sequence or a 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 an embodiment of the present invention, a 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), also referred to as a "mutation," compared to the wild type OMNI-50 nuclease shown in SEQ ID NO:1.

[0177] The terms "non-natural," "non-naturally occurring," or "modified" 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 found in nature.

[0178] The terms "mutant" or "variant" are used interchangeably and refer to a non-naturally occurring or altered molecule.

[0179] As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0180] As used herein, "genomic DNA" refers to a sequence of linear and / or chromosomal DNA and / or plasmid or other extrachromosomal DNA 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.

[0181] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.

[0182] In this specification, the term "modified cell" refers to a cell that has undergone a double-strand break by a complex of an RNA molecule and a CRISPR nuclease as a result of hybridization with a target sequence, i.e., on-target hybridization. The term "modified cell" may further include a cell in which a mutation has been repaired or corrected after a double-strand break by a variant. A modified cell may be any cell, such as a eukaryotic or prokaryotic cell, in any environment, isolated or not, or maintained in culture, in vitro, ex vivo, in vivo, or in planta.

[0183] The present invention provides modified cells obtained by the variants or methods described herein. In some aspects, these modified cells are capable of giving rise to progeny cells. In some aspects, these modified cells are capable of giving rise to progeny cells after engraftment. By way of non-limiting example, the modified cells may be hematopoietic stem cells (HSCs) or cells suitable for allogeneic or autologous cell transplantation. The variants and methods described herein may also be utilized to generate chimeric antigen receptor T (CAR-T) cells.

[0184] 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 the same, comprising combining the cells with a pharma- ceutically acceptable carrier.

[0185] 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.

[0186] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a nucleotide sequence of a target DNA that is located adjacent to the target DNA sequence and is recognized by a CRISPR nuclease. The PAM sequence may vary depending on the nuclease. For example, wild-type Streptococcus pyogenes Cas9 recognizes the PAM sequence "NGG". Those skilled in the art will appreciate that a single guide RNA molecule or a crRNA:tracrRNA complex can be complexed with a CRISPR nuclease to bind, for example, to a target genomic DNA sequence of interest next to the protospacer adjacent motif. The nuclease then cleaves the target DNA to generate a double-stranded break in the protospacer.

[0187] As used herein, a sequence or molecule has X% "sequence identity" with respect to another sequence or molecule if X% of the bases 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 bases in the same relative positions as the other sequence.

[0188] The terms "nuclear localization sequence" and "NLS" are used interchangeably to refer to an amino acid sequence / peptide that directs the transport of the associated protein from the cytoplasm across the nuclear membrane barrier. The term "NLS" is intended to encompass not only a specific peptide nuclear localization sequence, but also derivatives thereof that can direct the translocation of cytoplasmic polypeptides across the nuclear membrane barrier. An NLS can direct nuclear translocation of a polypeptide when attached to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptide. Additionally, polypeptides with an NLS linked at the N-terminus or C-terminus to the side chain of an amino acid randomly located in the amino acid sequence of the polypeptide are translocated. Typically, an NLS is composed of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, although other types of NLS are known. Non-limiting examples of NLSs include NLS sequences derived from SV40 virus large T antigen, nucleoplasmin, c-myc, hRNPA1 M9 NLS, the IBB domain from importin alpha, sarcoma T protein, human p53, mouse c-abl IV, influenza virus NS1, hepatitis virus delta antigen, mouse Mx1 protein, human poly(ADP-ribose) polymerase and steroid hormone receptor (human) glucocorticoid.

[0189] The term "CRISPR system" refers to a CRISPR endonuclease system that includes a CRISPR nuclease protein, such as a mutant or variant described herein, and an appropriate guide RNA molecule or guide RNA complex (e.g., a single guide RNA or crRNA:tracrRNA complex) to direct the CRISPR nuclease protein to a desired target DNA sequence based on complementarity between a portion of the guide RNA molecule or guide RNA complex and the target DNA sequence. The term "wild-type CRISPR endonuclease system" refers to a CRISPR endonuclease system that includes a wild-type CRISPR protein and an appropriate guide RNA molecule or guide RNA complex (e.g., a single guide RNA or crRNA:tracrRNA complex) to direct the wild-type CRISPR nuclease protein to a desired target DNA sequence based on complementarity between a portion of the guide RNA molecule or guide RNA complex and the target DNA sequence.

[0190] In this invention, "maintains on-target editing activity" refers to the ability of the OMNI-50 variant to target a DNA target site targeted by a guide RNA molecule associated with the OMNI-50 variant and thereby programming the OMNI-50 variant. In some embodiments, the OMNI-50 variant maintains on-target editing activity on a DNA target at or above the editing rate of wild-type OMNI-50 nuclease. In some embodiments, the OMNI-50 variant maintains on-target editing activity on a DNA target at at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40% or 30% of the editing rate of wild-type OMNI-50 nuclease.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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

[0197] Example 1 Variant Selection To select OMNI-50 nuclease variants with increased specificity for the target site (e.g., increased ratio between on-target and off-target cleavage), amino acid substitutions were introduced into the open reading frame of the wild-type OMNI-50 sequence (SEQ ID NO: 1) as shown in Table 1.

[0198] [Table 4]

[0199] [Table 5]

[0200] Example 2 OMNI-50 mutants show allele-specific editing of ELANE downstream components We demonstrated the improved specificity of the OMNI-50 nuclease variants compared to wild-type OMNI-50 nuclease by examining the ability of the nuclease to discriminate between the variant and base sequences of rs1683564 using the g62alt and g62ref RNA guide sequences (see Figures 1-6C).

[0201] Editing with sgRNA-564DS-alt depicted a high degree of specificity using wild-type OMNI-50 nuclease, whereas sgRNA-564DS-ref showed a lack of discrimination displayed by editing of both prototype and mutant alleles. Allele specificity was tested by excision with four OMNI-50 nuclease variants v3942, v2902, v3954, and v3795 in HSCs.

[0202] ddPCR revealed improved specificity for sgRNA-564DS-ref compared to OMNI-50 while maintaining discrimination of the sgRNA-564DS-alt allele for all variants of OMNI-50 (Figure 4A). In addition to allele discrimination, one off-target for sgRNA-564DS-ref, sgRNA-564DS-alt, and sg-constant was validated and showed a higher degree of variant fidelity compared to OMNI-50, characterized by less than 0.5% editing activity (Figure 4B and Figure 4C).

[0203] These results highlight the relatively high fidelity of the OMNI-50 nuclease variants.

[0204] Example 3 Variant allele knockout using novel CRISPR nucleases enhances myelopoiesis in ELANE neutropenia Representative ELANE mutations and individual treatment strategies Severe congenital neutropenia (SCN) is associated with multiple heterozygous mutations in the ELANE gene (Figure 7A). The current study presents a novel approach for the removal of mutant ELANE alleles by targeting heterozygous sites of SNPs flanking the majority of ELANE-mediated SCN mutations, instead of editing each pathogenic mutation individually. Among hundreds of possible SNPs in the ELANE gene, three SNPs were identified that are frequently heterozygous in healthy populations and were retrieved from the health database 1000 Genomes Project Consortium, herein named rs1683564, rs10414837, and rs3761005 (Figure 7B upper panel and Figure 10A). Analysis of healthy and patient populations revealed similar heterozygous frequencies between the two populations at each of the SNPs. Approximately 76-85% of each of the populations (healthy and patient, respectively) were heterozygous for at least one of the SNPs, indicating the applicability of our strategy to more than 75% of the SCN patient population. (Figure 10B, Supplementary Table 1). For each of the three SNPs, we developed ribonucleoprotein (RNP) editing compositions containing two different guides and a novel optimized CRISPR / Cas9 nuclease named OMNI variant 3795. One guide (herein named sgRNA constant) is common to all three compositions and cleaves both ELANE alleles in intron 4. The second guide targets the heterozygous form of one of the three SNPs and therefore cleaves only one allele. The composition targeting SNP rs1683564 excises exon 5 and the entire 3'UTR, causing degradation of the destabilized mRNA transcript (Figure 7B, I). Compositions targeting either SNP rs10414837 or SNP rs3761005 result in excision of the coding region and a large portion of the promoter, thus preventing transcription of the mutant allele (Figure 7B, II and III). The current study focuses on composition I, which targets SNP rs1683564, the most frequent form of which is cytosine, referred to herein as the reference allele.Adenosine is a relatively infrequent form of the SNP, referred to herein as the variant (alt) allele. Prior to treatment, patient cells are genotyped to determine whether the mutation and SNP are on the same allele or different alleles, in a method referred to as linkage determination (Figure 11). If the pathogenic mutation is linked to the prototype allele, a nuclease-guide RNA composition is selected that includes a guide (sgRNA(ref)) that targets the cytosine form of the SNP (referred to herein as RNP(ref)). If the pathogenic mutation is linked to the variant allele, a composition is selected that includes a guide (sgRNA(alt)) that targets the adenosine form of the SNP (referred to herein as RNP(alt)). The guides differ by only one nucleotide and, when used with the sgRNA constant, produce the same editing results (Figure 12).

[0205] Allele specificity and excision efficiency using OMNI variant 3795 nuclease in a SNP-based knockout strategy Unlike most CRISPR-related editing strategies that cut the target gene in both alleles, our approach aims to remove the mutant allele without affecting the wild-type functional allele. To demonstrate the feasibility of our single allele editing strategy, HSCs heterozygous for SNP rs1683564 from healthy individuals and SCN patients were excised with either RNP(ref) or RNP(alt) (depending on their lineage) or left untreated (NT), and HSCs were cultured on CD34 for 3 days. + They were allowed to recover in growth medium and subsequently cultured for 7 days in the presence of IL-3, SCF, GM-CSF and G-CSF for proliferation and myeloid progenitor differentiation, followed by stimulation with G-CSF for an additional 7 days for neutrophil differentiation (Figure 8A).

[0206] Fractions of cells were harvested on days 6 and 14 of differentiation for genomic DNA or RNA extraction. Allele specificity was determined by two competitive probes that bind either the mutant or prototype alleles. (See Supplementary Methods and Figures 19A and 19B for probe specificity). To test the RNP(ref) composition, we used HSCs from a P41 SCN patient (SCN-P41) carrying a mutation on the prototype allele and HSCs from a V3 healthy individual (HD-V3), both heterozygous for the prototype form of the SNP. In HD-V3 and SCN-P41 HSCs, approximately 40% and approximately 20% of the prototype alleles were intact, respectively, revealing that editing by RNP(ref) was specific (Figure 8B). However, the mutant allele was not affected by the RNP(ref) composition, as shown by high levels similar to those of untreated cells (NT) (Figure 8B). These results demonstrated that treatment with the RNP(ref) composition resulted in allele-specific editing.

[0207] Next, we evaluated the excision efficiency at days 6 and 14 of neutrophil differentiation. The excision was determined by amplification of two regions in the ELANE gene with two differently labeled probes, one for exon 1, which is not affected by the current excision strategy, and the second for exon 5, which is degraded upon excision (Figure 6B, I). The ratio between the signals of the two probes was interpreted as the excision efficiency. Treatment with RNP(ref) resulted in about 13% excision in HD-V3 HSCs and about 25% excision in SCN-P41 HSCs (Figure 8C). Given the high specificity of the nuclease (Figure 8B), this excision was associated with about 50% of the cell population undergoing excision at the prototype allele in SCN-P41 HSCs. RNP(ref) treatment also included about 6% inversion events in SCN-P41 HSCs as measured by EvaGreen staining (Figures 13A and 13B). Notably, other experiments evaluating RNP(ref)-mediated excision levels in HSCs from healthy individuals showed higher excision levels than reported above, which were comparable to those measured in HSCs from SCN-P41 (Figure 14). In addition, NGS analysis of cDNA from SCN-P41 cells targeting exon 4, which harbors the mutation, showed a 1:1 ratio between wild-type and mutant alleles in untreated cells. RNP(ref) treatment shifted the ratio of wild-type to mutant alleles to 3:1 by differentially reducing mutant allele transcripts, thereby enriching for the wild-type allele (Figure 8D).

[0208] To evaluate the specificity and efficiency of the RNP(alt) composition, we used HSCs from a P55 SCN patient (SCN-P55), who carries a mutation on the mutant allele and is heterozygous for the SNP variant.

[0209] RNP(alt)-based treatment resulted in approximately 90% editing of the mutant allele (only 10% remained intact), and the prototype allele remained intact at day 6 of differentiation (Figure 8E). The excision level in SCN-P55 HSCs was approximately 23% at days 6 and 14 of neutrophil differentiation (Figure 8F), indicating that approximately 46% of the cell population had undergone excision at the mutant allele. RNP(alt) treatment resulted in 7.6% reversal events in SCN-P55 HSCs as measured by EvaGreen staining (Figure 13C). The specificity and excision efficiency of RNP(alt)-edited HSCs from healthy individuals were tested and found to be comparable to those obtained from patient-derived cells (Figure 14). In addition, NGS analysis of cDNA from SCN-P55 cells targeting exon 5 carrying the mutation showed enrichment of the wild-type allele (Figure 8G). ELANE mRNA levels were decreased after ablation in cells from SCN-P41 and SCN-P55 patients (Figure 8H). Given the increased WT:mutant allele ratios obtained after ablation (Figures 8D and 8G), the reduced mRNA levels were primarily the result of degradation of the mutant transcript.

[0210] Next, we demonstrated that RNP(alt)-based ablation targets a subpopulation of HSCs (CD34 + / CD90 + We confirmed that this occurs in 3T3 cells (Figures 15A to 15C).

[0211] HSCs from both P41 and P55 edited with RNP(ref) and RNP(alt), respectively, were analyzed by amplicon NGS to measure editing to the off-targets identified for the constant guide (SgRNA(constant)), prototype guide (SgRNA(ref)), and variant guide (SgRNA(alt)).

[0212] Specifically, an unbiased survey (GUIDE-seq) of genome-wide off-target cleavage using 3795 nuclease variants and each of the constant guide (SgRNA(constant)), prototype guide (SgRNA(ref)), and mutant guide (SgRNA(alt)) resulted in no identified off-targets (≦4 mismatches) (Figures 20A-C).

[0213] In addition, in silico off-target analysis was performed for each of the guides, identifying several possible off-targets, none of which were validated by rhAmpSeq analysis performed on edited HSCs from patients SCN-P41 and SCN-P55 (Figure 20D), demonstrating the high fidelity of the novel nuclease composition.

[0214] Taken together, the results so far provide an active and highly accurate nuclease that can target mutant alleles while leaving wild-type functional alleles unaffected.

[0215] Editing promoted by OMNI variant 3795 enhances neutrophil differentiation and maturation in vitro To demonstrate the functional outcome of our SNP-based single allele editing approach, we assessed the neutrophil differentiation and maturation capacity of in vitro RNP(ref)-edited and untreated healthy and patient-derived HSCs on day 14 of differentiation. Flow cytometry analysis showed that approximately 74% of HD-V3 HSCs subjected to the differentiation protocol were neutrophilic (CD66b + ; the two right quadrants of the dot plot (Q2+Q3)). In contrast, only about 15% of HD-V3 HSCs were monocytes (CD14 + / CD66b -; upper left quadrant (Q1) of the dot plot). These observations are consistent with the characteristic hematopoietic defects in SCN patients (Figure 9A-C; NT: HD-V3 vs. SCN-P41). SCN-P41-derived HSCs treated with RNP (ref) differentiated into neutrophils (CD66b + 74% increase in monocyte subsets (CD14 + / CD66b - ) (Figure 9A-9C; SCN-P41:NT vs. RNP(ref)). A similar increase in neutrophil counts was observed in the CD11b + / CD15 + Flow cytometric analysis of cells was observed in SCN-P41-derived edited HSCs (Figures 16A and 16B). RNP(ref)-mediated editing in HD-V3 HSCs did not affect monocyte and neutrophil subsets, supporting the safety of this composition (Figures 9A-C; HD-V3: NT vs. RNP(ref)).

[0216] After demonstrating that our allele-specific editing approach significantly ameliorated SCN-associated cellular abnormalities, we next assessed neutrophil function in RNP(ref)-treated and untreated healthy (V3) and patient (P41) HSC-derived neutrophils. In vitro phagocytic capacity was tested by measuring phagosomal uptake of zymosan green particles by neutrophils from the different groups. Flow cytometry analysis revealed comparable levels of phagocytosis in RNP(ref)-treated and untreated neutrophils derived from both HD-V3 and SCN-P41 (Figure 9D). In addition, anti-bacterial killing capacity was examined by incubating neutrophils derived from untreated healthy HSCs (HD-V3, NT) and RNP(ref)-treated patient HSCs (SCN-P41, RNP(ref)) with E. coli expressing a bacterial luciferase gene and tracking real-time changes in luminescence expressed as relative light units (RLU). The two groups were compared to bacteria only (E. coli control without neutrophils). Healthy donor neutrophils and RNP(ref)-treated patient neutrophils efficiently killed bacteria as indicated by a 23-25% reduction in bacterial units (RLU) compared to the bacteria only control. Experiments were performed with 50,000 and 100,000 HSCs (Figure 9E).

[0217] Experiments with the RNP(ref) composition were performed on cells from another patient (SCN-P42) depicting similar ablation, differentiation and functional results, including histological staining demonstrating restoration of neutrophil differentiation (Figures 21A-21J). Thus, specific ablation of the ELANE mutant allele with the RNP(ref) composition improved the abnormal phenotype of reduced differentiation into neutrophils and preserved basic neutrophil functions. Similar analysis was performed on the RNP(alt) composition. Flow cytometry analysis demonstrated that neutrophil (CD66b + Approximately 83% of HD-V4-derived HSCs differentiated into the two right quadrants (Q2+Q3) of the dot plot, and monocytes (CD14 + / CD66b -; the upper left quadrant (Q1) of the dot plot) showed approximately 7% differentiation. SCN-P55-derived HSCs showed low differentiation into neutrophils (approximately 53%) and high differentiation into monocytes (approximately 29%) (Figure 9F-H; NT: HD-V4 vs. SCN-P55), demonstrating the typical SCN hematopoietic defect. SCN-P55-derived HSCs treated with RNP(alt) showed low differentiation into neutrophils (CD66b + cells) and monocyte subsets (CD14 + / CD66b - ) (Figure 9F-H; SCN-P41:NT vs. RNP(alt)). A similar increase in neutrophil subsets was observed in the CD11b + / CD15 + Flow cytometric analysis of cells was observed in SCN-P55-derived edited HSCs (Figure 16C and Figure 16D). Diff-Quik staining of HSCs derived from SCN-P55 treated with RNP(alt) or electroporated without nuclease composition (SCN-P55 Mock) revealed a relatively large number of cells with classic polymorphonuclear neutrophil morphology in the RNP(alt)-treated group compared to the mock group (Figure 9I). Flow cytometric analysis of zymosan green particles revealed a slightly higher level of phagocytosis in RNP(alt)-treated neutrophils compared to untreated neutrophils derived from SCN-P55 (Figure 9J). In addition, untreated neutrophils from healthy donors (HD-V4, NT) and patient RNP(alt)-treated neutrophils (SCN-P55, RNP(alt)) efficiently killed bacteria, as indicated by a significant 30% reduction in bacterial units (RLU) compared to bacteria-only controls (Figure 9K).

[0218] Experiments using the RNP(alt) composition were performed on cells from another patient (SCN-P12) depicting similar excision, differentiation and functional outcomes (Figures 22A-I). Furthermore, another experiment performed on patient cells (SCN-P56) carrying a mutation on exon 2 located upstream of the mutation found in the previous patient (exons 4 and 5) showed similar results (Figures 23A-I). The results described herein demonstrate that single allele knockout of the ELANE mutant allele using the RNP(ref) and RNP(alt) novel nuclease compositions is safe and effective in both enhancing neutrophil differentiation and maintaining essential neutrophil core functions.

[0219] OMNI variant 3795 showed high fidelity without traceable off-target. This is the first report to demonstrate specific single-allelic gene editing that is not mediated by the acquisition of PAM sequence. This unique feature of our novel nuclease can be performed in various indications, including dominant, dominant-negative, and compound heterozygotes, covering the majority of genetic disorders that other technologies cannot address.

[0220] The OMNI variant 3795 composition showed an excision efficiency of about 25%. Notably, given the high allele specificity of this nuclease composition, it is estimated that about 50% of the cell population underwent excision at the mutant allele. With regard to functional aspects, ELANE single allele excision significantly promoted neutrophil differentiation in vitro. The abnormal number of monocytes, consistent with the hematopoietic defects of SCN patients, was also reduced. The excised neutrophils showed normal phagocytosis and bacterial killing capacity, indicating that the editing was effective and safe.

[0221] Thus, this example presents a novel CRISPR / Cas9-based strategy for specific single-allele excision that was found to be efficient, functional, precise and safe.

[0222] Example 3 Materials and Methods Human HSC isolation HSCs were isolated from the bone marrow of SCN patients and from mobilized peripheral blood of healthy donors.

[0223] CRISPR / Cas9 OMNI variant 3795 ELANE gene editing The OMNI variant 3795 was used in a ribonucleoprotein (RNP) system with a molar ratio of 1:2.5 nuclease:sgRNA. + Cells were electroporated using the CA-137 program (Lonza 4D, Nucleofector™).

[0224] Digital droplet PCR (excision, allele specificity) Excision and allele specificity were measured using Digital Droplet PCR™ on genomic DNA. For the excision reaction, amplification of two regions in the ELANE gene, exon 1 and exon 5, was performed using two different probes, FAM (X1) and HEX (X1), respectively. The ratio between the probe signals was interpreted as the excision efficiency. For allele specificity, a FAM probe (binding to the mutant allele) and a HEX probe (binding to the prototype allele) were used (FAM+HEX). The ratio between the two probes was normalized to the endogenous gene.

[0225] Assessment of mutant:wild-type allele ratios cDNA was mapped using next-generation sequencing (NGS) targeting exons 4 and 5, which harbor the S126L and R220Q mutations, respectively, in patients 41 and 55. The relative ratios of mutant:wild-type alleles in treated cells were calculated and compared with those in untreated cells.

[0226] Differentiation assay Edited and untreated HSCs were subjected to a differentiation protocol adapted from Nasri et al.. On day 14, cells were differentiated into monocytes (CD14 + / CD66b - ) and neutrophils (CD66b + ) subsets were analyzed by flow cytometry.

[0227] Bacterial killing assay The bacterial killing capacity of day 13 differentiated HSCs was assessed as described by JT Atosuo. Relative light units (RLU) were measured over a 5-h period. The final time point presented is when the RLU levels reached a plateau. Wells without differentiated HSCs (only E. coli) and containing the phagocytosis inhibitor cytochalasin D (data not shown) served as controls.

[0228] Phagocytosis assay Phagocytic capacity was assessed using the EZCell™ Phagocytosis Assay Kit (Green Zymosan), (BioVision, Cat no. K397). Cells were analyzed by flow cytometry for internalization of opsonized fluorescent Zymosan Green particles.

[0229] Details regarding FACS antibodies, probes, sequences, assays, protocols and additional methods are provided in the Supplementary Methods below.

[0230] Example 3 Supplementary Methods Human SCN patient HSC isolation 3-6 mL of freshly harvested bone marrow was shipped overnight at ambient temperature. Hematopoietic stem and progenitor cells, HSPCs, were first enriched using RosetteSep Human Bone Marrow Progenitor Cell Pre-Enrichment Cocktail (Cat. no. 15027) and Lymphoprep (Cat. no. 07801) according to the manufacturer's protocol. The HSC-enriched cell population was cultured in CD34 medium supplemented with 1% Penn Strep (Cat. no 03-031-1B, Biological Industries), 1x StemSpan CD34 Proliferation Supplement (10x) (Cat. no. 02691), and 1.0 μM UM729 (Cat. no. 72332) at 37 °C and 5% CO2. + The cells were expanded by culturing in a proliferation medium (StemSpan SFEMII medium (Cat. no. 09655)) for 4 days. After expansion, CD34 +The cells were further enriched using the EasySep Human CD34 Positive Selection Kit II (Cat. no. 17856) according to the manufacturer's protocol. + Cells were cultured at 1 × 10 in Cryostor CS10 (cat. no. 07931). 6 Cells were stored frozen in cells / mL. Cells were stored in the vapor phase of liquid nitrogen. All catalog numbers refer to material from StemCell Technologies unless otherwise indicated.

[0231] Healthy human HSC isolation Cryopreserved healthy human CD34 derived from mobilized peripheral blood + Progenitor cells were obtained from Lonza (Cat no. 4Y-101C). Cells were cultured at 50,000 cells / mL with CD34 + Cells were suspended in growth medium and grown for 4 days at 37° C., 5% CO 2 before electroporation.

[0232] ELANE gene editing by CRISPR / Cas9 OMNI variant 3795 Editing of HSCs was performed using a ribonucleoprotein (RNP) system containing 17 μg nuclease and 262 pmol of each guide at a molar ratio of 1:2.5 (nuclease:sgRNA). The nuclease and sgRNA complex was incubated for 10 min at 25°C. Human CD34 + The cells were washed once with PBS. 5 CD34 + Cells were suspended in 20 μL of P3 electroporation buffer (Lonza P3 Kit S) and added to the RNP mix. After electroporation using the CA-137 program (Lonza 4D, Nucleofector™), cells were diluted to 1.25.0×10 5 Prewarmed CD34 cells / mL + The guides were manufactured by Agilent. The guide sequences are summarized in the table below:

[0233] [Table 6]

[0234] Digital droplet PCR for excision rate and allele specificity Excision rates and allele specificity were measured using Digital Droplet PCR™ (ddPCR™, Bio-Rad, Hercules, CA, USA) on genomic DNA extracted using the QIAamp DNA Micro Kit, Qiagen (Cat no. 56304), following the manufacturer's protocol.

[0235] The ddPCR reactions contained 1x dUTP-free ddPCR Supermix for probe (#1863024), 25-100ng DNA digested with HindIII (diluted to 4U / μL in X1 Cutsmart buffer) and appropriate primers / probes. For the excision reactions, amplification of two regions in the ELANE gene, exon 1 and exon 5, was performed using two different probes labeled with FAM (X1) and HEX (X1), respectively. The ratio between the HEX and FAM signals was read as the excision efficiency.

[0236] For allele specificity, two competitive probes were used: a FAM probe that binds to the mutant allele, and a HEX probe that binds to the prototype allele (FAM+HEX). The ratio between the two concentrations in heterozygous untreated cells is 1, normalized to the endogenous genes RPP30 and STAT1 for each gDNA sample. The total reaction volume was 22 μL. Binding of each probe to DNA extracted from cells of healthy individuals who were homozygous for either the prototype or the mutant form of the SNP was measured by ddPCR to confirm that the probes did not cross-react (Figure 19A). Furthermore, healthy cells homozygous for the prototype of the SNP depicted efficient excision when treated with the RNP(ref) composition, compared to treatment with the RNP(alt) composition, which produced excision levels comparable to untreated cells. This further demonstrates the specific targeting of the sgRNA (Figure 19B).

[0237] Genomic DNA in the ddPCR mixture was split into individual droplets using a QX100 droplet generator, transferred to a 96-deep-well PCR plate, and amplified in a Bio-Rad PCR thermocycler. Experiments were read and analyzed using a Bio-Rad droplet reader and QuantaSoft software according to the manufacturer's guidelines (Bio-Rad).

[0238] Primers and probes were from Bio-Rad and are described in the following table:

[0239] [Table 7]

[0240] Assessment of mutant:wild-type allele ratios cDNA from treated HSCs with either RNP(ref) or RNP(alt) was mapped using next generation sequencing (NGS) targeting exons 4 and 5 harboring the S126L and R220Q mutations in patients 41 and 55, respectively. Raw FASTQ files were analyzed and BAM files (a text-based format for storing biological sequences) were generated using the FASTQ to BAM script. The relative ratio of mutant alleles to wild-type alleles was calculated and compared to untreated cells in both patients. Primers are detailed in the table below:

[0241] [Table 8]

[0242] Differentiation assay Edited and unprocessed HSCs were cultured using CD34 + After recovery in growth medium for 3 days, Nasri et al. 23The cells were subjected to a differentiation protocol adapted from the previous study. Briefly, HSCs were cultured for 7 days in RPMI (Cat.no 11875093, Gibco™) supplemented with 1% Glutamax (Cat.no 35050061, Gibco™), 10% FBS (Cat.no 04-001-1A, Biological Industries), 5 ng / mL IL-3 (Cat.no 200-03), SCF (Cat.no 300-07), GM-CSF (Cat.no 300-03) and 10 ng / mL G-CSF (Cat.no 300-23), all from PeproTech, for proliferation and myeloid progenitor differentiation, and then cultured for 7 days in RPMI, 1% Glutamax, 10% FBS, 1% Penn Strep (Cat.no 03-031-1B, Biological Industries), 10 ng / mL IL-3 (Cat.no 200-03), SCF (Cat.no 300-07), GM-CSF (Cat.no 300-03) and 10 ng / mL G-CSF (Cat.no 300-23), all from PeproTech, for neutrophil differentiation and maturation. The cells were cultured in G-CSF for 7 days. On day 14, the cells were cultured as monocytes (CD14 + / CD66b - ) and neutrophils (CD66b + ) subsets were analyzed by flow cytometry using CD66b anti-human, Pacific Blue (Cat No. 305112, Biolegend) and CD14, anti-human, APC (Cat No. 130-110-520, Miltenyi Biotec).

[0243] Cytospin staining 8 x 10 on day 15 of differentiation 4 HSCs were centrifuged onto Cytoslide microscope slides (ThermoFisher) using a Cytospin 4 low-speed cytocentrifuge (Thermo Scientific) and stained with Diff-Quick staining system (MilliporeSigma) according to the manufacturer's recommendations. Photomicrographs were obtained on a LEITZ LABORLUX S polarizing microscope at 400x magnification using a Nikon DSLR digital camera.

[0244] Bacterial killing assay The bacterial killing capacity of day 13 differentiated HSCs (subjected to the differentiation protocol adopted from Nasri et al.) derived from healthy controls and SCN patients (edited with either RNP(ref), RNP(alt) or untreated) was assessed as described by JT Atosuo. Briefly, 100,000 differentiated HSCs per well were incubated in the presence of 200,000 luciferase-expressing bacterial cells pAKLUX2 (Addgene, Cat No. 14080). Cells were cultured at 37°C in 200 μL HBSS++, 10% FBS. Luminescence was measured at 30 min intervals by transferring the plate to a luminometer (Berthold CentroXS3 LB960) and measuring luminescence for 0.5 s per well. Relative light units (RLU), the real-time change in luminescence, were measured over a 5-h period. The final time point presented is when RLU levels reached a plateau. Wells containing no differentiated HSCs (only E. coli ) but 10 ng / mL of the phagocytosis inhibitor cytochalasin D (Santa Cruz Biotec, Cat No. sc-20144) (data not shown) served as controls.

[0245] Phagocytosis assay Phagocytic ability was assessed using the EZCell™ Phagocytosis Assay Kit (Green Zymosan), (BioVision, Cat no. K397, according to the manufacturer's protocol). Day 14 differentiated HSCs (subjected to the differentiation protocol adopted from Nasri et al.) from healthy controls and SCN patients (edited with either RNP(ref), RNP(alt) or untreated) were cultured in HBSS++ / 10% FBS (0.5×10 6The cells were resuspended in 1000 mM NaCl (100 mM NaCl / mL) and incubated for 1.5 h at 37° C. in the presence of 5 mL of opsonized Alexa Fluor 488-conjugated zymosan particles per 200 mL of suspended cells. As a negative control, cells were incubated with 10 ng / mL of the phagocytosis inhibitor cytochalasin D (Santa Cruz Biotec, Cat No. sc-201442) 1 h prior to and during incubation with the Zymosan Green reagent. Cells were then washed and incubated in quencher solution according to the kit instructions to remove fluorescence from non-internalized particles. Cells were then analyzed for internalization of opsonized fluorescent Zymosan Green particles using flow cytometry.

[0246] Invert Inversion events were detected and quantified by digital droplet PCR (ddPCR) mutation assay. First, complete inversion was mimicked using SnapGene software and verified by NGS. Subsequently, total inversion events were quantified by ddPCR using EvaGreen dye (BIO-RAD, Cat. No. 186-4034, according to manufacturer's protocol), a fluorescent DNA-binding dye that binds to dsDNA. Specific primers were designed to amplify the inverted variations of the excised fragment (see diagram in FIG. 11). Fluorescence signals were normalized to the amplification of the ELANE exon 1 region that was not affected by the excision (performed by two different sets of primers; averaged normalized data are presented). Primers are detailed in the following table:

[0247] [Table 9]

[0248] Ablation levels in long-term HSC populations HSCs from two healthy individuals (heterozygous for MLP1 (3055934); SNP variant and homozygous for MLP2 (3055940); SNP variant) isolated from leukopaks purchased from AllCells were edited the day after thawing according to the "CRISPR / Cas9 OMNI variant 3795 ELANE gene editing" section above with minor modifications. Cell number was 2M cells / electroporation. Scaling up of guides and nuclease was performed accordingly. A molar ratio of 1:2.5 (nuclease:sgRNA) was used with 85 μg nuclease and 1310 pmol of each guide. Nucleofection was performed in P3 Nucleofection Solution (Lonza) and Lonza 4D-Nucleofector™ X Kit L (program CA-137). Three days after editing, cells were sorted using CD90-APC-Vio770, human (130-114-863 Milteny) on a FACS ARIA™ II SORP flow cytometer cell (BD). Sorted cells were incubated for 7 days in growth medium according to Nasri et al. + , CD90 - and excision levels in the total population were assessed by ddPCR as described in the ddPCR section.

[0249] Mutation-SNP linkage First, ELANE mutations and possible SNPs were identified in cells from SCN patients by targeted short-read NGS. A portion of the gene encompassing both the mutation and the SNP was then amplified by PCR reaction using linked primers and cloned into bacteria. Each clone carries the amplicon from one allele. Plasmids from multiple clones were Sanger sequenced (using T7 and SP6 primers) for the mutation and SNP regions. If the mutation and SNP were in cis, they were found in the same clone, and if they were in trans, the mutation and SNP were found in different clones. The primers are detailed in the table below:

[0250] [Table 10]

[0251] Frequency of heterozygosity for SNPs in healthy and affected populations Variant call files encompassing the ELANE gene region (±3 kb of the ELANE gene) were downloaded from the 1000 Genomes Project Consortium (Phase 3) using the Data Slicer tool and analyzed in the R statistical computing environment. 3501 genotypes were available from 3501 individuals. Family relationships were omitted from the analysis, resulting in 2407 genotypes from unrelated individuals. Allele frequencies (>1% MAF) for all common polymorphisms were calculated. Three SNPs were selected to optimize population coverage for allele-specific ELANE knockout (rs3761005, rs1683564, and rs10414837 polymorphisms). The proportion of the population heterozygous for at least one of the three selected SNPs was calculated.

[0252] Fifty-three patient samples were sequenced for pathogenic mutations and the three selected SNPs. A total of 46 bone marrow samples and 7 iPSC lines were used. 44 of the samples were from SCN patients, 9 of which were from patients with cyclic neutropenia. The heterozygosity frequency for each of the three selected SNPs and the proportion of the population heterozygous for at least one of them were calculated.

[0253] statistical methods Two-sample T-tests for individual samples or ANOVA models, where appropriate, were applied to test the statistical significance of differences in continuous variables between treatment groups. Two-way ANOVA with repeated measures was used to analyze killing assays. 2The squared test or Fisher's exact test, where appropriate, was applied to test the statistical significance of differences in heterozygosity between healthy and affected populations. All tests were two-sided, and a p-value of 5% or less was considered statistically significant. Data were analyzed using Prism software (GraphPad version 9.0.2).

[0254] [Table 11]

[0255] Appendix Table 1. Coverage of the three SNPs by patient population. Sequencing results of the three SNPs in the ELANE gene in samples from SCN and cyclic neutropenia (CyN) patients. Pathogenic variants in the ELANE gene were also verified by sequencing. Green cells depict heterozygous SNPs.

[0256] Example 4 Contribution of each of the variant 3795 mutations to nuclease specificity and fidelity Variant 3795 is a specific variant of CRISPR-based nuclease with extremely low off-target activity (<0.05%) and the ability to discriminate between two alleles of the same gene with a single mismatch between them. The variant contains four mutations: R478I, Y545H, Q803V and L805I. To investigate the effect and contribution of each mutation found in OMNI-50 V3795 to activity and specificity, three of the four single mutant variants and four triple mutant variants in which one of the four mutations is absent in each variant were expressed and purified (Table A). RNP complexes were prepared with WT OMNI-50 nuclease, the V3795 variant, and the seven variants by assembly with sgRNA g62-Ref. These RNP complexes were electroporated into LCL cells heterozygous for the guide sequence target (Table B). Editing levels of the targeted allele (REF) and non-targeted allele (ALT) were measured by NGS. As can be seen from Figures 17A-B, discrimination of V3795 against the targeted allele (REF) is mainly conferred by the R478I mutation (Table E). Only variants containing the R478I mutation (e.g. V6864, V6865, V6866, V3795) showed a clear preference for the targeted allele, and removing this mutation abolished discrimination (e.g. V6867).

[0257] The specificity effect was verified by measuring the editing levels at two known off-target sites of g62 by NGS (g62OT1 and g62OT2, Table C). OMNI-50 V3795 has very low levels of editing at these sites compared to WT OMNI-50 nuclease (Figure 18A-B, Table F). Lower than WT editing levels at off-target sites were observed in all three single mutants, indicating the contribution of the three mutations to the reduction in off-target. However, the Y545H mutation had the most significant reduction in off-target editing (OT1 0.5% and OT2 0.04%, Table F). All triple variants also showed very low off-target activity similar to the V3795 nuclease variant. Surprisingly, when the Y545H mutation was excluded from the set of mutations (e.g., V6866), off-target editing increased slightly (OT1 1.90%, OT2 0.2%, Figures 18A-B, Table F). Overall, these results led us to conclude that the high level of specificity of the V3795 nuclease variant is due to the R478I and Y545H mutations.

[0258] Once it was established that R478I and Y545H were responsible for the observed specificity of the V3795 nuclease variant, the effect of other substitutions was examined. For each of these positions, RNPs were generated using several nuclease variants containing substitutions from different biochemical groups in the background of the g62-Ref sgRNA and the other three mutations (Table A). Discrimination and off-target effects were measured as previously described (Figures 17A-B and 18A-B).

[0259] For position 478: charged amino acids such as aspartic acid (D) in variant V7087 abolish discrimination and increase off-target editing (Figure 18B). A similar effect on discrimination is seen for arginine (R, reference amino acid) in triple variant V6867 (Figures 17A-B). However, large hydrophobic amino acids such as valine (V) in V7086 and isoleucine (I) in V3795 retain high discrimination and reduced off-targets. Aromatic amino acids such as histidine (H) in V7085 and polar uncharged amino acids such as serine (S) in V7088 also support a phenotype similar to OMNI-50 V3795.

[0260] For position 545: substitution with amino acids such as phenylalanine (F) in V7093 and histidine (H) in V3795 retains high discrimination and low off-target activity similar to variant V3795 (Figures 17A-B and 18A-B). Alanine (A) is a small hydrophobic residue and is used as a substitute in V7094, which also showed a similar phenotype to V3795 (Figures 17B and 18B).

[0261] Example 4 Materials and Methods HTP protein expression and purification Wild-type OMNI-50 nuclease and its variants were cultured in autoinduction TB medium at 37°C, 350 rpm agitation for 3.5 h, followed by transfer to 18°C ​​for 17–20 h. Cells were harvested by centrifugation at 4000×g and stored at -80°C. OMNI-50 variants and WT OMNI-50 cell pellets were thawed and incubated in lysis buffer for 30 min. Crude lysates were clarified by centrifugation at 4000×g for 1 h at 4°C. The clarified protein lysates were incubated with Sepharose 6 Ni-NTA resin (Cytiva). Protein-bound Ni-NTA resin was loaded into a 96-well filter well plate and washed with buffer (HEPES 20 mM, NaCl 0.6 M, imidazole 60 mM) to remove contaminants. Proteins were eluted from the resin using a high concentration imidazole buffer (HEPES 20 mM, NaCl 0.6 M, imidazole 0.4 M). Eluted proteins were desalted using a 96-well filter plate containing 1800 μL of G-25 Sephadex resin equilibrated with storage buffer. Desalted proteins were then concentrated (Amicon-ultra 0.5 ml 50 kDa, Millipore) and sterile filtered (Ultrafree-MC 0.22 μm PVDF filter). Purified variants were then stored at −80° C. and analyzed for concentration, purity and in vitro activity before transfection into cells.

[0262] RNP electroporation of LCL cells The RNP mixture was prepared by mixing 124 pmol of sgRNA and 105 pmol of nuclease. LCL cells were centrifuged at 300 × g for 5 min at room temperature and washed with PBS. The pellet was resuspended in an appropriate volume of Lonza Nucleofection SG electroporation solution and transferred to the RNP mixture. Electroporation was performed using a 4D-Nucleofector device (Lonza Bioscience). Pre-warmed BLCL medium was added to the cuvette immediately after electroporation. Cells were incubated for 2-4 days (37 °C, 5% CO2).

[0263] NGS analysis At 72 hours, cells were harvested and their genomic DNA content was used in PCR reactions that amplified the corresponding putative genomic targets. The amplicons were subjected to next-generation sequencing (NGS), and the resulting sequences were subsequently used to calculate the percentage of editing events at each target site. Short insertions or deletions (indels) around the cleavage site are a typical result of DNA repair after DNA cleavage by nucleases. Thus, the calculation of percent editing was estimated from the percentage of indel-containing sequences within each amplicon.

[0264] [Table 12]

[0265] [Table 13]

[0266] [Table 14]

[0267] [Table 15-1]

[0268] [Table 15-2]

[0269] [Table 16-1]

[0270] [Table 16-2]

[0271]

Table 17-1

[0272]

Table 17-2

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Claims

1. A non-naturally occurring OMNI-50 nuclease variant having a wild-type OMNI-50 protein sequence (SEQ ID NO: 1) containing an amino acid substitution at at least one of the following positions: R478, Y545, R61, Y437, A493, G606, K688, L690, E695, L718, R788, Q803, L805, L844, V981, K965, and K1036.

2. 2. The OMNI-50 nuclease variant of claim 1, comprising an amino acid substitution at position R478 and / or Y545, preferably at both positions R478 and Y545.

3. 3. The OMNI-50 nuclease variant of claim 1, comprising an amino acid substitution at each of positions R478 and Y545.

4. the amino acid substitution at position R478 is any one of R478D, R478E, R478S, R478T, R478N, R478Q, R478G, R478P, R478C, R478A, R478V, R478I, R478L, R478M, R478F, R478Y, or R478W, preferably any one of R478V, R478H, R478L, R478M, R478P, R478F, R478W, R478Y, R478S, R478C, R478T, R478N, or R478Q; and / or the amino acid substitution is at position R478 and the amino acid substituting the arginine is an amino acid with a negatively charged or uncharged R group; and / or the amino acid substitution is at position R478 and the amino acid substituting the arginine is a polar amino acid or a non-polar amino acid; and / or The OMNI-50 nuclease variant of claim 1 or 2, wherein the amino acid substitution is at position R478 and the amino acid substituting for arginine is a non-polar amino acid.

5. 3. The OMNI-50 nuclease variant of claim 1, wherein the amino acid substitution at position Y545 is any one of Y545D, Y545E, Y545S, Y545T, Y545N, Y545Q, Y545G, Y545P, Y545C, Y545A, Y545V, Y545I, Y545L, Y545M, Y545F, Y545R, Y545K, Y545H, or Y545W, preferably any one of Y545W, Y545F, Y545H, Y545V, or Y545G.

6. the amino acid substitution is at position Y545 and the amino acid substituting the tyrosine is an amino acid with a negatively charged R group or a positively charged R group; or 3. The OMNI-50 nuclease variant of claim 1, wherein the amino acid substitution is at position Y545 and the amino acid substituting the tyrosine is a non-polar amino acid.

7. 3. The OMNI-50 nuclease variant of claim 1, wherein the amino acid substitution is at position Y545, and the amino acid substituting the tyrosine lacks a phenyl ring or is phenylalanine. (A) the amino acid substitutions are R478I and Y545H; (B) the amino acid substitution is any one of R478I, Y545H, Q803V, L805I, R61A, Y437W, R788K, L844N, V981M, G606P, L690V, E695Q, R478T, A493G, K688E, L718C, K965V, and K1036V; (C) the amino acid substitutions are at positions R478, Y545, Q803, and L805, respectively, and optionally the amino acid substitutions are R478I, Y545H, Q803V, and L805I; (D) the amino acid substitutions are at each of positions R61, Y437, R788, L844, and V981, and optionally the amino acid substitutions are R61A, Y437W, R788K, L844N, and V981M; (E) the amino acid substitutions are at each of positions G606, L690, and E695, and optionally the amino acid substitutions are G606P, L690V, and E695Q; (F) the amino acid substitutions are at each of positions R478, A493, K688, L718, K965, and K1036, and optionally the amino acid substitutions are R478T, A493G, K688E, L718C, K965V, and K1036V; (G) the amino acid substitution is at position Q803, and optionally the amino acid substitution is Q803V; (H) the amino acid substitution is at position Y545, and optionally the amino acid substitution is Y545H; (I) the amino acid substitution is at position L805, and optionally the amino acid substitution is L805I; (J) the amino acid substitutions are at positions R478, Y545, and Q803, respectively, and optionally the amino acid substitutions are R478I, Y545H, and Q803V; (K) the amino acid substitutions are at positions R478, Y545, and L805, respectively, and optionally the amino acid substitutions are R478I, Y545H, and L805I; (L) the amino acid substitutions are at positions R478, Q803, and L805, respectively, and optionally the amino acid substitutions are R478I, Q803V, and L805I; (M) the amino acid substitutions are at positions Y545, Q803, and L805, respectively, and optionally the amino acid substitutions are R478I, Y545H, and L805I; or (N) The OMNI-50 nuclease variant according to claim 1, wherein the OMNI-50 nuclease variant has an amino acid sequence represented by a sequence number selected from the group consisting of sequence numbers 2 to 5, 63 to 70, and 89 to 97.

9. has at least 80% sequence identity to the wild-type OMNI-50 protein sequence (SEQ ID NO: 1); and / or and / or further comprising a nuclear localization sequence (NLS); 9. The OMNI-50 nuclease variant of any one of claims 1, 2 or 8, wherein the variant exhibits increased specificity for a DNA target site when complexed with a guide RNA molecule that directs the variant to the DNA target site compared to a wild-type OMNI-50 nuclease complexed with the guide RNA molecule.

10. 10. A CRISPR system comprising the OMNI-50 nuclease variant of any one of claims 1, 2 or 8 complexed with a guide RNA molecule that targets a DNA target site, wherein the CRISPR system has reduced off-target editing activity compared to a wild-type CRISPR system comprising a wild-type OMNI-50 nuclease protein and the guide RNA molecule.

11. A method of gene editing with reduced off-target editing activity, comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-50 nuclease variant according to any one of claims 1, 2 or 8, Optionally, the active CRISPR system has reduced off-target editing activity compared to a wild-type CRISPR system comprising a wild-type OMNI-50 nuclease protein; and / or the gene editing is performed in a eukaryotic or prokaryotic cell, optionally wherein the eukaryotic cell is a plant cell or a mammalian cell, optionally wherein the mammalian cell is a human cell; and / or The DNA target site is located within or near a pathogenic allele of a gene, and optionally the DNA target site is selected from the group consisting of ELANE, CXCR4, EMX, RyR2, KNCQ1, KCNH2, SCN5a, GBA1, GBA2, rhodopsin, GUCY2D, IMPDH1, FGA, BEST1, PRPH2, KRT5, KRT14, ApoA1, STAT3, STAT1, ADA2, R located within a gene selected from the group consisting of PS19, SBDS, GATA2, RPE65, LDLR, ANGPTL3, B2M, TRAC, TCF4, TGFBi, PAX6, C3, LRRK2, SARM1, SAMD9, SAMD9L, HAVCR2, CD3E, APLP2, CISH, TIGIT, TNNT2, TNN, MYH7, RPP30 and HLA-E; and / or The DNA target is repaired by an exogenous donor molecule; and / or the off-target editing activity is reduced by at least 2-fold, 10-fold, 10-fold, 10-fold, 10-fold, 10-fold, 10-fold, or 10-fold; method.

12. A modified cell obtained by a gene editing method having reduced off-target editing activity, the method comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-50 nuclease variant according to any one of claims 1, 2 or 8; Optionally, the cells are capable of engraftment, and / or the cells are capable of giving rise to progeny cells after engraftment; and / or the cells are capable of giving rise to progeny cells after autologous engraftment; and / or the cells are capable of giving rise to progeny cells for at least 12 months or at least 24 months after engraftment; and / or The modified cells, wherein the cells are selected from the group consisting of hematopoietic stem cells, progenitor cells, CD34+ hematopoietic stem cells, bone marrow cells, and peripheral mononuclear cells.

13. A composition comprising modified cells obtained by a gene editing method with reduced off-target editing activity and a pharmaceutically acceptable carrier, said method comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-50 nuclease variant described in any one of claims 1, 2 or 8.

14. An in vitro or ex vivo method for producing a composition, comprising mixing modified cells obtained by a gene editing method with reduced off-target editing activity with a pharmaceutically acceptable carrier, said method comprising contacting a DNA target site with an active CRISPR system comprising an OMNI-50 nuclease variant described in any one of claims 1, 2 or 8.

15. A polynucleotide molecule encoding the OMNI-50 nuclease variant of any one of claims 1, 2 or 8.

Images