Crispr-related methods and compositions targeting angiopoietin like 3 (angptl3)

Abstract

The present disclosure relates to CRISPR-related genome editing systems and components for targeting, editing and / or modulating the expression of a target nucleic acid sequence of interest, for example, an ANGPTL3 target nucleic acid sequence encoding the ANGPTL3 protein. The present disclosure is also directed to methods and applications thereof in connection with the treatment and / or management of metabolic disease.

Description

Attorney Docket No.: 084177.0330CRISPR-RELATED METHODS AND COMPOSITIONS TARGETINGANGIOPOIETIN LIKE 3 (ANGPTL3)CROSS-REFERENCE TO RELATED APPLICATIONSThe present application claims priority from U.S. Provisional Application No. 63 / 718,364, filed November 8, 2024, and U.S. Provisional Application No. 63 / 752,847, filed February 2, 2025, the entire contents of which are incorporated herein by reference for all purposes.SEQUENCE LISTINGThe present specification makes reference to a Sequence Listing (submitted as an .xml file named 084177.0330_ST26. xml). The XML file was generated on November 7, 2025, and is 740,000 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.FIELDThe present disclosure is directed to CRISPR-r elated genome editing systems and components for targeting, editing and / or modulating the expression of an angiopoietin like 3 (ANGPTL3') target nucleic acid sequence of interest, e.g., an ANGPTL3 target nucleic acid sequence encoding the ANGPTL3 protein. The present disclosure is also directed to methods and applications thereof in connection with the treatment and / or management of metabolic disease.BACKGROUNDCRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complementary to the viral genome, mediates targeting of a Cas protein to a target sequence in the viral genome. The Cas protein, in turn, cleaves and thereby silences the viral target. Naturally occurring CRISPR systems are organized evolutionarily into two classes and five types. Cas 12a (also known as Cpfl) represents a Class 2, Type V CRISPR / Cas system that has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double strand breaks (DSBs) into the targetedAttorney Docket No.: 084177.0330 sequence allows for knocking out a gene through the formation of an indel through endogenous DNA repair mechanisms, for example non-homologous end-joining (NHEJ). The introduction of site-specific DSBs into the targeted sequence can also facilitate gene conversion or gene correction through the incorporation of an exogenous or endogenous homologous sequence with a repair template, for example homology-directed repair (HDR).Hyperlipidemia, characterized by high levels of Lp(a) (>150 mg / dL) has been associated with the formation of atherosclerotic plaques in the blood vessels that lead to major adverse cardiovascular events (MACE) including, heart attack, stroke, aortic stenosis, peripheral vascular disease, and renal dysfunction. An Lp(a) particle is formed when a single LDL particle covalently attaches to a single Apo(a) protein. The current standard of care (SOC) for hyperlipidemia is lipoprotein apheresis, mitigation of other MACE risk factors, and drugs (e.g., statins, PCSK9 inhibitors), all of which yield only moderate to low reductions in Apo(a). Therapies employing antisense oligonucleotides (ASO) and small interfering RNA (siRNA) have met with many challenges, including tolerability, dosing inconsistencies, area under-the-curve (AUC) reductions and poor medication adherence by the patient. There is therefore an unmet need in the art for the treatment and / or management of metabolic disease, e.g., alternate approaches that address hyperlipidemia to reduce the risk of MACE in an individual or treat an individual who has suffered from a MACE.The human ANGPTL3 gene is located on Chromosome 1. The ANGPTL3 transcript ENST00000371129.4 comprises seven exons that encode the ANGPTL3 protein. Expression of ANGPTL3 impedes lipoprotein lipase (LPL) activity, thereby increasing V / LDL levels, and thereby increasing Lp(a) levels. In contrast, ANGPTL3 deficiencies are associated with reduction of all plasma lipoproteins. Heterozygous carriers of ANGPTL3 loss of function variants have reduced plasma levels of total cholesterol and triglycerides and are at lower risk of developing atherosclerotic cardiovascular disease, as compared to non-carriers. In view of such outcomes, treatment with an ANGPTL3 -blocking antibody (evinacumab) has been evaluated and found to reduce both triglycerides and LDL-C.SUMMARYThe presently disclosed subject matter is directed, in certain embodiments, to a genome editing system comprising (a) a gRNA molecule comprising a targeting domain that targets a sequence of an ANGPT 3 gene, and (b) an RNA-guided nuclease, or a nucleic acid (e.g., an RNA) encoding the RNA-guided nuclease thereof.Attorney Docket No.: 084177.0330In certain embodiments, the target sequence of the ANGPTL3 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-34. In certain embodiments, the target sequence of the ANGPTL3 gene comprises the nucleotide sequence set forth in SEQ ID NO: 26, or SEQ ID NO: 31. In certain embodiments, the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45. In certain embodiments, the targeting domain comprises the nucleotide sequence set forth in SEQ ID NO: 37, or SEQ ID NO: 42.In certain embodiments, the RNA-guided nuclease is selected from the group consisting of Cas9 (e.g., SpCas9, SaCas9, (KKH) SaCas9, eSpCas9, Cas9-HF1, HypaCas9, dCas9-Fokl, Sniper-Cas9, xCas9, evoCas9, SpCas9-NG, VRQR, VRER, NmeCas9, CjCas9), Casl2a (also known as Cpfl; e.g., AsCasl2a, LbCasl2a), Casl2b (e.g., AaCasl2b, BhCasl2b, BhCasl2bV4), Cast 2c (e.g., Casl2cl, Casl2c2), Casl2h (e.g., Casl2hl), Casl2i e.g., Casl2il), CasX, CasY, and Cas . In certain embodiments, the RNA-guided nuclease is a Casl2a protein.In certain embodiments, the RNA-guided nuclease is a modified Casl2a protein. In certain embodiments, the modified Casl2a protein is an activity enhanced Casl2a protein. In certain embodiments, the RNA-guided nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 66-70 and 72-78. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease is an RNA. In certain embodiments, the RNA encoding the RNA-guided nuclease comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 80-84. In certain embodiments, the RNA-guided nuclease is AsCasl2a, for example an AsCasl2a comprising the amino acid sequence set forth in SEQ ID NO: 70.In certain embodiments, the gRNA molecule further comprises a Casl2a stem loop.In certain embodiments, the gRNA molecule further comprises a nucleotide extension. In certain embodiments, the nucleotide extension is a 5’ extension, a 3’ extension, or a combination thereof. In certain embodiments, the nucleotide extension comprises one or more RNA bases, one or more DNA bases, or a combination thereof. In certain embodiments, the gRNA molecule contains one or more modifications. In certain embodiments, the one or more modifications is selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage and a combination thereof. In certain embodiments, the one or more modifications comprises the 5’ inverted thymidine (idT) modification and the 3’ idT modification. In certain embodiments, the one or moreAttorney Docket No.: 084177.0330 modifications comprises the 2’ fluoro modification. In certain embodiments, the one or more modifications comprises one or more 2’ fluoro modifications, and each of the 2’ fluoro modifications modifies a nucleotide internal to the gRNA molecule.In certain embodiments, the nucleotide extension is a 5’ extension comprising or consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1- 23. In certain embodiments, the nucleotide extension is a 5’ extension comprising the nucleotide sequence set forth in SEQ ID NO: 7.In certain embodiments, the gRNA molecule comprises a DNA / RNA oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 35-45. In certain embodiments, the gRNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 37, SEQ ID NO: 48, or SEQ ID NO: 58. In certain embodiments, the gRNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 42, SEQ ID NO: 53, or SEQ ID NO: 103. In certain embodiments, the gRNA molecule comprises the sequence set forth in SEQ ID NO: 91. In certain embodiments, the gRNA molecule comprises the sequence set forth in SEQ ID NO: 92.The presently disclosed subject matter is also directed to a ribonucleoprotein (RNP) complex comprising the genome editing systems disclosed herein.The presently disclosed subject matter is also directed to a delivery system for delivering the genome editing systems disclosed herein.In certain embodiments, the delivery system comprises a DNA sequence encoding the gRNA molecule. In certain embodiments, the delivery system comprises a DNA sequence encoding the RNA-guided nuclease. In certain embodiments, the delivery system comprises an RNA sequence encoding the gRNA molecule. In certain embodiments, the delivery system comprises an RNA sequence encoding the RNA-guided nuclease.In certain embodiments, the gRNA is conjugated to one or more N- acetylgalactosamine (GalNAc) or GalNAc derivatives. In certain embodiments, the GalNAc or GalNAc derivatives is attached to the gRNA via a linker.In certain embodiments, the conjugate targets the gRNA to liver cells, e.g., hepatocytes.In certain embodiments, the delivery system comprises a lipid nanoparticle (LNP).In certain embodiments, the LNP comprises ionizable lipids, polyethylene glycol (PEG) lipids, helper lipids, sterols, or combinations thereof.In certain embodiments, the ionizable lipid is selected from the group consisting of ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), andAttorney Docket No.: 084177.03308-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102).In certain embodiments, the PEG lipid is selected from the group consisting of a dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG), a distearoyl-sn- glycerol-3 -methoxypolyethylene glycol (DSG-PEG), and a distearoyl-sn-glycero-3- phosphoethanolamine-N-methoxypolyethylene glycol (DSPE-PEG).In certain embodiments, the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC) and a l,2-dioleoyl-sn-glycero-3- phosphoethanolamine. (DOPE).In certain embodiments, the sterol is selected from the group consisting of cholesterol and sitosterol.In certain embodiments, the LNP comprises ((4- Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315), DMG- PEG, DSPC, and cholesterol.In certain embodiments, the LNP comprises a targeting modification. In certain embodiments, the targeting modification is N-acetylgalactosamine (GalNAc) or GalNAc derivatives. In certain embodiments, the GalNAc or GalNAc derivatives is attached to the LNP via a linker.In certain embodiments, the GalNAc or GalNAc derivatives targets the LNP to liver cells, e.g., hepatocytes.The presently disclosed subject matter is also directed to a method of editing an ANGPTL3 gene in a target cell comprising contacting the target cell with a genome editing system, an RNP complex, and / or a delivery system disclosed herein.In certain embodiments, the target cell is a cell involved in metabolism. In certain embodiments, the target cell is a hepatocyte.In certain embodiments, the target cell is an in vivo target cell.The presently disclosed subject matter is also directed to a method of treating a disease or disorder comprising administering to a subject in need thereof, a genome editing system, an RNP complex, and / or a delivery system disclosed herein.In certain embodiments, the disease or disorder is a hyperlipidemia.In certain embodiments, the subject is suffering from an atherosclerotic cardiovascular disease (ASCVD).Attorney Docket No.: 084177.0330In certain embodiments, the subject is identified to be at a high risk for a major adverse cardiovascular event (MACE) or has suffered from a MACE.In certain embodiments, the subject has an Apo(a) or Lp(a) level (e.g., a serum or plasma Apo(a) or Lp(a) level) >150 mg / dL.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces an ANGPTL3 expression level in a cell (e.g., a hepatocyte) from the subject by at least about 50% to at least about 95% relative to asxANGPTL3 expression in a cell from the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system reduces an ANGPTL3 expression level in a cell (e.g., a hepatocyte) from the subject by at least 90% relative to an ANGPTL3 expression in a cell from the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces an ANGPTL3 level in the subject, or in a cell, tissue, or fluid in the subject, by at least about 50% to at least about 95% relative to the ANGPTL3 level prior to administration, or relative to the ANGPTL3 level in a control subject, cell, tissue, or fluid. For example, _in certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma ANGPTL3 level in the subject by at least about 50% to at least about 95% relative to the serum or plasma ANGPTL3 level in the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma ANGPTL3 level in the subject by at least 90% relative to the serum or plasma ANGPTL3 level in the subject prior to administration.In certain embodiments, reducing the ANGPTL3 expression level in a cell from the subject or reducing the serum or plasma ANGPTL3 level in the subject reduces the serum or plasma level of LDL and / or Lp(a).In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma Lp(a) level in the subject by at least about 50% to at least about 95% relative to the serum or plasma Lp(a) level in the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma Lp(a) level inAttorney Docket No.: 084177.0330 the subject by at least 90% relative to the serum or plasma Lp(a) level in the subject prior to administration.The presently disclosed subject matter is also directed to a method of editing an ANGPTL3 gene in a target cell comprising contacting the target cell with the genome editing system, the RNP complex, and / or the delivery system disclosed herein, further comprising administering to the subject a standard of care (SOC) for hyperlipidemia.In certain embodiments, the SOC is Apo(a) apheresis and / or at least one pharmacological agent. In certain embodiments, the pharmacological agent is selected from the group consisting of a statin, a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, and LY3473329, or a combination thereof.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces an ANGPTL3 expression level in a cell (e.g., a hepatocyte) from the subject by at least about 50% to at least about 95% relative to an ANGPTL3 expression in a cell from the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system reduces an ANGPTL3 expression level in a cell (e.g., a hepatocyte) from the subject by at least 90% relative to an ANGPTL3 expression in a cell from the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces an ANGPTL3 level in the subject, or in a cell, tissue, or fluid in the subject, by at least about 50% to at least about 95% relative to the ANGPTL3 level prior to administration, or relative to the ANGPTL3 level in a control subject, cell, tissue, or fluid. For example, _in certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma ANGPTL3 level in the subject by at least about 50% to at least about 95% relative to the serum or plasma ANGPTL3 level in the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma ANGPTL3 level in the subject by at least 90% relative to the serum or plasma ANGPTL3 level in the subject prior to administration.In certain embodiments, reducing the ANGPTL3 expression level in a cell from the subject or reducing the serum or plasma ANGPTL3 level in the subject reduces the serum or plasma level of LDL and / or Lp(a).Attorney Docket No.: 084177.0330In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma Lp(a) level in the subject by at least about 50% to at least about 95% relative to the serum or plasma Lp(a) level in the subject prior to administration.In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system to the subject reduces the serum or plasma Lp(a) level in the subject by at least 90% relative to the serum or plasma Lp(a) level in the subject prior to administration.The presently disclosed subject matter is also directed to a method of treating an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a formulation comprising a lipid nanoparticle (LNP), an mRNA encoding a Casl2a nuclease, and a gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene of the subject, wherein the mRNA and the gRNA are encapsulated within the LNP.In certain embodiments, the target sequence of the ANGPTL3 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-34.In certain embodiments, the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.In certain embodiments, the gRNA molecule comprises the sequence set forth in SEQ ID NO: 35.In certain embodiments, the gRNA molecule comprises the sequence and modifications set forth in SEQ ID NO: 91.The presently disclosed subject matter is also directed to a gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene of the subject, wherein the gRNA molecule comprises one or more modifications selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage and a combination thereof.In certain embodiments, the one or more modifications are on nucleotides positioned outside of the targeting domain. In certain embodiments, the gRNA comprises a 5’ DNA extension. In certain embodiments, the 5’ DNA extension comprises or consists of the sequence set forth in SEQ ID NO: 7. In certain embodiments, the gRNA molecule comprises the sequence and modifications set forth in SEQ ID NO: 91.In certain embodiments, the gRNA molecule comprises the following sequence:Attorney Docket No.: 084177.0330 / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2F A / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2F A / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 95); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.In certain embodiments, the gRNA molecule comprises the following sequence: mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2F U / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 96); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45, and wherein the 3 ’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArUrUrUrCrUrArCrUrCr UrUrGrUrArGrArUx / 3InvdT / (SEQ ID NO: 97); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.In certain embodiments, the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT (SEQ ID NO: 98); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.In certain embodiments, the gRNA molecule comprises the following sequence: mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 99); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45, and wherein the 3’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.BRIEF DESCRIPTION OF THE DRAWINGSThe accompanying drawings are intended to provide illustrative, and schematic rather than comprehensive, examples of certain aspects and embodiments of the present disclosure. The drawings are not intended to be limiting or binding to any particular theory or model. Without limiting the foregoing, nucleic acids and polypeptides can be depicted asAttorney Docket No.: 084177.0330 linear sequences, or as schematic two- or three-dimensional structures; these depictions are intended to be illustrative rather than limiting or binding to any particular model or theory regarding their structure.FIGs. 1A-1E depict exemplary results of lipid nanoparticle (LNP)-mediated ANGPTL3 editing. FIGs. 1A-1B depict ANGPTL3 editing in primary human hepatocytes (PHHs; FIG. 1A) and HepG2 cells (FIG. IB) treated with LNPs containing AsCasl2a mRNA and VG 7Z3-targeting gRNA of SEQ ID NO: 91 (ANGPTL3 496) or SEQ ID NO: 92 (ANGPTL3 497). FIGs. 1C-1E depict ANGPTL3 gene editing and ANGPTL3 protein knockdown measured in PHHs from multiple donors treated with LNPs containing AsCasl2a mRNA and ANGPTL3 -targeting gRNA of SEQ ID NO: 91FIG. 2 depicts exemplary results for in vivo ANGPTL3 editing using LNPs in the liver of wild type C57B1 / 6 (WT) mice. A surrogate guide targeting mouse ANGPTL3 was formulated into LNPs with engineered AsCasl2a mRNA using an ALC-0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k), and administered at the specified dose to mice by IV tail vein injection.FIGs. 3A-3B illustrate exemplary results for in vivo mouse ANGPTL3 protein knockdown in WT mice treated with LNPs encapsulating AsCasl2a mRNA plus surrogate gRNA targeting mouse ANGPTL3. FIG. 3A depicts the serum concentration of mouse ANGPTL3 treated WT mice at different LNP doses. FIG. 3B depicts the data of FIG. 3A as percent knockdown normalized to the vehicle control.FIG. 4 depicts exemplary results for in vivo ANGPTL3 editing using LNPs in the liver of humanized ANGPTL3 transgenic mice. A guide targeting human ANGPTL3 was formulated into LNPs with engineered AsCasl2a mRNA using an ALC-0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k), and administered at the specified dose to mice by IV tail vein injection.FIGs. 5A-5B depict exemplary results for human ANGPTL3 detection in humanized ANGPTL3 transgenic mice. FIG. 5A depicts human and mouse ANGPTL3 protein in plasma of WT (+ / +) and humanized ANGPTL3 transgenic mice (H / H). FIG. 5B depicts human ANGPTL3 protein in plasma of pre-dose humanized ANGPTL3 transgenic mice, quantified by a low limit of quantification (LoQ) ELISA.FIGs. 6A-6B depict exemplary results for in vivo ANGPTL3 editing using LNPs in the liver of humanized ANGPTL3 transgenic mice. A guide targeting human ANGPTL3 was formulated into LNPs with engineered AsCasl2a mRNA using an ALC-0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k),Attorney Docket No.: 084177.0330 and administered at the specified dose to mice by IV tail vein injection. FIG. 6A depicts indel generation in human ANGPTL3 and knockdown of human ANGPTL3 protein in liver tissue following LNP administration. FIG. 6B depicts human ANGPTL3 protein concentration in liver tissue of pre-dose and terminal mice.FIG. 7 depicts the results of an RNP nucleofection screen for AsCasl2a gRNAs targeting ANGPTL3 in HepG2 cells. Briefly, AsCasl2a protein was complexed with gRNA at a 1 :2 molar ratio (RNP complex) and delivered to HepG2 cells via nucleofection. Three days after nucleofection, gDNA was extracted from both cell types and the target sites were amplified by PCR and evaluated by next generation sequencing (NGS).FIGs. 8A-8B depict exemplary results for in vivo ANGPTL3 editing using LNPs in the liver of nonhuman primates (“NHPs”). FIG. 8A depicts indel generation frequency at ANGPTL3 in liver tissue and knockdown of ANGPTL3 protein in serum following LNP administration. FIG. 8B depicts knockdown of ANGPTL3 protein in serum following LNP administration. The maximum level of ANGPTL3 reduction over the 4-week study is shown (FIGs 8A-8B)DETAILED DESCRIPTIONThe presently disclosed subject matter relates to RNA-guided nuclease-related, e.g., CRISPR / Cas-related, genome editing systems, compositions, delivery vehicles, and methods for targeting an ANGPTL3 nucleic acid sequence, editing a target ANGPTL3 nucleic acid sequence, or modulating expression of a target ANGPTL3 nucleic acid sequence, and applications thereof. The presently disclosed subject matter also provides genome editing systems, compositions, vectors, and methods for editing cells using CRISPR / Cas-related components to edit a target ANGPTL3 gene. The presently disclosed subject matter also provides lipid nanoparticle (LNP) facilitated delivery of genome editing systems and methods for editing cells using CRISPR / Cas-related components delivered via LNP to edit a target ANGPTL3 gene. Some aspects of the present disclosure provide pharmaceutical compositions, cells, cell populations, methods, strategies, and treatment modalities that are useful in the context of treating and / or managing a metabolic disease, e.g., hyperlipidemia or hypercholesterolemia.The subject matter of the present disclosure is described with reference to the Figures. It should be understood that numerous specific details, relationships, and methods are set forth in this Detailed Description, Examples, and accompanying Figures to provide a more complete understanding of the subject matter disclosed herein. For purposes ofAttorney Docket No.: 084177.0330 clarity of disclosure and not by way of limitation, the Detailed Description is divided into the following subsections:1. Definitions and Abbreviations2. Genome editing systems3. Guide RNA (gRNA) molecules4. Guide RNA design5. RNA-guided nucleases6. Genome editing strategies7. Implementation of genome editing systems: delivery, formulations, and routes of administration8. Exemplary Embodiments9. Examples1. Definitions and AbbreviationsUnless otherwise specified, each of the following terms has the meaning associated with it in this section.The indefinite articles “a” and “an” refer to at least one of the associated noun and are used interchangeably with the terms “at least one” and “one or more.” For example, “a module” means at least one module, or one or more modules.The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.The conjunctions “or” and “and / or” are used interchangeably as non-exclusive disjunctions.The phrase “consisting essentially of’ means that the species recited are the predominant species, but that other species can be present in trace amounts or amounts that do not affect structure, function or behavior of the subject composition. For instance, a composition that consists essentially of a particular species will generally comprise 90%, 95%, 96%, or more of that species.Attorney Docket No.: 084177.0330“Domain” is used to describe a segment of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.An “indel” is an insertion and / or deletion in a nucleic acid sequence. An indel can be the product of the repair of a DNA double strand break, such as a double strand break formed by a genome editing system of the present disclosure. An indel is most commonly formed when a break is repaired by an “error prone” repair pathway such as the NHEJ pathway described below.“Gene conversion” refers to the alteration of a DNA sequence by incorporation of an endogenous homologous sequence (e.g., a homologous sequence within a gene array). “Gene correction” refers to the alteration of a DNA sequence by incorporation of an exogenous homologous sequence, such as an exogenous single-or double stranded donor template DNA. Gene conversion and gene correction are products of the repair of DNA double-strand breaks by HDR pathways such as those described below.Indels, gene conversion, gene correction, and other genome editing outcomes are typically assessed by sequencing (most commonly by “next-gen” or “sequencing-by- synthesis” methods, though Sanger sequencing can still be used) and are quantified by the relative frequency of numerical changes (e.g., ±1, ±2 or more bases) at a site of interest among all sequencing reads. DNA samples for sequencing can be prepared by a variety of methods known in the art and can involve the amplification of sites of interest by polymerase chain reaction (PCR), the capture of DNA ends generated by double strand breaks, as in the GUIDE-seq process described in Tsai et al. (Nat. Biotechnol. 34(5): 483 (2016), incorporated by reference herein) or by other means well known in the art. Genome editing outcomes can also be assessed by in situ hybridization methods such as the FiberComb™ system commercialized by Genomic Vision (Bagneux, France), and by any other suitable methods known in the art.“Alt-HDR,” “alternative homology-directed repair,” or “alternative HDR” are used interchangeably to refer to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g., a sister chromatid, or an exogenous nucleic acid, e.g., a template nucleic acid). Alt-HDR is distinct from canonical HDR in that the process utilizes different pathways from canonical HDR, and can be inhibited by the canonical HDR mediators, RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of a single-stranded or nicked homologous nucleic acid template, whereas canonical HDR generally involves a double-stranded homologous template.Attorney Docket No.: 084177.0330“Canonical HDR,” “canonical homology-directed repair” or “cHDR” refer to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g., a sister chromatid, or an exogenous nucleic acid, e.g., a template nucleic acid). Canonical HDR typically acts when there has been significant resection at the double strand break, forming at least one single stranded portion of DNA. In a normal cell, cHDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. The process requires RAD51 and BRCA2, and the homologous nucleic acid is typically double stranded.Unless indicated otherwise, the term “HDR” as used herein encompasses both canonical HDR and alt-HDR.“Non-homologous end joining” or “NHEJ” refers to ligation mediated repair and / or non-template mediated repair including canonical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), which in turn includes microhomology-mediated end joining (MMEJ), singlestrand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).“Replacement” or “replaced,” when used with reference to a modification of a molecule (e.g., a nucleic acid or protein), does not require a process limitation but merely indicates that the replacement entity is present.“Knock-out” or “knockout” refers to an inactivating mutation in a target gene, wherein the product of the target gene comprises a loss of function.“Gene product” refers to biochemical products resulting from the expression of the gene and includes the RNA or protein that is encoded by the gene.“On-target site” refers to the exact genomic sequence or locus within the gene of interest for which the guide RNA was designed to target. “Off-target site” refers to a genomic sequence or locus that is not within the gene of interest and is found to be edited by the RNA guided nucleases.As used herein, the term “hairpin” or “hairpin region” refers to a nucleic acid secondary structure comprising a stem-loop structure. A guide-RNA of the present disclosure may comprise one or more hairpins, or hairpin regions, that are not part of its targeting domain.As used herein, the term “pseudoknot” refers to a nucleic acid secondary structure comprising at least two stem-loop structures in which half of one stem is intercalatedAttorney Docket No.: 084177.0330 between the two halves of another stem. Exemplary pseudoknot sequences are bolded in the two gRNA sequences shown below;TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrCrUrGrArGrArUrGr CrCrArGrCrUrGrUrCrCrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU [SEQ ID NO: 100]; mU*rArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrCrUrGrArGrArUrG rCrCrArGrCrUrGrUrCrCrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrA*mU [SEQ ID NO: 101],As used herein, the term “locked nucleic acid” or “LNA” refers to a modified RNA nucleotide in which the ribose moiety is modified with a bridge connecting the 2' oxygen and 4' carbon. An exemplary LNA is shown below, where the “+” indicates a locked nucleotide; / 5IdT / +A+T+G+T+G+T+T+T+T+T+G+T+C+A+A+A+A+G+A+C+C+T+T+T+T rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArArCrCrUrCrCrUrGrGrCrCrArG rArUrUrCrUrC / 3InvdT / [SEQ ID NO: 102],“Subject” means a human or non-human animal. A human subject can be any age e.g., an infant, child, young adult, or adult), and can suffer from a disease, or can be in need of alteration of a gene. Alternatively, the subject can be an animal, which term includes, but is not limited to, mammals, birds, fish, reptiles, amphibians, and more particularly non- human primates (NHP), rodents (such as mice, rats, hamsters, etc.), rabbits, guinea pigs, dogs, cats, and so on. In certain embodiments of this disclosure, the subject is livestock, e.g., a cow, a horse, a sheep, or a goat. In certain embodiments, the subject is poultry.As used herein a “therapeutically effective amount” refers to the amount of a cell and / or composition that when administered to a subject for treating a disease, is sufficient to beneficially affect such treatment for the disease.“Treat,” “treating,” and “treatment” mean the treatment of a disease in a subject (e.g., a human subject), including one or more of inhibiting the disease, i.e., arresting or preventing its development or progression; relieving the disease, i.e., causing regression of the disease state; relieving one or more symptoms of the disease; and curing the disease.“Prevent,” “preventing,” and “prevention” refer to the prevention of a disease in a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease.Attorney Docket No.: 084177.0330A “Kit” refers to any collection of two or more components that together constitute a functional unit that can be employed for a specific purpose. By way of illustration (and not limitation), one kit according to this disclosure can include a guide RNA complexed or able to complex with an RNA-guided nuclease, and accompanied by (e.g., suspended in, or suspendable in) a pharmaceutically acceptable carrier. The kit can be used to introduce the complex into, for example, a cell or a subject, for the purpose of causing a desired genomic alteration in such cell or subject. The components of a kit can be packaged together, or they can be separately packaged. Kits according to this disclosure also optionally include directions for use (DFU) that describe the use of the kit e.g., according to a method of this disclosure. The DFU can be physically packaged with the kit, or it can be made available to a user of the kit, for instance by electronic means.The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA and mean any chain of two or more nucleotides. The polynucleotides, nucleotide sequences, nucleic acids etc. can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. They can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically carries genetic information, including, but not limited to, the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.Conventional IUPAC notation is used in nucleotide sequences presented herein, as shown in Table 1, below (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10; 13 (9): 3021-30, incorporated by reference herein). It should be noted, however, that “T” denotes “Thymine or Uracil” in those instances where a sequence can be encoded by either DNA or RNA, for example in a gRNA, for example in a gRNA targeting domain.Table 1: IUPAC nucleic acid notationAttorney Docket No.: 084177.0330The terms “protein,” “peptide” and “polypeptide” are used interchangeably to refer to a sequential chain of amino acids linked together via peptide bonds. The terms include individual proteins, groups or complexes of proteins that associate together, as well as fragments or portions, variants, derivatives and analogs of such proteins. Peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left, and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three-letter abbreviations can be used.The term “variant” refers to an entity such as a polypeptide, polynucleotide or small molecule that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity.As used herein, the term “promoter” refers to a region (z.e., a DNA sequence) of a genome that initiates the transcription of a gene.The term “endogenous,” as used herein in the context of nucleic acids (e.g., genes, protein-encoding genomic regions, promoters), refers to a native nucleic acid or protein in its natural location, e.g., within the genome of a cell. In contrast, the term “exogenous,” as used herein in the context of nucleic acids, e.g, expression constructs, cDNAs, indels, and nucleic acid vectors, refers to nucleic acids that have artificially been introduced into the genome of a cell using, for example, gene-editing or genetic engineering techniques, e.g, CRISPR-based editing techniques.The terms “RNA-guided nuclease” and “RNA-guided nuclease molecule” are used interchangeably herein. In some embodiments, the RNA-guided nuclease is an RNA-guidedAttorney Docket No.: 084177.0330DNA endonuclease enzyme. In some embodiments, the RNA-guided nuclease is a CRISPR nuclease. Non-limiting examples of RNA-guided nucleases are listed in Table 2 below, and the methods and compositions disclosed herein can use any combination of RNA-guided nucleases disclosed herein, or known to those of ordinary skill in the art. Those of ordinary skill in the art will be aware of additional nucleases and nuclease variants suitable for use in the context of the present disclosure, and it will be understood that the present disclosure is not limited in this respect.Table 2: Exemplary RNA-Guided NucleasesAttorney Docket No.: 084177.0330Additional suitable RNA-guided nucleases, e.g., Cas9 and Cast 2 nucleases, will be apparent to the skilled artisan in view of the present disclosure, and the disclosure is not limited by the exemplary suitable nucleases provided herein. In some embodiment, a suitable nuclease is a Cas9 or Casl2a (Cpfl) nuclease. In some embodiments, the disclosure also embraces nuclease variants, e.g., Cas9 or Casl2a nuclease variants. A nuclease variant refers to a nuclease comprising an amino acid sequence characterized by one or more amino acid substitutions, deletions, or additions as compared to the wild type (WT) amino acid sequence of the nuclease. Suitable nucleases and nuclease variants may also comprise purification tags (e.g., polyhistidine tags) and signaling peptides, e.g., comprising or consisting of a nuclear localization signal sequence (NLS). Some non-limiting examples of suitable nucleases and nuclease variants are described in more detail elsewhere herein, and also comprise those described in PCT application PCT / US2019 / 22374, filed March 14, 2019, and entitled “ Systems and Methods for the Treatment of Hemoglobinopathies,” the entire contents of which are incorporated herein by reference.In some embodiments, the RNA-guided nuclease is an Acidaminococcus sp. Casl2a (Cpfl) variant (also known as AsCasl2a or AsCpfl variant). Suitable Casl2a nuclease variants, including suitable AsCasl2a variants will be known or apparent to those of ordinary skill in the art based on the present disclosure, and include, but are not limited to, the AsCasl2a variants disclosed herein or otherwise known in the art. For example, in some embodiments, the RNA-guided nuclease is an Acidaminococcus sp. Casl2a RR variant (AsCasl2a-RR). In certain embodiments, the RNA-guided nuclease is a Casl2a RVR variant. These and other variants are described in PCT patent application PCT / US2017 / 028420 and Guo et al., Nat Biotechnol. 2017 Aug; 35(8): 789-792, each of which is incorporated by reference herein for all purposes. Additionally, or alternatively, suitable Casl2a variants include those having an M537R substitution, an H800A substitution, and / or an F870L substitution, or any combination thereof (numbering scheme according to AsCasl2a wild-type sequence).Attorney Docket No.: 084177.0330In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a DNA modifying enzyme for targeted nucleotide alteration, commonly referred to as a “base editor.” In certain embodiments, the base editor is a cytosine base editor. In certain embodiments, the base editor is an adenosine base editor.In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a DNA modifying enzyme fused to a reverse transcriptase for targeted nucleotide insertion, deletion, or substitution, commonly referred to as a “prime editor.”2. Genome editing systemsVarious genome editing systems known in the art can be used for the methods disclosed herein. Non-limiting examples of genome editing systems that can be used with the presently disclosed subject matter include, but are not limited to CRISPR systems, zinc- finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, meganuclease (MN) systems, MegaTAL systems, other targeted endonuclease systems, and other chimeric endonuclease systems.In certain embodiments, the genome editing system has RNA-guided DNA editing activity. In certain embodiments, the genome editing system includes at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex that is capable of associating with a specific nucleic acid sequence and optionally editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and / or a point mutation.Naturally occurring CRISPR systems are organized evolutionarily into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun; 9(6): 467-477 (Makarova), incorporated by reference herein), and while genome editing systems of the present disclosure can adapt components of any type or class of naturally occurring CRISPR system, the embodiments presented herein are generally adapted from Class 2, and type II or V CRISPR systems. Class 2 systems, which encompass types II and V, are characterized by relatively large, multidomain RNA-guided nuclease proteins (e.g., Cas9 or Cast 2a) and one or more guide RNAs (e.g., a crRNA and, optionally, a tracrRNA) that form ribonucleoprotein (RNP) complexes that associate with ( / .< ., target) and cleave specific loci complementary to a targeting (or spacer) sequence of the crRNA. Genome editing systems according to the present disclosure similarly target and optionally edit cellular DNA sequences but differ significantly from CRISPR systems occurring in nature. For example,Attorney Docket No.: 084177.0330 the unimolecular guide RNAs described herein do not occur in nature, and both guide RNAs and RNA-guided nucleases according to this disclosure can incorporate any number of non- naturally occurring modifications.Genome editing systems disclosed herein can be delivered into a cell by electroporation. Other non-viral approaches can also be employed for genome editing of target cells disclosed herein. For example, a nucleic acid molecule can be introduced into cells / subjects by administering the nucleic acid by lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101 :512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247: 1465, 1990). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Lipid nanoparticles (LNPs) or liposomes are also contemplated for delivery of nucleic acid molecules into a cell. In some embodiments, the genome editing systems disclosed herein are delivered in vivo to a subject by administration of lipid nanoparticles (LNPs) containing one or more components of the genome editing system.Genome editing system disclosed herein can be delivered into subjects or cells using viral vectors, e.g., retroviral vectors, gamma-retroviral vectors or lentiviral vectors. Combinations of a retroviral vector and an appropriate packaging line are suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virusproducing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art. Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80: 1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and poly cations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89: 1817.Genome editing systems can be implemented (e.g., administered or delivered to a cell or a subject) in a variety of ways, and different implementations can be suitable for distinct applications. For instance, a genome editing system is implemented, in certainAttorney Docket No.: 084177.0330 embodiments, as a protein / RNA complex (a ribonucleoprotein complex, or RNP complex), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and / or an encapsulating agent, such as a lipid or polymer micro- or nanoparticle, micelle, liposome, etc. In certain embodiments, a genome editing system is implemented as one or more nucleic acids encoding the RNA-guided nuclease and / or guide RNA components described above (optionally with one or more additional components). In certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus. In certain embodiments, the genome editing system is implemented as a combination of any of the foregoing. Additional or modified implementations that operate according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.It should be noted that the genome editing systems of the present disclosure can be targeted to a single specific nucleotide sequence or can be targeted to — and capable of editing in parallel — two or more specific nucleotide sequences through the use of two or more guide RNAs. The use of multiple gRNAs is referred to as “multiplexing” throughout this disclosure, and can be employed to target multiple, unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain and, in some cases, to generate specific edits within such target domain. For example, International Patent Publication No. WO 2015 / 138510 by Maeder et al. (Maeder), which is incorporated by reference herein, describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene that results in the creation of a cryptic splice site, which in turn reduces or eliminates the function of the gene. The genome editing system of Maeder utilizes two guide RNAs targeted to sequences on either side of (i.e., flanking) the point mutation, and forms DSBs that flank the mutation. This, in turn, promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site, and restoring normal gene function.Genome editing systems can, in some instances, form double strand breaks that are repaired by cellular DNA double-strand break mechanisms such as NHEJ or HDR. These mechanisms are described throughout the literature, for example by Davis & Maizels, PNAS, l l l(10):E924-932, March 11, 2014 (Davis) (describing Alt-HDR); Frit et al. DNA Repair 17(2014) 81-97 (Frit) (describing Alt-NHEJ); and lyama and Wilson III, DNA Repair (Amst.) 2013-Aug; 12(8): 620-636 (lyama) (describing canonical HDR and NHEJ pathways generally).Attorney Docket No.: 084177.0330Where genome editing systems operate by forming DSBs, such systems optionally comprise one or more components that promote or facilitate a particular mode of doublestrand break repair or a particular repair outcome. For instance, Cotta-Ramusino also describes genome editing systems in which a single stranded oligonucleotide “donor template” is added; the donor template is incorporated into a target region of cellular DNA that is cleaved by the genome editing system and can result in a change in the target sequence.In certain embodiments, genome editing systems modify a target sequence, or modify expression of a gene in or near the target sequence, without causing double-strand breaks, e.g., by causing single-strand breaks or no cleavage (i.e., no strand breaks). For example, a genome editing system can comprise an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression. As one example, an RNA-guided nuclease can be connected to (e.g., fused to) a cytidine deaminase functional domain, and can operate by generating targeted C-to-A substitutions. An RNA-guided nuclease can also, for example, be connected to (e.g. fused to) an adenosine deaminase functional domain. Exemplary nuclease / deaminase fusions are described in Komor et al. Nature 533, 420-424 (19 May 2016) (“Komor”) and Kantor et al., Int. J. Mol. Sci. 21(17) 6240 (2020), which are hereby incorporated by reference in their entirety. Further non-limiting examples of suitable base editors, variants thereof, and strategies for preparing RNA-guided nucleases comprising the same are described in PCT applications: PCT / US2020 / 016664, filed February 4, 2020; PCT / US2020 / 018192, filed February 13, 2020; PCT / US2020 / 049975, field September 9, 2020; PCT / US2022 / 012054, filed January 11, 2022; and PCT / US2022 / 078655, filed October 25, 2022, the entire contents of each of which are incorporated herein by reference.Alternatively, a genome editing system can utilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a dead Cas9 (dCas9), and can operate by forming stable complexes on one or more targeted regions of cellular DNA, thereby recruiting other functional domains and / or interfering with functions involving the targeted region(s) including, without limitation, mRNA transcription, chromatin remodeling, among others.In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a polymerase domain (e.g., a reverse transcriptase domain). In certain embodiments, the RNA-guided nuclease may use a gRNA with a primer binding sequence and / or a template for the polymerase domain.Attorney Docket No.: 084177.0330In certain embodiments, the RNA-guided nuclease may be a prime editor (PE), where the PE is an RNA-guided nuclease with nickase activity that is fused to a reverse transcriptase domain. In certain embodiments, the PE may use a prime editing gRNA (pegRNA), where the pegRNA is a gRNA with a primer binding sequence (PBS) and a donor template, e.g., added at one of the termini, e.g., the 3' end. In certain embodiments, a PE:pegRNA complex binds to the target DNA, and the nickase domain of the prime editor nicks only one strand, generating a flap. The PBS, located on the pegRNA, binds to the DNA flap and the edited RNA sequence is reverse transcribed using the reverse transcriptase domain of the prime editor. The edited strand is incorporated into the DNA at the end of the nicked flap, and the target DNA is repaired with the new reverse transcribed DNA. The original DNA segment is removed by a cellular endonuclease. Additional methods employing RNA-guided nucleases and polymerases for template mediated gene editing are described in PCT publications: WO 2020 / 191233, WO 2020 / 191248, WO 2021226558, WO2023283246, WO 2023 / 235501, and WO 2023 / 076898, each of which are incorporated by reference for all purposes herein.3. Guide RNA (gRNA) moleculesThe terms “guide molecule,” “guide RNA” and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 or a Casl2a (Cpfl) to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing). gRNAs and their component parts are described throughout the literature, for instance in Briner et al. (Molecular Cell 56(2), 333- 339, October 23, 2014 (Briner), which is incorporated by reference), and in Cotta- Ramusino. The guide molecule can be an RNA molecule. The guide molecule can also comprise one or more nucleotides other than RNA nucleotides, for example, the guide molecule can be a DNA / RNA hybrid molecule, and / or the guide molecule can comprise one or more modified nucleotides (including, but not limited to, one or more modified DNA or RNA nucleotides).In bacteria and archaea, type II CRISPR systems generally comprise an RNA-guided nuclease protein such as Cas9, a CRISPR RNA (crRNA) that comprises a 5’ region that is complementary to a foreign sequence, and a trans-activating crRNA (tracrRNA) thatAttorney Docket No.: 084177.0330 comprises a 5’ region that is complementary to, and forms a duplex with, a 3’ region of the crRNA. This duplex can facilitate the formation of — and is necessary for the activity of — the Cas / gRNA complex. As type II CRISPR systems were adapted for use in gene editing, it was discovered that the crRNA and tracrRNA could be joined into a single unimolecular or chimeric guide RNA, in one non-limiting example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3’ end) and the tracrRNA (at its 5’ end). (Mali et al. Science. 2013 Feb 15; 339(6121): 823-826 (“Mali”); Jiang et al. Nat Biotechnol. 2013 Mar; 31(3): 233-239 (“Jiang”); and Jinek et al., 2012 Science Aug. 17; 337(6096): 816-821 (“Jinek”), all of which are incorporated by reference herein.)Guide RNAs, whether unimolecular or modular, comprise a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. Targeting domains are referred to by various names in the literature, including without limitation “guide sequences” (Hsu et al., Nat Biotechnol. 2013 Sep; 31(9): 827-832, (“Hsu”), incorporated by reference herein), “complementarity regions” (Cotta-Ramusino), “spacers” (Briner) and generically as “crRNAs” (Jiang). Irrespective of the names they are given, targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5’ terminus of in the case of a Cas9 gRNA, and at or near the 3’ terminus in the case of a Cast 2a gRNA.In addition to the targeting domains, gRNAs typically (but not necessarily, as discussed below) comprise a plurality of domains that can influence the formation or activity of gRNA / Cas9 complexes. For instance, as mentioned above, the duplexed structure formed by first and secondary complementarity domains of a gRNA (also referred to as a repeat: anti-repeat duplex) interacts with the recognition (REC) lobe of Cas9 and can mediate the formation of Cas9 / gRNA complexes. (Nishimasu et al., Cell 156, 935-949, February 27, 2014 (Nishimasu 2014) and Nishimasu et al., Cell 162, 1113-1126, August 27, 2015 (Nishimasu 2015), both incorporated by reference herein). It should be noted that the first and / or second complementarity domains can contain one or more poly-A tracts, which can be recognized by RNA polymerases as a termination signal. The sequence of the first and second complementarity domains are, therefore, optionally modified to eliminate these tracts and promote the complete in vitro transcription of gRNAs, for instance through the use of A-G swaps as described in Briner, or A-U swaps. These and other similarAttorney Docket No.: 084177.0330 modifications to the first and second complementarity domains are within the scope of the present disclosure.Along with the first and second complementarity domains, Cas9 gRNAs typically comprise two or more additional duplexed regions that are involved in nuclease activity in vivo but not necessarily in vitro. (Nishimasu 2015). A first stem-loop near the 3’ portion of the second complementarity domain is referred to variously as the “proximal domain,” (Cotta-Ramusino) “stem loop 1” (Nishimasu 2014 and 2015) and the “nexus” (Briner). One or more additional stem loop structures are generally present near the 3’ end of the gRNA, with the number varying by species: S. pyogenes gRNAs typically comprise two 3’ stem loops (for a total of four stem loop structures including the repeat: anti -repeat duplex), while S. aureus and other species have only one (for a total of three stem loop structures). A description of conserved stem loop structures (and gRNA structures more generally) organized by species is provided in Briner.While the foregoing description has focused on gRNAs for use with Cas9, it should be appreciated that other RNA-guided nucleases have been (or can in the future be) discovered or invented which utilize gRNAs that differ in some ways from those described to this point. For instance, Casl2a (also known as Cpfl; “CRISPR from Prevotella and Franciscella 1”) is an RNA-guided nuclease that does not require a tracrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771 October 22, 2015 (Zetsche I), incorporated by reference herein). A gRNA for use in a Casl2a genome editing system generally comprises a targeting domain and a complementarity domain (alternately referred to as a “handle”). It should also be noted that, in gRNAs for use with Casl2a, the targeting domain is usually present at or near the 3’ end, rather than the 5’ end as described above in connection with Cas9 gRNAs (the handle is at or near the 5’ end of a Casl2a gRNA).Those of skill in the art will appreciate that, although structural differences can exist between gRNAs from different prokaryotic species, or between Cast 2a and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that comprises one or more chemical modifications and / or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs can be described solely in terms of their targeting domain sequences.Attorney Docket No.: 084177.0330More generally, skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA- guided nuclease, and not only those gRNAs that are compatible with a particular RNA- guided nuclease, e.g., a particular species of Cas9 or Cast 2a. By way of illustration, the term gRNA can, in certain embodiments, comprise a gRNA for use with any RNA-guided nuclease occurring in a Class 2 CRISPR system, such as a type II or type V CRISPR system, or an RNA-guided nuclease derived or adapted therefrom.In some embodiments, the gRNA scaffold sequence (e.g., SEQ ID NO: 57; Tables 7A-7B) is the same sequence as the gRNA hairpin region e.g., SEQ ID NO: 59; Table 8). In some embodiments, the guide RNA used comprises a modification as compared to the standard gRNA scaffold. Such modifications may comprise, for example, chemical modifications of a part of the gRNA, e.g, of a nucleobase or backbone moiety. In some embodiments, such a modification may also comprise the presence of one or more DNA nucleotide within the gRNA, e.g, within or outside of the targeting domain. In some embodiments, the modification may comprise an extension of the gRNA scaffold, e.g., by addition of 1-100 nucleotides, including RNA and / or DNA nucleotides at the 3’ or the 5’ terminus of the guide RNA, e.g., at the terminus distal to the targeting domain.In certain embodiments, a gRNA complexed to an unmodified or modified Cast 2a protein may be modified to increase the editing efficiency of a target nucleic acid. In certain embodiments, the modified gRNA may comprise one or more modifications including a phosphorothioate (PS2) linkage modification, a 2’-O-methyl modification (non-limiting exemplary modifications are illustrated in FIG. 3), one or more or a stretch of additional nucleotides (e.g., RNA or deoxyribonucleic acid (DNA) nucleotides) not found in a corresponding native gRNA (also referred herein as a “gRNA extension”), or combinations thereof.In some embodiments, a gRNA used herein comprises one or more or a stretch of additional ribonucleic acid or deoxyribonucleic acid (DNA) bases outside of the spacer region, also referred to herein as a “gRNA extension.” In some embodiments, a gRNA used herein comprises a gRNA extension that comprises one or more or a stretch of DNA bases, referred to herein as a “DNA extension”. In some embodiments, a gRNA used herein comprises a DNA extension at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof. In certain embodiments, the DNA extension may be 1, 2, 3, 4, 5, 6, 7,Attorney Docket No.: 084177.03308, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 DNA bases long. For example, in certain embodiments, the DNA extension may be 1, 2, 3, 4, 5, 10, 15,20, or 25 DNA bases long. In certain embodiments, the DNA extension may comprise one or more DNA bases selected from adenine (A), guanine (G), cytosine (C), or thymine (T). In certain embodiments, the DNA extension comprises the same DNA bases. For example, the DNA extension may comprise a stretch of adenine (A) bases. In certain embodiments, the DNA extension may comprise a stretch of thymine (T) bases. In certain embodiments, the DNA extension comprises a combination of different DNA bases. In certain embodiments, a DNA extension may comprise or consist of a sequence set forth in Table 3. In certain embodiments, a gRNA used herein comprises a DNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3 ’ end of the gRNA, or combinations thereof. In certain embodiments, a gRNA including a DNA extension may comprise a sequence set forth in Table 3 that comprises a DNA extension. Without wishing to be bound by theory, it is contemplated that any DNA extension may be used herein, so long as it does not hybridize to the target nucleic acid being targeted by the gRNA. In some embodiments the DNA extension additionally exhibits an increase in editing efficiency, e.g., via changes to gRNA stability, uptake, and / or activity, at the target nucleic acid site relative to a gRNA which does not comprise such a DNA extension.In some embodiments, a gRNA used herein comprises a gRNA extension that comprises one or more or a stretch of ribonucleic acid (RNA) bases, also referred to herein as an “RNA extension.” In some embodiments, a gRNA used herein comprises an RNA extension at the 5’ end of the gRNA, the 3’ end of the gRNA, or a combination thereof. In certain embodiments, the RNA extension may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 RNA bases long. For example, in certain embodiments, the RNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 RNA basesAttorney Docket No.: 084177.0330 long. In certain embodiments, the RNA extension may comprise one or more RNA bases selected from adenine (rA), guanine (rG), cytosine (rC), or uracil (rU), in which the “r” represents RNA, 2’-hydroxy. In certain embodiments, the RNA extension comprises the same RNA bases. For example, the RNA extension may comprise a stretch of adenine (rA) bases. In certain embodiments, the RNA extension comprises a combination of different RNA bases. In certain embodiments, an RNA extension may comprise or consist of a sequence set forth in Table 3. In certain embodiments, a gRNA used herein comprises an RNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof. In certain embodiments, a gRNA including an RNA extension may comprise a sequence set forth in Table 3 that comprises an RNA extension. gRNAs including an RNA extension at the 5’ end of the gRNA may comprise a sequence disclosed herein. gRNAs including an RNA extension at the 3’ end of the gRNA may comprise a sequence disclosed herein.It is contemplated that gRNAs used herein may also comprise an RNA extension and a DNA extension. In certain embodiments, the RNA extension and DNA extension may both be at the 5’ end of the gRNA, the 3’ end of the gRNA, or a combination thereof. In certain embodiments, the RNA extension is at the 5’ end of the gRNA and the DNA extension is at the 3’ end of the gRNA. In certain embodiments, the RNA extension is at the 3’ end of the gRNA and the DNA extension is at the 5’ end of the gRNA.It is further contemplated that gRNAs used herein may comprise a gRNA extension that is a hybrid extension that comprises both deoxyribonucleic acid and ribonucleic acid moieties.In some embodiments, a gRNA which comprises a modification, e.g., a DNA extension at the 5’ end, is complexed with a RNA-guided nuclease, e.g., an AsCasl2a nuclease, to form an RNP complex, (such RNP complex formation occurring either prior to delivery of a composition described herein to a subject or following such delivery, e.g., in a cell after expression of an mRNA encoding the RNA-guided nuclease), which then edits a target cell (e.g., a liver cell). Exemplary suitable 5’ extensions for guide RNAs, e.g., Cast 2a guide RNAs are provided in the table below:Table 3: gRNA 5’ ExtensionsAttorney Docket No.: 084177.0330All bases are in upper case Lowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bonds except as noted: represents phosphorothioate modification“PS” represents phosphorothioate modificationAdditional suitable gRNA modifications will be apparent to those of ordinary skill in the art based on the present disclosure. Suitable gRNA modifications comprise, for example, those described in PCT application PCT / US2018 / 054027, filed on Oct. 2, 2018, and entitled “MODIFIED CPF1 GUIDE RNA;” in PCT application PCT / US2015 / 000143,Attorney Docket No.: 084177.0330 filed on Dec. 3, 2015, and entitled “GUIDE RNA WITH CHEMICAL MODIFICATIONS;” in PCT application PCT / US2016 / 026028, filed Apr. 5, 2016, and entitled “CHEMICALLY MODIFIED GUIDE RNAS FOR CRISPR / C AS -MEDIATED GENE REGULATION;” and in PCT application PCT / US2016 / 053344, filed on Sep.23, 2016, and entitled “NUCLEASE-MEDIATED GENOME EDITING OF PRIMARY CELLS AND ENRICHMENT THEREOF;” the entire contents of each of which are incorporated herein by reference. Without being bound by theory, in certain embodiments of genome editing systems of the present disclosure, the one or more modifications of the gRNA enhance binding affinity of the gRNA molecule to RNA-guided nuclease of the genome editing system, e.g., a Cast 2a nuclease.4. Guide RNA designMethods for selection and validation of target sequences as well as off-target analyses have been described previously, e.g., in Mali; Hsu; Fu et al., 2014 Nat Biotechnol 32(3): 279-84, Heigwer et al., 2014 Nat methods 11(2): 122-3; Bae et al. (2014) Bioinformatics 30(10): 1473-5; and Xiao A et al. (2014) Bioinformatics 30(8): 1180-1182. Each of these references is incorporated by reference herein. In certain non-limiting embodiments, gRNA design can involve the use of a software tool to optimize the choice of potential target sequences corresponding to a user’s target sequence, e.g., to minimize total off-target activity across the genome. These and other guide selection methods are described in detail in Maeder and Cotta-Ramusino.In certain embodiments, one or more or all of the nucleotides in a gRNA are modified. Strategies for modifying a gRNA are described in WO2019 / 152519, published Aug. 8, 2019, the entire contents of which are expressly incorporated herein by reference.Non-limiting examples of guide RNAs suitable for certain embodiments embraced by the present disclosure are provided herein, for example, in the Tables below. Those of ordinary skill in the art will be able to envision suitable guide RNA sequences for a specific nuclease, e.g., a Cas9 or Casl2a nuclease, from the disclosure of the targeting domain sequence, either as a DNA or RNA sequence. For example, a guide RNA comprising a targeting sequence consisting of RNA nucleotides would comprise the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and thus contain uracil instead of thymidine nucleotides. For example, a guide RNA comprising a targeting domain sequence consisting of RNA nucleotides and described by the DNA sequence GCCAATGGCCTCCTTCAGTTG (SEQ ID NO: 26) would have a targetingAttorney Docket No.: 084177.0330 domain of the corresponding RNA sequence rGrCrCrArArUrGrGrCrCrUrCrCrUrUrCrArGrUrUrG (SEQ ID NO: 37). As will be apparent to the skilled artisan, such a targeting sequence would be linked to a suitable guide RNA scaffold, e.g., a crRNA scaffold sequence or a chimeric crRNA / tracrRNA scaffold sequence. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For AsCasl2a, for example, a suitable scaffold sequence comprises the sequence rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU (SEQ ID NO: 57), added to the 5’- terminus of the targeting domain. In the example above, this would result in a Casl2a guide RNA of the sequence; rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrCrCrArArUrGrGrCrCrUrCrCrUr UrCrArGrUrUrG (SEQ ID NO: 48). Those of skill in the art would further understand how to modify such a guide RNA. For example, adding a 25-mer DNA extension (e.g., SEQ ID NO: 7) would result, for example, in a guide RNA of the sequence ATGTGTTTTTGTCAAAAGACCTTTTrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGr ArUrGrCrCrArArUrGrGrCrCrUrCrCrUrUrCrArGrUrUrG (SEQ ID NO: 58). It will be understood that the exemplary targeting sequences provided herein are not limiting, and additional suitable sequences, e.g., variants of the specific sequences disclosed herein, will be apparent to the skilled artisan based on the present disclosure in view of the general knowledge in the art.In some embodiments, the gRNA for use in the disclosure is a gRNA targeting ANGPTL3 (ANGPTL3 gRNA). In some embodiments, the target sequence of an ANGPTL3 gene comprises or consists of a nucleotide sequence set forth in SEQ ID NOs: 24-34 (Table 4). In certain embodiments, the target sequence of an ANGPT 3 gene comprises or consists of the nucleotide sequence set forth in SEQ ID NOs: 26 or 31. In some embodiments, the targeting domain of the gRNA molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NOs: 35-45 (Table 5). In certain embodiments, the targeting domain of the gRNA targeting an ANGPTL3 gene is SEQ ID NO: 37, or SEQ ID NO: 42. In some embodiments, the gRNA molecule targeting an ANGPTL3 gene comprises or consists of a nucleotide sequence set forth in SEQ ID NOs: 46-56 (Table 6). In certain embodiments, the gRNA molecule targeting asxANGPTL3 gene comprises or consists of the sequence set forth in SEQ ID NO: 48, or SEQ ID NO: 53. An exemplary ANGPTL3 gene target sequence, gRNA targeting domain, scaffold sequence, and DNA extension are set forth in Table 7A- 7BAttorney Docket No.: 084177.0330Table 4: ANGPTL3 Target Sequences’Chromosome 1, ANGPTL3 transcript = ENST00000371129.4Table 5: ANGPTL3 Targeting SequencesTable 6'. ANGPTL3 gRNA sequencesAttorney Docket No.: 084177.0330All bases are in upper caseLowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNA All linkages between the nucleotides are standard phosphodiester groups.All bases are in upper caseLowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNA All linkages between the nucleotides are standard phosphodiester groups.In some embodiments, the targeting domain of the gRNA molecule has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a nucleotide sequence setAttorney Docket No.: 084177.0330 forth in SEQ ID NOs: 35-45. In some embodiments, the targeting domain of the gRNA molecule has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a nucleotide sequence set forth in SEQ ID NOs: 35-45. In some embodiments, the targeting domain of the gRNA molecule has less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a nucleotide sequence set forth in SEQ ID NOs: 35-45.4.1 Guide RNA modi ficationsThe activity, stability, or other characteristics of gRNAs can be altered through the incorporation of certain modifications. As one example, transiently expressed or delivered nucleic acids can be prone to degradation by, e.g., cellular nucleases. Accordingly, the gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not wishing to be bound by theory it is also believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into cells. Those of skill in the art will be aware of certain cellular responses commonly observed in cells, e.g., mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which can comprise induction of cytokine expression and release and cell death, can be reduced or eliminated altogether by the modifications presented herein.Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5’ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5’ end) and / or at or near the 3’ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3’ end). In some cases, modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Casl2a gRNA, and / or a targeting domain of a gRNA.As one example, the 5’ end of a gRNA can comprise a eukaryotic mRNA cap structure or cap analog (e.g., a G(5 )ppp(5 )G cap analog, a m7G(5 )ppp(5 )G cap analog, or a 3 ’-O-Me-m7G(5 )ppp(5 )G anti reverse cap analog (ARC A)), as shown below:Attorney Docket No.: 084177.0330The cap or cap analog can be included during either chemical synthesis or in vitro transcription of the gRNA.Along similar lines, the 5’ end of the gRNA can lack a 5’ triphosphate group. For instance, in vitro transcribed gRNAs can be phosphatase-treated (e.g., using calf intestinal alkaline phosphatase) to remove a 5’ triphosphate group.Another modification involves the addition, at the 3’ end of a gRNA, of a plurality (e.g., 1-10, 10-20, or 25-200) of adenine (A) residues referred to as a polyA tract. The polyA tract can be added to a gRNA during chemical synthesis, following in vitro transcription using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase), or in vivo by means of a polyadenylation sequence, as described in Maeder.It should be noted that the modifications described herein can be combined in any suitable manner, e.g., a gRNA, whether transcribed in vivo from a DNA vector, or in vitro transcribed gRNA, can comprise either or both of a 5’ cap structure or cap analog and a 3’ polyA tract.Guide RNAs can be modified at a 3’ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:wherein “U” can be an unmodified or modified uridine.The 3’ terminal U ribose can be modified with a 2’3’ cyclic phosphate as shown below:wherein “U” can be an unmodified or modified uridine.Guide RNAs can contain 3’ nucleotides which can be stabilized against degradation, e.g, by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridines can be replaced with modified uridines, e.g, 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein;Attorney Docket No.: 084177.0330 adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.In certain embodiments, sugar-modified ribonucleotides can be incorporated into the gRNA, e.g., wherein the 2’ OH-group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate (PhTx) group. In certain embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-O-methyl, 2’-O-methoxyethyl, or 2’-Fluoro modified including, e.g., 2’-F (as illustrated in FIG. 3) or 2’-O-methyl, adenosine (A), 2’-F or 2’-O-methyl, cytidine (C), 2’-F or 2’-O-methyl, uridine (U), 2’-F or 2’-O-methyl, thymidine (T), 2’-F or 2’-O-methyl, guanosine (G), 2’ -O-m ethoxy ethyl-5-methyluri dine (Teo), 2’ -O-methoxy ethyladenosine (Aeo), 2’-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.Guide RNAs can also comprise “locked” nucleic acids (LNA) in which the 2’ OH- group can be connected, e.g., by a Cl -6 alkylene or Cl -6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar. Any suitable moiety can be used to provide such bridges, comprise without limitation methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH2)n-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).In certain embodiments, a gRNA can comprise a modified nucleotide which is multi cyclic (e.g., tri cyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl- (3’-2’)).Generally, gRNAs comprise the sugar group ribose, which is a 5-membered ring with an oxygen atom. Exemplary modified gRNAs can comprise, without limitation,Attorney Docket No.: 084177.0330 replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). Although the majority of sugar analog alterations are localized to the 2’ position, other sites are amenable to modification, including the 4’ position. In certain embodiments, a gRNA comprises a 4’-S, 4’-Se or a 4’-C-aminomethyl-2’-O-Me modification.In certain embodiments, deaza nucleotides, e.g., 7-deaza-ad enosine, can be incorporated into the gRNA. In certain embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl adenosine, can be incorporated into the gRNA. In certain embodiments, one or more or all of the nucleotides in a gRNA are deoxynucleotides.In certain embodiments, a gRNA comprises one or more 2’F modifications. In certain embodiments, a 2’F modification is positioned on a nucleotide internal to the gRNA (which may be represented herein as “i2F”). In certain embodiments, a gRNA comprises multiple 2’F modifications in an aggressive pattern or a conservative pattern. In certain embodiments, a gRNA hairpin region has the same sequence as the gRNA scaffold sequence. For example, a Casl2a gRNA can have hairpin sequence of SEQ ID NO: 59 (Table 8) and a scaffold sequence of SEQ ID NO: 57 (Tables 7A-7B). Herein a Casl2a gRNA with an “aggressive” pattern of hairpin 2’F modifications comprises a hairpin comprising 2’F modifications at each of nucleotide positions 7 and 8 of the hairpin. In certain embodiments, a gRNA comprising an aggressive pattern of hairpin 2’F modifications comprises a hairpin of SEQ ID NO: 63. Herein a Casl2a gRNA with a “conservative” pattern of hairpin 2’F modifications comprises a hairpin lacking 2’F modifications at each of nucleotide positions 7 and 8 of the hairpin. In certain embodiments, a gRNA comprising a conservative pattern of hairpin 2’F modifications comprises a hairpin of SEQ ID NO: 60.In certain embodiments, gRNAs as used herein may be modified or unmodified gRNAs. In certain embodiments, a gRNA may include one or more modifications. In certain embodiments, the one or more modifications may include a phosphorothioate linkage modification, a phosphorodithioate (PS2) linkage modification, a 2’-O-methyl modification, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinationsAttorney Docket No.: 084177.0330 thereof. In general, any combination of modifications may be used. In non-limiting examples, patterns of modification comprising, a combination of 5’ extension; 5’3’ idT; hairpin 2’F in an aggressive pattern (see, e.g., the relevant gRNAs in Table 8 for illustration of an exemplary hairpin_2’F aggressive pattern); a combination of 5’3’ idT and hairpin 2’F in an aggressive pattern; or a combination of 5’3’ idT and 5’ extension combination can be used. The pattern of modification of the gRNA, e.g., the modifications illustrated in Fig. 3 or described elsewhere herein, can be applied to guides for any targeting domain (e.g., ANGPTL3) and / or for targeting genes in a tissue specific manner.Table 8. gRNA modification pattern**Underlined = 2’ F modified nucleotide ; No underline = unmodified nucleotideIn certain embodiments, a modified gRNA as described herein comprises one or more modifications of the gRNA in the hairpin region, the targeting domain, or both. For example, but not by way of limitation, the hairpin region of such a modified gRNA can comprise SEQ ID NO: 59. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 2’Fluorine modifications (e.g., see SEQ ID NOs: 60 or 63 in Table 8). In certain embodiments, the hairpin region of a modified gRNA comprises a DNA extension at the 5’ end of the hairpin region. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 2’0-methyl modifications. For example, in certain embodiments, a modified gRNA comprises a IxPSOMe modification on a 5’ terminusAttorney Docket No.: 084177.0330 and / or a 3’ terminus; in certain embodiments, a modified gRNA comprises a 3xPSOMe modification on a 5’ terminus and / or a 3’ terminus. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 5’ inverted dT modifications. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 3’ inverted dT modifications. In certain embodiments, the hairpin region of a modified gRNA comprises a 3’ or 4’ pseudoknot. In certain embodiments, the hairpin region of a modified gRNA comprises a 3’ pseudoknot. In certain embodiments, the hairpin region of a modified gRNA comprises a locked nucleic acid (LNA). In certain embodiments, the hairpin region of a modified gRNA comprises a LNA with a 5’ extension.In certain embodiments, the hairpin region of a modified gRNA can comprise one or more of 5’ extensions, 2’Fluorine modifications, 2’O-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications, a pseudoknot, or an LNA. For example, in certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a IxPSOMe modification on 5’ and 3’ ends. In certain embodiments, the hairpin region of a modified gRNA comprises a IxPSOMe modification on the 5’end and a 3’ pseudoknot. In certain embodiments, the hairpin region of the modified gRNAs comprises a 5’ extension and IxPSOMe modification on the 3 ’end only. In certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a conservative pattern of 2’F modifications. In certain embodiments, the hairpin region of the modified gRNAs comprises a 5’ extension and an aggressive pattern of 2’F modifications. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and inverted dT modifications at the 5’ and 3’ termini. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension, inverted dT modifications at the 5’ and 3’ termini, and an LNA. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and an aggressive pattern of 2’ modifications, and IxPSOMe modifications on 5’ and 3’ termini. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and an aggressive pattern of 2’F modifications, and inverted dT modifications at the 5’ and 3’ termini. In certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a 2’OMe modification.In certain embodiments, the targeting domain of the gRNA can comprise one or more of 5’ extensions, 2’Fluorine modifications, 2’O-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications, a pseudoknot, or an LNA. In certain embodiments the targeting domain of a gRNA comprises 2’F modifications at nucleotideAttorney Docket No.: 084177.0330 positions 1, 8, 9, 10, 11, 12, 17, 19 and optionally 20 and 21. In certain embodiments the targeting domain of a gRNA comprises 2’F modifications at nucleotide positions 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 and optionally 20 and 21. In certain embodiments, the targeting domain of a gRNA comprises one or more of 2’Fluorine modifications, 2’0- m ethyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications.In any of the above embodiments, a gRNA can comprise any pattern of 2’F modifications in the hairpin (e.g., a conservative pattern or an aggressive pattern) and any pattern of 2’F modifications in the targeting domain (e.g., 2’F modifications at nucleotide positions 1, 8, 9, 10, 11, 12, 17, 19 of the targeting domain; 2’F modifications at nucleotide positions 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 of the targeting domain; or no 2’F modifications in the targeting domain).In certain embodiments, a genome editing system described herein comprises a gRNA comprising an RNA portion comprising a 2’F modification. In certain embodiments, the RNA portion comprises a 5’ hairpin and a 3’ targeting domain. In certain embodiments, a gRNA further comprises an extension region at the 5’ end of the hairpin (e.g., a DNA extension). In certain embodiments, a gRNA comprises a hairpin comprising a 2’F modification at one or more of nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin (z.e., counting from the 5’ end of the hairpin, e.g., having SEQ ID NO: 59). In certain embodiments, a gRNA comprises a hairpin comprising multiple 2’F modifications. In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin with a pattern of 2’F modifications that consists of 2’F modifications at nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin (exemplifying an aggressive pattern of 2’F modifications). In certain embodiments, a gRNA comprises a hairpin that does not include a 2’F modification on at least one of nucleotide positions 7 or 8 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at each of nucleotide positions 7 and 8 of the hairpin. In certain embodiments, a— gRNA comprises a hairpin comprising 2’F modifications at nucleotide positions 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpinAttorney Docket No.: 084177.0330 with a pattern of 2’F modifications that consists of 2’F modifications at nucleotide positions 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin (exemplifying a conservative pattern of 2’F modifications). In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at one or more of nucleotide positions 2, 3, 4, 11 and 15 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at each of nucleotide positions 2, 3, 4, 11 and 15 of the hairpin.Without wishing to be bound by theory, it is contemplated that, in certain embodiments, chemical modification of the gRNA can improve editing potency by enhancing binding affinity of the modified gRNA for an RNA-guided enzyme (e.g., Casl2a).5. RNA-guided nucleasesRNA-guided nucleases according to the present disclosure comprise, but are not limited to, naturally occurring Class 2 CRISPR nucleases such as Cas9, and Cast 2a, as well as other nucleases derived or obtained therefrom. In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g. complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that comprises (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations can exist between individual RNA- guided nucleases that share the same PAM specificity or cleavage activity. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease having a certain PAM specificity and / or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs. Casl2a), species (e.g., S. pyogenes vs. S. aureus) or variation (e.g., full-length vs. truncated or split; naturally occurring PAM specificity vs. engineered PAM specificity, etc.) of RNA-guided nuclease.The PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary to gRNA targeting domains (or “spacers”). Together with protospacer sequences, PAM sequences define target regions or sequences for specific RNA-guided nuclease / gRNA combinations.Attorney Docket No.: 084177.0330Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. For example, Cas9 nucleases recognize PAM sequences that are 3’ of the protospacer, while Cast 2a, on the other hand, generally recognizes PAM sequences that are 5’ of the protospacer.In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3’ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. And F. novicida Casl2a recognizes a TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described by Shmakov et al., 2015, Molecular Cell 60, 385-397, November 5, 2015. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of reference molecules (for instance, in the case of an engineered RNA-guided nuclease, the reference molecule can be the naturally occurring variant from which the RNA-guided nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to the engineered RNA-guided nuclease).In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally-occurring RNA-guided nucleases typically form DSBs in target nucleic acids but engineered variants have been produced that generate only SSBs (discussed above) Ran & Hsu, et al., Cell 154(6), 1380-1389, September 12, 2013 (Ran), incorporated by reference herein), or that that do not cut at all.5.1 Cas9Crystal structures have been determined for S. pyogenes Cas9 (Jinek 2014), and for S. aureus Cas9 in complex with a unimolecular guide RNA and a target DNA (Nishimasu 2014; Anders 2014; and Nishimasu 2015).A naturally occurring Cas9 protein comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which comprise particular structural and / or functional domains. The REC lobe comprises an arginine-rich bridge helix (BH) domain, and at least one REC domain (e.g., a RECI domain and, optionally, a REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain. While not wishing to be bound by any theory, mutational analyses suggest specific functional roles for the BH and REC domains: the BH domain appears to play a role in gRNA:DNA recognition, whereas the REC domain is thought to interact withAttorney Docket No.: 084177.0330 the repeat: anti-repeat duplex of the gRNA and to mediate the formation of the Cas9 / gRNA complex.The NUC lobe comprises a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves the non-complementary ( / .< ., bottom) strand of the target nucleic acid. It can be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in S. pyogenes and S. aureus). The HNH domain, meanwhile, is structurally similar to HNN endonuclease motifs, and cleaves the complementary (i.e., top) strand of the target nucleic acid. The PI domain, as its name suggests, contributes to PAM specificity.While certain functions of Cas9 are linked to (but not necessarily fully determined by) the specific domains set forth above, these and other functions can be mediated or influenced by other Cas9 domains, or by multiple domains on either lobe. For instance, in S. pyogenes Cas9, as described in Nishimasu 2014, the repeat: antirepeat duplex of the gRNA falls into a groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem loop structure also interact with amino acids in multiple domains (PI, BH and RECI), as do some nucleotides in the second and third stem loops (RuvC and PI domains).5.2 Casl2a (formerly known as Cpfl)The crystal structure of Acidaminococcus sp. Cast 2a in complex with crRNA and a double-stranded (ds) DNA target including a TTTN PAM sequence has been solved by Yamano et al. (Cell. 2016 May 5; 165(4): 949-962 (Yamano), incorporated by reference herein). Casl2a, like Cas9, has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe. The REC lobe comprises RECI and REC2 domains, which lack similarity to any known protein structures. The NUC lobe, meanwhile, comprises three RuvC domains (RuvC-I, -II and -III) and a BH domain. However, in contrast to Cas9, the Cast 2a REC lobe lacks an HNH domain, and comprises other domains that also lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I, -II and -III), and a nuclease (Nuc) domain.While Cas9 and Cast 2a share similarities in structure and function, it should be appreciated that certain Cast 2a activities are mediated by structural domains that are not analogous to any Cas9 domains. For instance, cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which differs sequentially and spatially from the HNH domain of Cas9. Additionally, the non-targeting portion of Casl2aAttorney Docket No.: 084177.0330 gRNA (the handle) adopts a pseudoknot structure, rather than a stem loop structure formed by the repeat: antirepeat duplex in Cas9 gRNAs.Non-limiting examples of RNA-guided nucleases include, Cas9 e.g., SpCas9, SaCas9, (KKH) SaCas9, eSpCas9, Cas9-HF1, HypaCas9, dCas9-Fokl, Sniper-Cas9, xCas9, evoCas9, SpCas9-NG, VRQR, VRER, NmeCas9, CjCas9), Casl2a (also known as Cpfl; e.g., AsCasl2a, LbCasl2a), Cast 2b (e.g., AaCasl2b, BhCasl2b, BhCasl2bV4), Cast 2c (e.g., Casl2cl, Casl2c2), Casl2h (e.g., Casl2hl), Casl2i (e.g., Casl2il), CasX, CasY, and Cas .5.3 Modifications of RNA-guided nucleasesThe RNA-guided nucleases described above have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that RNA-guided nucleases can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.Turning first to modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe have been described above. Exemplary mutations that can be made in the RuvC domains, in the Cas9 HNH domain, or in the Cast 2a Nuc domain are described in Ran and Yamano, as well as in Cotta-Ramusino. In general, mutations that reduce or eliminate activity in one of the two nuclease domains result in RNA-guided nucleases with nickase activity, but it should be noted that the type of nickase activity varies depending on which domain is inactivated. As one example, inactivation of a RuvC domain or of a Cas9 HNH domain results in a nickase.Modifications of PAM specificity relative to naturally occurring Cas9 reference molecules have been described by KI einstiver et al. for both S. pyogenes (KI einstiver et al., Nature. 2015 Jul 23;523(7561):481-5 (Kleinstiver I)) and S. aureus (Kleinstiver et al., Nat Biotechnol. 2015 Dec; 33(12): 1293-1298 (Klienstiver II)). Kleinstiver et al. have also described modifications that improve the targeting fidelity of Cas9 (Nature, 2016 January 28; 529, 490-495 (Kleinstiver III)). Each of these references is incorporated by reference herein.RNA-guided nucleases have been split into two or more parts, as described by Zetsche et al. (Nat Biotechnol. 2015 Feb;33(2): 139-42 (Zetsche II), incorporated by reference), and by Fine et al. (Sci Rep. 2015 Jul 1;5: 10777 (Fine), incorporated by reference).RNA-guided nucleases can be, in certain embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the nuclease while still retainingAttorney Docket No.: 084177.0330 gRNA association, target and PAM recognition, and cleavage activities. In certain embodiments, RNA guided nucleases are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary bound nucleases and linkers are described by Guilinger et al., Nature Biotechnology 32, 577-582 (2014), which is incorporated by reference for all purposes herein.RNA-guided nucleases also optionally comprise a tag, such as, but not limited to, a nuclear localization signal (NLS) to facilitate movement of RNA-guided nuclease protein into the nucleus. In certain embodiments, the RNA-guided nuclease can incorporate C- and / or N-terminal nuclear localization signals, e.g., SEQ ID NO: 66, presented herein. Nuclear localization sequences are known in the art and are described in Maeder and elsewhere.The foregoing list of modifications is intended to be exemplary in nature, and the skilled artisan will appreciate, in view of the instant disclosure, that other modifications can be possible or desirable in certain applications. For brevity, therefore, exemplary systems, methods and compositions of the present disclosure are presented with reference to particular RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used can be modified in ways that do not alter their operating principles. Such modifications are within the scope of the present disclosure.Exemplary suitable nuclease variants comprise, but are not limited to, AsCasl2a variants comprising an M537R substitution, an H800A substitution, and / or an F870L substitution, or any combination thereof (numbering scheme according to AsCasl2a wildtype sequence). Other suitable modifications of the AsCasl2a amino acid sequence are known to those of ordinary skill in the art. Some non-limiting exemplary sequences of wildtype AsCasl2a and AsCasl2a variants are as follows:His-AsCpfl-sNLS-sNLS H800A amino acid sequence:MGHHHHHHGSTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATY RNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENA LLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAV PSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKI KGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSF CKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALY ERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHA ALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKAttorney Docket No.: 084177.0330LEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQL KAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKG YREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQR IAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQ RVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNRE KERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSK RTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMG TQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGD FILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHR FTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQ MRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLL NHLKESKDLKLQNGISNQDWLAYIQELRNGSPK KKRKVGSPKKKRKV [SEQ ID NO: 66]Caslla variant 1 amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRI YKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIG RTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNL AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRN ENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPT TLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLS FYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLG IMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQT HTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWID FTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAV ETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYR PKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEAR ALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPAttorney Docket No.: 084177.0330ETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQA WSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVY QQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNR NLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATG EDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDL KLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSG GSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 67]AsCaslla variant 2 amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRI YKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIG RTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNL AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPT TLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLS FYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYL GIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHF QTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAEL FYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDE ARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKE HPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQ AWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAV YQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNR NLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLY PANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATG EDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLAttorney Docket No.: 084177.0330KLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 68]AsCaslla variant 3 amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDR IYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFT TYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV LNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKT LLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRIS ELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLE MEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVK NGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLK AVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGY REALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRI AEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFN QRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDN REKER VAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVK TGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIE NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRS VLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKK RKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 69]AsCaslla variant 4 amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDR IYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTAttorney Docket No.: 084177.0330TYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV LNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKT LLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRIS ELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLE MEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVK NGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLK AVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGY REALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRI AEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFN QRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDN REKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVK TGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIE NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRS VLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKK RKV [SEQ ID NO: 70]AsCaslla variant 4 nucleotide sequence:ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCG TTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCT TCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGAT TATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGC TGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAAC CGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCC ATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAA ACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCA AAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACT GCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGAttorney Docket No.: 084177.0330CAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGAttorney Docket No.: 084177.0330GCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCAGCTCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCCTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTT [SEQ ID NO: 71]AsCaslla variant 5 amino acid sequence:Attorney Docket No.: 084177.0330MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDR IYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFT TYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV LNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKT LLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRIS ELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLE MEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVK NGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLK AVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGY REALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRI AEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFN QRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDN REKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVK TGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIE NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRS VLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKK RKV [SEQ ID NO: 72]AsCaslla variant 6 amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDR IYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFT TYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV LNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKT LLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISAttorney Docket No.: 084177.0330ELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLE MEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVK NGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLK AVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGY REALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRI AEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFN QRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDN REKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVK TGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIE NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWL AYIQELRNGRS SDDEAT AD SQHAAPPKKK RKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH [SEQ ID NO: 73]AsCaslla variant 7 amino acid sequence:MGRDPGKPIPNPLLGLDSTAPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFEL IPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENL SAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGL FKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIST AIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFY NQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFI PLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHE DINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYS VEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSF EPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPL EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTT SIDES SLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNAttorney Docket No.: 084177.0330KDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAH RLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEV SHEIIKDRRFT SDKFFFHVPITLNYQ AANSP SKFNQRVNAYLKEHPETPIIGIDRGE RNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKD LKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLI DKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKID PLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGL PGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIAL LEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGI SNQDWLAYIQELRNPKKKRKVKLAAALEHHHHHH [SEQ ID NO: 74]Exemplary AsCaslla wild-type amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDR IYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFT TYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV LNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKT LLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRIS ELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLE MEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVK NGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGY REALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRI AEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQ RVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNR EKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKS KRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVK TGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEAttorney Docket No.: 084177.0330NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRS VLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRN [SEQ ID NO: 75]AsCasl2a-MHFRR amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRI YKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIG RTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNL AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRN ENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPT TLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLS FYNKARNYATKKPYSVEKFKLNFQRPTLARGWDVNKEKNNGAILFVKNGLYYL GIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPRCSTQLKAVTAHF QTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIM DAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAEL FYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDE ARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKE HPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQ AWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAV YQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYV PAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNR NLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLY PANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATG EDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDL KLQNGISNQDWLAYIQELRNGGSPAAKRVKLDGGSPAAKRVKLD [SEQ ID NO: 76]AsCasl2a-MHFRR amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGAttorney Docket No.: 084177.0330RTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNL AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRN ENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPT TLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLS FYNKARNYATKKPYSVEKFKLNFQRPTLARGWDVNKEKNNGAILFVKNGLYYL GIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPRCSTQLKAVTAHF QTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIM DAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAEL FYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDE ARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKE HPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQ AWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAV YQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYV PAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNR NLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLY PANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATG EDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDL KLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKV [SEQ ID NO: 109]AsCasl2a-MHFRVR amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRI YKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIG RTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNL AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRN ENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPT TLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSAttorney Docket No.: 084177.0330FYNKARNYATKKPYSVEKFKLNFQRPTLARGWDVNVEKNRGAILFVKNGLYYL GIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHF QTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIM DAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAEL FYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDE ARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKE HPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQ AWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAV YQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYV PAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNR NLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLY PANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATG EDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDL KLQNGISNQDWLAYIQELRNGGSPAAKRVKLDGGSPAAKRVKLD [SEQ ID NO: 77]AsCasl2a-MHFRVR amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDR IYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFI GRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFT TYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV LNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKT LLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRIS ELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLE MEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLARGWDVNVEKNRGAILFVK NGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLK AVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGY REALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRI AEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNAttorney Docket No.: 084177.0330QRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDN REKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVK TGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIE NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKK RKV [SEQ ID NO: 110]AsCasl2a-MHF amino acid sequence:MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTT YFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRN ENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLS FYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLG IMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWID FTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAV ETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEAR ALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHP ETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVY QQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVP APYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLY PANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATG EDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLAttorney Docket No.: 084177.0330KLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKV [SEQ ID NO: 78]AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGGUUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUCUGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUCAUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACCCCGAGAttorney Docket No.: 084177.0330UUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGAGAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGAAUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCUGAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUCCAGGAUGAAGAGGAUGGCACACCGGCUGGGAGAGAAGAUGCUGAACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGCUUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGACCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAUAAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCGUGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGCACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCAttorney Docket No.: 084177.0330GAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAU CACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGAC GUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCC UUCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAG AAGAACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAG AGAAUCGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGAC CUGUAUCCUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUG UUCAGGGAUGGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCU CACGCCAUCGACACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGG AACUCCAAUGCCGCCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAU CUGAAUGGCGUGUGCUUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUG GACGCCGAUGCCAAUGGCGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUG CUGAAUCACCUGAAGGAGAGCAAGGAUCUGAAGCUGCAGAACGGCAUCUCC AAUCAGGACUGGCUGGCCUACAUCCAGGAGCUGCGCAACGGUCGUAGCAGU GAUGAUGAAGCAACCGCAGAUAGCCAGCAUGCAGCACCCAAGAAGAAGAGG AAAGUCUAAUAGUGAAUGGUUUAUAUUGCGGCCGCUUAAUUAAGCUGCCUU CUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAA [SEQ ID NO: 79]

[0001] In some embodiments, an RNA-guided nuclease has at least 80%, at least 85%, at least 86%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a wild-type RNA-guided nuclease and / or an RNA-guided nuclease disclosed herein (e.g., an RNA-guided nuclease comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-70 and 72-78). In some embodiments, an RNA-guided nuclease has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type RNA-guided nuclease and / or an RNA-guided nuclease disclosed herein (e.g., an RNA-guided nuclease comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-70 and 72-78). In some embodiments, an RNA-guided nuclease has less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20Attorney Docket No.: 084177.0330 mutations relative to a wild-type RNA-guided nuclease and / or an RNA-guided nuclease disclosed herein (e.g., an RNA-guided nuclease comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-70 and 72-78).5.4 Nucleic acids encoding RNA-guided nucleases

[0002] Nucleic acids encoding RNA-guided nucleases, e.g., Cas9, Cast 2a or functional fragments thereof, are provided herein. Exemplary nucleic acids encoding RNA-guided nucleases have been described previously (see, e.g., Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

[0003] In some cases, a nucleic acid encoding an RNA-guided nuclease can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease is an RNA. In certain embodiments, the nucleic acid encoding the RNA-guided nuclease is an mRNA. In certain embodiments, an mRNA encoding an RNA-guided nuclease will have one or more (e.g., all) of the following properties: it can be capped; polyadenylated; and substituted with 5-methylcytidine and / or pseudouridine.

[0004] Synthetic nucleic acid sequences can also be codon optimized, e.g., at least one noncommon codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein. Examples of codon optimized Cas9 coding sequences are presented in Cotta-Ramusino.

[0005] In addition, or alternatively, a nucleic acid encoding an RNA-guided nuclease can comprise a nucleic acid encoding a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art and examples include but are not limited to the NLS sequences fused to the RNA-guided nuclease sequence as indicated in SEQ ID NO: 66, presented herein.

[0006] Some non-limiting exemplary RNA sequences encoding the RNA-guided nuclease are as follows: mRNA #l(5’ modification is CleanCap® AG; 3’Tail Modification is a 79A poly A sequence) AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG ACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGG UUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGC UUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAttorney Docket No.: 084177.0330AUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUCUGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUCAUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACCCCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGAGAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGAAUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCUGAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUAttorney Docket No.: 084177.0330UCAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUCCAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGCUUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGACCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAUAAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCGUGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGCACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUAttorney Docket No.: 084177.0330CACGCCAUCGACACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGGAUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAGGAGCUGCGCAACGGUCGUAGCAGUGAUGAUGAAGCAACCGCAGAUAGCCAGCAUGCAGCACCGCCCAAGAAGAAGAGGAAAGUCUAAUAGUGAAUGGUUUAUAUUGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 80] mRNA #2(5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = N1 -Methyl-Pseudo UTP; 3’ Tail Modification = 79A tail)AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGGUUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUCUGGCCAUCCAGAAGAAUGAUGAGAAttorney Docket No.: 084177.0330CAGCCCACAUCAUCGCCUCCCUGCCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUCAUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACCCCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGAGAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGAAUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCUGAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUCCAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGCUUUACCAGCGACAAGUUCUAttorney Docket No.: 084177.0330UGUUCCACGUGCCUAUCACACUGAACUAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGACCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAUAAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCGUGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGCACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGGAUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAGGAGCUGCGCAACGGUCGUAGCAGUGAUGAUGAAGCAACCGCAGAUAGCCAGCAUGCAGCACCGCCCAAGAAGAAGAGGAAAGUCUAAUAGUGAAUGGUUUAUAUUGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 81] mRNA #3Attorney Docket No.: 084177.0330(5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = N1 -Methyl-Pseudo UTP; 3’ Tail Modification = 79A tail)AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGGUUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUCUGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUCAUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACCCCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGAGAttorney Docket No.: 084177.0330AAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCCGUGGCUGGGACGUGAAUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCUGAUGCCGCCAAGAUGAUCCCAAGGUGCAGCACCCAGCUGAAGGCCGUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUCCAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGCUUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGACCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAUAAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCGUGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGCACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACAttorney Docket No.: 084177.0330GUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGGAUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAGGAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGGUAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 82] mRNA #4(5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = N1 -Methyl-PseudoUTP; 3’ Tail Modification = 79A tail)AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGGUUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAGAACAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAttorney Docket No.: 084177.0330AUCCCACACCGCAUCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUCUGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUCAUCUCUGCCGCAGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACCCCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGAGAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCCGUGGCUGGGACGUGAAUGUGGAGAAGAACCGUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCUGAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCAttorney Docket No.: 084177.0330UGUUUUCUCCAGAGAACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUCCAGGAUGAAGAGGAUGGCAGCCCGGCUGGGAGAGAAGAUGCUGAACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGCUUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGACCGGCAUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAUAAGCUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCGUGCUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGCACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUCGAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAUCACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGACGUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCCUUCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAGAAGAACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAGAGAAUCGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGACCUGUAUCCUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUGUUCAGGGAUGGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCUCACGCCAUCGACACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGGAACUCCAAUGCCGCCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAUCUGAAUGGCGUGUGCUUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUGGACGCCGAUGCCAAUGGCGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUGCUGAAUCACCUGAAGGAGAGCAAGGAUCUGAAGCUGCAGAACGGCAUCUCCAAUCAGGACUGGCUGGCCUACAUCCAGGAGCUGCGCAACGGCGGAUCCCCUGCUGCUAAACGUGUUAAGCUUGAUGGGGGUAGCCCGGCAGCCAAGAGAGUCAAACUCGACUAGAUGGUUUAUAUUGCGGCCGCUUAAUUAAGCUGCCUUCUGAttorney Docket No.: 084177.0330CGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 83] mRNA #5(5’ Modification = CleanCap® AG 3’0me; Uridine Modifications = N1 -Methyl-Pseudo UTP; 3’ Tail Modification = 79A tail)AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGACACAGUUCGAGGGCUUUACCAACCUGUAUCAGGUGAGCAAGACACUGCGGUUUGAGCUGAUCCCACAGGGCAAGACCCUGAAGCACAUCCAGGAGCAGGGCUUCAUCGAGGAGGACAAGGCCCGCAAUGAUCACUACAAGGAGCUGAAGCCCAUCAUCGAUCGGAUCUACAAGACCUAUGCCGACCAGUGCCUGCAGCUGGUGCAGCUGGAUUGGGAGAACCUGAGCGCCGCCAUCGACUCCUAUAGAAAGGAGAAAACCGAGGAGACAAGGAACGCCCUGAUCGAGGAGCAGGCCACAUAUCGCAAUGCCAUCCACGACUACUUCAUCGGCCGGACAGACAACCUGACCGAUGCCAUCAAUAAGAGACACGCCGAGAUCUACAAGGGCCUGUUCAAGGCCGAGCUGUUUAAUGGCAAGGUGCUGAAGCAGCUGGGCACCGUGACCACAACCGAGCACGAGAACGCCCUGCUGCGGAGCUUCGACAAGUUUACAACCUACUUCUCCGGCUUUUAUGAAAACAGGAAGAACGUGUUCAGCGCCGAGGAUAUCAGCACAGCCAUCCCACACCGCAUCGUGCAGGACAACUUCCCCAAGUUUAAGGAGAAUUGUCACAUCUUCACACGCCUGAUCACCGCCGUGCCCAGCCUGCGGGAGCACUUUGAGAACGUGAAGAAGGCCAUCGGCAUCUUCGUGAGCACCUCCAUCGAGGAGGUGUUUUCCUUCCCUUUUUAUAACCAGCUGCUGACACAGACCCAGAUCGACCUGUAUAACCAGCUGCUGGGAGGAAUCUCUCGGGAGGCAGGCACCGAGAAGAUCAAGGGCCUGAACGAGGUGCUGAAUCUGGCCAUCCAGAAGAAUGAUGAGACAGCCCACAUCAUCGCCUCCCUGCCACACAGAUUCAUCCCCCUGUUUAAGCAGAUCCUGUCCGAUAGGAACACCCUGUCUUUCAUCCUGGAGGAGUUUAAGAGCGACGAGGAAGUGAUCCAGUCCUUCUGCAAGUACAAGACACUGCUGAGAAACGAGAACGUGCUGGAGACAGCCGAGGCCCUGUUUAACGAGCUGAACAGCAUCGACCUGACACACAUCUUCAUCAGCCACAAGAAGCUGGAGACAAUCAGCAGCGCCCUGUGCGACCACUGGGAUACACUGAGGAAUGCCCUGUAUGAGCGGAGAAUCUCCGAGCUGACAGGCAAGAUCACCAAGUCUGCCAAGGAGAAGGUGCAGCGCAGCCUGAAGCACGAGGAUAUCAACCUGCAGGAGAUCAUCUCUGCCGCAttorney Docket No.: 084177.0330AGGCAAGGAGCUGAGCGAGGCCUUCAAGCAGAAAACCAGCGAGAUCCUGUCCCACGCACACGCCGCCCUGGAUCAGCCACUGCCUACAACCCUGAAGAAGCAGGAGGAGAAGGAGAUCCUGAAGUCUCAGCUGGACAGCCUGCUGGGCCUGUACCACCUGCUGGACUGGUUUGCCGUGGAUGAGUCCAACGAGGUGGACCCCGAGUUCUCUGCCCGGCUGACCGGCAUCAAGCUGGAGAUGGAGCCUUCUCUGAGCUUCUACAACAAGGCCAGAAAUUAUGCCACCAAGAAGCCCUACUCCGUGGAGAAGUUCAAGCUGAACUUUCAGCGGCCUACACUGGCCUCUGGCUGGGACGUGAAUAAGGAGAAGAACAAUGGCGCCAUCCUGUUUGUGAAGAACGGCCUGUACUAUCUGGGCAUCAUGCCAAAGCAGAAGGGCAGGUAUAAGGCCCUGAGCUUCGAGCCCACAGAGAAAACCAGCGAGGGCUUUGAUAAGAUGUACUAUGACUACUUCCCUGAUGCCGCCAAGAUGAUCCCAAAGUGCAGCACCCAGCUGAAGGCCGUGACAGCCCACUUUCAGACCCACACAACCCCCAUCCUGCUGUCCAACAAUUUCAUCGAGCCUCUGGAGAUCACAAAGGAGAUCUACGACCUGAACAAUCCUGAGAAGGAGCCAAAGAAGUUUCAGACAGCCUACGCCAAGAAAACCGGCGACCAGAAGGGCUACAGAGAGGCCCUGUGCAAGUGGAUCGACUUCACAAGGGAUUUUCUGUCCAAGUAUACCAAGACAACCUCUAUCGAUCUGUCUAGCCUGCGGCCAUCCUCUCAGUAUAAGGACCUGGGCGAGUACUAUGCCGAGCUGAAUCCCCUGCUGUACCACAUCAGCUUCCAGAGAAUCGCCGAGAAGGAGAUCAUGGAUGCCGUGGAGACAGGCAAGCUGUACCUGUUCCAGAUCUAUAACAAGGACUUUGCCAAGGGCCACCACGGCAAGCCUAAUCUGCACACACUGUAUUGGACCGGCCUGUUUUCUCCAGAGAACCUGGCCAAGACAAGCAUCAAGCUGAAUGGCCAGGCCGAGCUGUUCUACCGCCCUAAGUCCAGGAUGAAGAGGAUGGCAGCUCGGCUGGGAGAGAAGAUGCUGAACAAGAAGCUGAAGGAUCAGAAAACCCCAAUCCCCGACACCCUGUACCAGGAGCUGUACGACUAUGUGAAUCACAGACUGUCCCACGACCUGUCUGAUGAGGCCAGGGCCCUGCUGCCCAACGUGAUCACCAAGGAGGUGUCUCACGAGAUCAUCAAGGAUAGGCGCUUUACCAGCGACAAGUUCUUGUUCCACGUGCCUAUCACACUGAACUAUCAGGCCGCCAAUUCCCCAUCUAAGUUCAACCAGAGGGUGAAUGCCUACCUGAAGGAGCACCCCGAGACACCUAUCAUCGGCAUCGAUCGGGGCGAGAGAAACCUGAUCUAUAUCACAGUGAUCGACUCCACCGGCAAGAUCCUGGAGCAGCGGAGCCUGAACACCAUCCAGCAGUUUGAUUACCAGAAGAAGCUGGACAACAGGGAGAAGGAGAGGGUGGCAGCAAGGCAGGCCUGGUCUGUGGUGGGCACAAUCAAGGAUCUGAAGCAGGGCUAUCUGAGCCAGGUCAUCCACGAGAUCGUGGACCUGAUGAUCCACUACCAGGCCGUGGUGGUGCUGGAGAACCUGAAUUUCGGCUUUAAGAGCAAGAGGACCGGCAttorney Docket No.: 084177.0330AUCGCCGAGAAGGCCGUGUACCAGCAGUUCGAGAAGAUGCUGAUCGAUAAG CUGAAUUGCCUGGUGCUGAAGGACUAUCCAGCAGAGAAAGUGGGAGGCGUG CUGAACCCAUACCAGCUGACAGACCAGUUCACCUCCUUUGCCAAGAUGGGC ACCCAGUCUGGCUUCCUGUUUUACGUGCCUGCCCCAUAUACAUCUAAGAUC GAUCCCCUGACCGGCUUCGUGGACCCCUUCGUGUGGAAAACCAUCAAGAAU CACGAGAGCCGCAAGCACUUCCUGGAGGGCUUCGACUUUCUGCACUACGAC GUGAAAACCGGCGACUUCAUCCUGCACUUUAAGAUGAACAGAAAUCUGUCC UUCCAGAGGGGCCUGCCCGGCUUUAUGCCUGCAUGGGAUAUCGUGUUCGAG AAGAACGAGACACAGUUUGACGCCAAGGGCACCCCUUUCAUCGCCGGCAAG AGAAUCGUGCCAGUGAUCGAGAAUCACAGAUUCACCGGCAGAUACCGGGAC CUGUAUCCUGCCAACGAGCUGAUCGCCCUGCUGGAGGAGAAGGGCAUCGUG UUCAGGGAUGGCUCCAACAUCCUGCCAAAGCUGCUGGAGAAUGACGAUUCU CACGCCAUCGACACCAUGGUGGCCCUGAUCCGCAGCGUGCUGCAGAUGCGG AACUCCAAUGCCGCCACAGGCGAGGACUAUAUCAACAGCCCCGUGCGCGAU CUGAAUGGCGUGUGCUUCGACUCCCGGUUUCAGAACCCAGAGUGGCCCAUG GACGCCGAUGCCAAUGGCGCCUACCACAUCGCCCUGAAGGGCCAGCUGCUG CUGAAUCACCUGAAGGAGAGCAAGGAUCUGAAGCUGCAGAACGGCAUCUCC AAUCAGGACUGGCUGGCCUACAUCCAGGAGCUGCGCAACGGCGGAUCCCCU GCUGCUAAACGUGUUAAGCUUGAUGGGGGUAGCCCGGCAGCCAAGAGAGUC AAACUCGACUAGAUGGUUUAUAUUGCGGCCGCUUAAUUAAGCUGCCUUCUG CGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUC UUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUAGAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 84]5.5 Functional analysis of candidate moleculesCandidate RNA-guided nucleases, gRNAs, and complexes thereof, can be evaluated by standard methods known in the art. See, e.g., Cotta-Ramusino. The stability of RNP complexes can be evaluated by differential scanning fluorimetry, as described below.5.5.1 Differential Scanning Fluorimetry (DSF)The thermostability of ribonucleoprotein (RNP) complexes comprising gRNAs and RNA-guided nucleases can be measured via DSF. The DSF technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding RNA molecule, e.g, a gRNA.Attorney Docket No.: 084177.0330A DSF assay can be performed according to any suitable protocol, and can be employed in any suitable setting, including without limitation (a) testing different conditions (e.g. different stoichiometric ratios of gRNA: RNA-guided nuclease protein, different buffer solutions, etc.) to identify optimal conditions for RNP complex formation; and (b) testing modifications (e.g. chemical modifications, alterations of sequence, etc.) of an RNA-guided nuclease and / or a gRNA to identify those modifications that improve RNP complex formation or stability. One readout of a DSF assay is a shift in melting temperature of the RNP complex; a relatively high shift suggests that the RNP complex is more stable (and can thus have greater activity or more favorable kinetics of formation, kinetics of degradation, or another functional characteristic) relative to a reference RNP complex characterized by a lower shift. When the DSF assay is deployed as a screening tool, a threshold melting temperature shift can be specified, so that the output is one or more RNP complexes having a melting temperature shift at or above the threshold. For instance, the threshold can be 5- 10°C (e.g., 5°C, 6°C, 7°C, 8°C, 9°C, 10°C) or more, and the output can be one or more RNP complexes characterized by a melting temperature shift greater than or equal to the threshold.Two non-limiting examples of DSF assay conditions are set forth below:To determine the best solution to form RNP complexes, a fixed concentration (e.g., 2 pM) of RNA-guided nuclease (e.g., Cas9 or Casl2a) in water+lOx SYPRO Orange® (Life Technologies cat#S-6650) is dispensed into a 384 well plate. An equimolar amount of gRNA diluted in solutions with varied pH and salt is then added. After incubating at room temperature for 10’ and brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System Cl 000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1°C increase in temperature every 10 seconds.The second assay involves mixing various concentrations of gRNA with fixed a concentration (e.g., 2 pM) of RNA-guided nuclease (e.g., Cas9 or Casl2a) in optimal buffer from assay 1 above and incubating (e.g., at RT for 10’) in a 384 well plate. An equal volume of optimal buffer + lOx SYPRO Orange® (Life Technologies cat#S-6650) is added and the plate sealed with Microseal® B adhesive (MSB-1001). Following brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System Cl 000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1°C increase in temperature every 10 seconds.Attorney Docket No.: 084177.03306. Genome editing strategiesThe genome editing systems described above are used, in various embodiments of the present disclosure, to generate edits in (i.e., to alter) targeted regions of DNA within or obtained from a cell. Various strategies are described herein to generate particular edits, and these strategies are generally described in terms of the desired repair outcome, the number and positioning of individual edits (e.g., SSBs or DSBs), and the target sites of such edits.Genome editing strategies that involve the formation of SSBs or DSBs are characterized by repair outcomes including: (a) deletion of all or part of a targeted region; (b) insertion into or replacement of all or part of a targeted region; or (c) interruption of all or part of a targeted region. This grouping is not intended to be limiting, or to be binding to any particular theory or model and is offered solely for economy of presentation. Skilled artisans will appreciate that the listed outcomes are not mutually exclusive and that some repairs can result in other outcomes. The description of a particular editing strategy or method should not be understood to require a particular repair outcome unless otherwise specified.Replacement of a targeted region in certain embodiments involves the replacement of all or part of the existing sequence within the targeted region with a homologous sequence, for instance through gene correction or gene conversion, two repair outcomes that are mediated by HDR pathways. HDR is promoted by the use of a donor template, which can be single-stranded or double stranded, as described in greater detail below. Single or double stranded templates can be exogenous, in which case they will promote gene correction, or they can be endogenous (e.g., a homologous sequence within the cellular genome), to promote gene conversion. Exogenous templates can have asymmetric overhangs (i.e., the portion of the template that is complementary to the site of the DSB can be offset in a 3’ or 5’ direction, rather than being centered within the donor template), for instance as described by Richardson et al. (Nature Biotechnology 34, 339-344 (2016), (Richardson), incorporated by reference). In instances where the template is single stranded, it can correspond to either the complementary (top) or non-complementary (bottom) strand of the targeted region.Gene conversion and gene correction are facilitated, in some cases, by the formation of one or more nicks in or around the targeted region, as described in Ran and Cotta- Ramusino. In some cases, a dual-nickase strategy is used to form two offset SSBs that, in turn, form a single DSB having an overhang (e.g., a 5’ overhang).Attorney Docket No.: 084177.0330Interruption and / or deletion of all or part of a targeted sequence can be achieved by a variety of repair outcomes. As one example, a sequence can be deleted by simultaneously generating two or more DSBs that flank a targeted region, which is then excised when the DSBs are repaired, as is described in Maeder for the LCA10 mutation. As another example, a sequence can be interrupted by a deletion generated by formation of a double strand break with single-stranded overhangs, followed by exonucleolytic processing of the overhangs prior to repair.One specific subset of target sequence interruptions is mediated by the formation of an indel within the targeted sequence, where the repair outcome is typically mediated by NHEJ pathways (including Alt-NHEJ). NHEJ is referred to as an “error prone” repair pathway because of its association with indel mutations. In some cases, however, a DSB is repaired by NHEJ without alteration of the sequence around it (a so-called “perfect” or “scarless” repair); this generally requires the two ends of the DSB to be perfectly ligated. Indels, meanwhile, are thought to arise from enzymatic processing of free DNA ends before they are ligated that adds and / or removes nucleotides from either or both strands of either or both free ends.Because the enzymatic processing of free DSB ends can be stochastic in nature, indel mutations tend to be variable, occurring along a distribution, and can be influenced by a variety of factors, including the specific target site, the cell type used, the genome editing strategy used, etc. Even so, it is possible to draw limited generalizations about indel formation: deletions formed by repair of a single DSB are most commonly in the 1-50 bp range but can reach greater than 100-200 bp. Insertions formed by repair of a single DSB tend to be shorter and often comprise short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells.Indel mutations and genome editing systems configured to produce indels are useful for interrupting target sequences, for example, when the generation of a specific final sequence is not required and / or where a frameshift mutation would be tolerated. They can also be useful in settings where particular sequences are preferred, insofar as the certain sequences desired tend to occur preferentially from the repair of an SSB or DSB at a given site. Indel mutations are also a useful tool for evaluating or screening the activity of particular genome editing systems and their components. In these and other settings, indels can be characterized by (a) their relative and absolute frequencies in the genomes of cellsAttorney Docket No.: 084177.0330 contacted with genome editing systems and (b) the distribution of numerical differences relative to the unedited sequence, e.g., ±1, ±2, ±3, etc. As one example, in a lead-finding setting, multiple gRNAs can be screened to identify those gRNAs that most efficiently drive cutting at a target site based on an indel readout under controlled conditions. Guides that produce indels at or above a threshold frequency, or that produce a particular distribution of indels, can be selected for further study and development. Indel frequency and distribution can also be useful as a readout for evaluating different genome editing system implementations or formulations and delivery methods, for instance by keeping the gRNA constant and varying certain other reaction conditions or delivery methods.6.1 Multiplex StrategiesWhile exemplary strategies discussed above have focused on repair outcomes mediated by single DSBs, genome editing systems according to this disclosure can also be employed to generate two or more DSBs, either in the same locus or in different loci. Strategies for editing that involve the formation of multiple DSBs, or SSBs, are described in, for instance, Cotta-Ramusino.6.2 Donor template designDonor template design is described in detail in the literature, for instance in Cotta- Ramusino. DNA oligomer donor templates (oligodeoxynucleotides or ODNs), which can be single stranded (ssODNs) or double-stranded (dsODNs), can be used to facilitate HDR- based repair of DSBs, and are particularly useful for introducing alterations into a target DNA sequence, inserting a new sequence into the target sequence, or replacing the target sequence altogether.Whether single-stranded or double stranded, donor templates generally comprise regions that are homologous to regions of DNA within or near (e.g., flanking or adjoining) a target sequence to be cleaved. These homologous regions are referred to here as “homology arms,” and are illustrated schematically below:[5’ homology arm] — [replacement sequence] — [3’ homology arm].The homology arms can have any suitable length (including 0 nucleotides if only one homology arm is used), and 3’ and 5’ homology arms can have the same length or can differ in length. The selection of appropriate homology arm lengths can be influenced by a variety of factors, such as the desire to avoid homologies or microhomologies with certain sequences such as Alu repeats or other very common elements. For example, a 5’ homology arm can be shortened to avoid a sequence repeat element. In other embodiments, a 3’Attorney Docket No.: 084177.0330 homology arm can be shortened to avoid a sequence repeat element. In certain embodiments, both the 5’ and the 3’ homology arms can be shortened to avoid including certain sequence repeat elements. In addition, some homology arm designs can improve the efficiency of editing or increase the frequency of a desired repair outcome. For example, Richardson et al. Nature Biotechnology 34, 339-344 (2016) (Richardson), which is incorporated by reference, found that the relative asymmetry of 3’ and 5’ homology arms of single stranded donor templates influenced repair rates and / or outcomes.Replacement sequences in donor templates have been described elsewhere, including in Cotta-Ramusino et al. A replacement sequence can be any suitable length (including zero nucleotides, where the desired repair outcome is a deletion), and typically comprises one, two, three or more sequence modifications relative to the naturally occurring sequence within a cell in which editing is desired. One exemplary sequence modification involves the alteration of the naturally occurring sequence to repair a mutation that is related to a disease or condition of which treatment is desired. Another exemplary sequence modification involves the alteration of one or more sequences that are complementary to, or code for, the PAM sequence of the RNA-guided nuclease or the targeting domain of the gRNA(s) being used to generate an SSB or DSB, to reduce or eliminate repeated cleavage of the target site after the replacement sequence has been incorporated into the target site.Where a linear ssODN is used, it can be configured to (i) anneal to the nicked strand of the target nucleic acid, (ii) anneal to the intact strand of the target nucleic acid, (iii) anneal to the plus strand of the target nucleic acid, and / or (iv) anneal to the minus strand of the target nucleic acid. An ssODN can have any suitable length, e.g., about, at least, or no more than 150-200 nucleotides (e.g., 150, 160, 170, 180, 190, or 200 nucleotides).It should be noted that a template nucleic acid can also be a nucleic acid vector, such as a viral genome or circular double stranded DNA, e.g., a plasmid. Nucleic acid vectors comprising donor templates can comprise other coding or non-coding elements. For example, a template nucleic acid can be delivered as part of a viral genome (e.g., in an AAV or lentiviral genome) that comprises certain genomic backbone elements (e.g., inverted terminal repeats, in the case of an AAV genome) and optionally comprises additional sequences coding for a gRNA and / or an RNA-guided nuclease. In certain embodiments, the donor template can be adjacent to, or flanked by, target sites recognized by one or more gRNAs, to facilitate the formation of free DSBs on one or both ends of the donor template that can participate in repair of corresponding SSBs or DSBs formed in cellular DNA usingAttorney Docket No.: 084177.0330 the same gRNAs. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta-Ramusino.Whatever format is used, a template nucleic acid can be designed to avoid undesirable sequences. In certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.6.3 Quantitative measurements of on-target and off-target gene editingIt should be noted that the genome editing systems of the present disclosure allow for the detection and quantitative measurement of on-target and off-target gene editing outcomes. The compositions and methods described herein can rely on the use of PCR primer sequences to amplify the genomic locus comprising the expected cut site of the RNA- guided nuclease. In some embodiments, the primers comprise an adaptor tail for use in a two-step PCR amplification process to prepare amplicon libraries for Next Generation Sequencing (NGS) analysis. A non-limiting example for primers and amplification site for assessing the on-target genome editing efficiency of an ANGPTL3 target site set forth in [SEQ ID NO: 26] are found in Table 9. Another non-limiting example for the primers and amplification site used for assessing the on-target genome editing efficiency of an ANGPTL3 target site set forth in [SEQ ID NO: 31] are found in Table 10.Table 9: First Exemplary Primer and Amplicon Sequences for an On-Target cut site analysisAttorney Docket No.: 084177.0330Table 10: Second Exemplary Primer and Amplicon Sequences for an On-Target cut site analysisIn certain embodiments, the genome editing systems or RNP complexes disclosed herein have minimal or no off-target effects. In certain embodiments, the off-target effect of a genome editing system or RNP complex is measured by Digenome-seq analysis (Kim et al., Nature Methods (2015); 12:237-243). In certain embodiments, the off-target effect of a genome editing system or RNP complex is indicated by an off-target count as measured by Digenome-seq analysis. In certain embodiments, the off-target count is measured by Digenome-seq analysis at 1000 nM of the RNP complex. In certain embodiments, the off- target count is measured by the Digenome-seq analysis at 100 nM of the RNP complex.In certain embodiments, the off-target count as measured by Digenome-seq analysis of an RNP complex disclosed herein at 1000 nM is less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1. In certain embodiments, the off-target count of an RNP complex disclosed herein as measured by Digenome-seq atAttorney Docket No.: 084177.03301000 nM is zero or is about zero. In certain embodiments, the off-target count of an RNP complex disclosed herein as measured by Digenome-seq at 100 nM is less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1. In certain embodiments, the off-target count of an RNP complex disclosed herein as measured by Digenome-seq at 100 nM is zero or is about zero.In some embodiments, a quantitative method of assessing the on-target and off- target sites, e.g., GUIDE-Seq, includes the integration of an exogenous double stranded oligo nucleotide (dsODN) tag into the genome. For example, Tsai et al., 2016; Tsai et al., 2014; and Tycko et., 2016, which are incorporated by reference herein in their entirety, describe compositions and methods which allow for the quantitative analysis of off-target and on-target gene editing outcomes, by the integration of a dsODN into RNA guided nuclease (RGN) induced double strand breaks (DSBs). In some embodiments, the dsODN tag is a 34 bp, blunt, 5’ phosphorylated, phosphorothioate linked polynucleotide that becomes incorporated into double stand breaks. The dsODN tag contains a priming site that allows for the amplification, sequencing, and discovery of RNG induced double strand breaks. Non-limiting examples of the dsODN tag and primer are set forth in Table 11.Table 11: Exemplary Primer and Amplicon Sequences for an Off-Target cut site analysisP represents a 5' phosphorylation and * indicates a phosphorothioate linkage.Attorney Docket No.: 084177.03307. Implementation of genome editing systems: Delivery, Formulations, and Routes of AdministrationAs discussed above, the genome editing systems of this disclosure can be implemented in any suitable manner, meaning that the components of such systems, including without limitation the RNA-guided nuclease, gRNA, and optional donor template nucleic acid, can be delivered, formulated, or administered in any suitable form or combination of forms that results in the transduction, transfection, expression or introduction of a genome editing system and / or causes a desired repair outcome in a cell, tissue or subject. The genome editing systems according to this disclosure can incorporate multiple gRNAs, multiple RNA-guided nucleases (or polynucleotides (e.g., mRNA) encoding such RNA-guided nucleases), and other components such as proteins, and a variety of implementations will be evident to the skilled artisan based on the principles illustrated in systems of the disclosure. In some embodiments the genome editing system of the disclosure is delivered into cells as a ribonucleoprotein (RNP) complex. In some embodiments, one or more RNP complexes are delivered to the cell sequentially in any order, or simultaneously. In some embodiments the genome editing system described herein is delivered into cells (e.g., via an LNP) as a gRNA and an RNA encoding an RNA-guided nuclease protein. Tables 12 and 13 set forth several, non-limiting examples of genome editing system implementations. Those of skill in the art will appreciate, however, that these listings are not comprehensive, and that other implementations are possible. With reference to Table 12 in particular, the table lists several exemplary implementations of a genome editing system comprising a single gRNA and an optional donor template. However, genome editing systems according to this disclosure can incorporate multiple gRNAs, multiple RNA-guided nucleases, and other components such as proteins, and a variety of implementations will be evident to the skilled artisan based on the principles illustrated in the table. In the table, [N / A] indicates that the genome editing system does not comprise the indicated component.Table 12: Genome Editing System ComponentsAttorney Docket No.: 084177.0330Table 13 summarizes various delivery methods for the components of genome editing systems, as described herein. Again, the listing is intended to be exemplary rather than limiting.Attorney Docket No.: 084177.0330Attorney Docket No.: 084177.03307.1 Nucleic acid-based delivery of genome editing systemsNucleic acids encoding the various elements of a genome editing system according to the present disclosure can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, RNA-guided nuclease-encoding and / or gRNA-encoding DNA, as well as donor template nucleic acids can be delivered by, e.g., vectors (e.g., viral or non-viral vectors), non -vector-based methods (e.g., using naked DNA or DNA complexes), or a combination thereof. In some embodiments the genome editing system of the disclosure are delivered by AAV. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. In certain embodiments, the AAV serotypes can be matched to target cell types. For example, in certain non-limiting embodiments, AAV serotypes selected from the group consisting of AAV3, AAV5, AAV8, and AAV9 can be used to target liver cells.7.1.1 Non-Viral VectorsNon-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. In one embodiment, the vector is a lipid nanoparticle (LNP).In certain embodiments, the LNP can have a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 1000 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 500 nm. In some embodiments, the LNP has a size ranging from about 1 nm to about 250 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 200 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 100 nm. In some embodiments, the LNP has a size ranging from about 35 nm to about 75 nm. In some embodiments, the LNP has a size ranging from about 25 nm to about 60 nm.Attorney Docket No.: 084177.0330In certain embodiments, but not by way of any limitation, LNPs can be made from ionizable lipids (e.g., cationic lipids), neutral lipids, structural lipids, helper lipids, and PEGylated lipids, or a combination of these. Exemplary lipids and polymers that can be used in LNP formulations, and / or gene transfer. In some embodiments, fusogenic phospholipids (e.g., DOPE) and / or sterols (e.g., cholesterol), may be included in LNPs as ‘helper lipids’ to enhance transfection activity and / or LNP stability. Exemplary lipids for use in nanoparticle formulations, and / or gene transfer are shown in Table 14, and Table 15.Table 14: Lipids Used for LNPs and / or Gene TransferAttorney Docket No.: 084177.0330Table 15: Polymers Used for LNPs and / or Gene TransferAttorney Docket No.: 084177.0330In certain embodiments, the LNPs of the present disclosure comprise a cationic lipid, e.g., those cationic lipids disclosed in Table 14. In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA); 1,2- dilinolenyloxyN, N-dimethylaminopropane (DLenDMA); 2,2-dilinoleyl-4-(2-dimethyl- aminoethyl)-[l,3]dioxolane (DLin-K-C2-DMA.; '"XTC2"'); 2,2-dilinoleyl-4-(3-dimethyl- aminoopropyl)-[l,3]-di oxolane (DLin-K-C3-DMA); 2,2-dilinoieyl-4-(4- dimethylaminobutyl)-ll,3]-di oxolane (DLinK-C4-DMA); 2,2-dilinoleyl-5- dimethylaminomethyl-[J ,3]-dioxane (DLin-K6-DMA); 2,2-dilinoleyl-4-N- methylpepiazino-[l,3]-di oxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl- [l,3]-di oxolane (DLin-K-DMA); l,2-dilinoleylcarbamoyl-oxy-3 -dim. ethylaminopropane (DLin-C-DAP); 1,2-dilinoley oxy-3 -(dimethylamino) acetoxy-propane (DLin-MAC); 1,2- dilinoley oxy-3 -morpholinopropane (DLin-MA); L2-dilinoleoyl-3 -dimethylaminopropaneAttorney Docket No.: 084177.0330(DLinDAP), l,2-dilinoleylthio-3 -dimethylaminopropane (DLin-SDMA); l-linoleoyi-2- linoleyloxy-3 -dimethylaminopropane (DLin-2-DMAP); l,2-dilinoleyloxy-3- trirmethylaminopropane chloride salt (DLin-TMA Cl), 1 ,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP Cl); l,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ); 3-(N,N-dilinoleylamino)-l,2-propanediol (DLinAP); 3-(N,N-dioleylamino)-l ,2-propanedio (DOAP); l,2-dilinoleyloxo-3-(2- N,Ndimethylamino) ethoxypropane (DLin-EG-DMA); N,N-dioleyl-N,N- dimethylammonium chloride (DODAC); l,2-dioleyloxy-N,N-dimethylanrinopropane (DODMA); l,2-distearyloxyN,N-dimethylamino-propane (DSDMA); N-(l-(2,3-dioley loxy)propyl)-N,N,N-trirnethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB); N-(l-(2,3-dioleoyloxy) propyl)-N,N,N- trimethylammonium chloride (DOTAP); 3-(N - (N',N'-dimethylamino-ethane)- carbanloyl)cholesterol (DC-Chol); N-(l,2-dim.yristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE); 2,3- dioleyioxy-N-[2(spermine- carboxmnido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA); dioctadecyl-amidoglycyl spermine (DOGS); 3-dimethylamino-2-(cholest-5-en-3- betaoxybutan-4-oxy)-l-(cis,cis-9, 12-octadecadienoxy)propane (CLinDMA); 2-[ 5'- (cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy l-l-(cis,cis-9', l-2'-octadecadienoxy) propane (CpLinDMA); 1,2-Dilinoleoy Icarbamy 1-3-dimethyiaminopropane (DLinCDAP); N-dimethyi-3,4-dioleyloxybenzylamine (DMOBA); and l,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP).In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoase or a pharmaceutically acceptable salt thereof. In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: (9Z,9'Z,12Z,12'Z)-2-(((3-(4- methylpiperazin-l-yl)propanoyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z)-2-(((4-(pyrrolidin-l-yl)butanoyl)oxy)methyl)propane-l,3- diylbis(octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z)-2-(((4-(piperidin-l- yl)butanoyl)oxy)methyl)propane- 1,3 -diyl bis(octadeca-9,12-dienoate); (9Z,9Z,12Z,2'Z)-2- (((l,4-dimethylpiperidine-4-carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9,12- dienoate); (9Z,9'Z,12Z,12'Z)-2-(((l-(cyclopropylmethyl)piperidine-4- carbonyl)oxy)methyl)propane- 1,3 -diyl bis(octadeca-9,12-dienoate); (9Z,12Z)-3-((4,4-Attorney Docket No.: 084177.0330 bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl) propyl octadeca-9,12-di enoate; (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l- ethylpiperi din-3 -yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate; 2-((((2- (diethylamino)ethoxy)carbonyl)oxy)methyl)propane-l,3-diylbis(2 -heptylundecanoate); (9Z,12Z)-3-(((2-(diethylamino)ethoxy)carbonyl)oxy)-2-(((2-heptylundecanoyl)oxy) methyl)propyl octadeca-9, 12-di enoate; 2-((((3-(dimethylamino)propoxy)carbonyl) oxy)methyl)propane- 1 , 3 -diyl bi s(2-heptylundecanoate); (9Z, 12Z)-3 -(((3 -(diethylamino)propoxy)carbonyl)oxy)-2-(((2-heptylundecanoyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9Z, 12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)-2-(((3- octylundecanoyl)oxy)methyl)propyl octadeca-9, 12-di enoate; 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane- 1,3 -diyl bis(3-octylundecanoate);(9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3- octylundecanoyl)oxy)methyl )propyl octadeca-9, 12-di enoate; (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((7-hexyltridecaonoyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9Z, 12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((9- pentyltetradecanoyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9Z, 12Z)-3-(((3- (diethylamino)propoxy)cabonyl)oxy)-2-(((5-heptyldodecanoyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9, 12Z)-3-(2,2-bis(heptyloxy)acetoxy)-2-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate; (9Z, 12Z)-3- ((6,6-bis(octyloxy)hexanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate; 2-(3-ethyl- l l-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)-8,14-dioxo-7,9,13-trioxa-3- azaheptadecan- 17 -yl)propane- 1 ,3 -diyl dioctanoate; (9Z,9'Z, 12Z, 12Z)-2-((((3 -(dimethylamino)propoxy)carbonyl)oxy)methyl)propane- 1,3 -diyl bis(octadeca-9,12- dienoate); (9Z,9'Z,12Z,12'Z)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane- 1,3 -diyl bis(octadeca-9,12- dienoate); (9Z,9'Z,12Z,12'Z)-2-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)propane-l,3-diyl bis(octadeca-9,12- dienoate); 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z, 12Z)-octadeca-9, 12- dienoyloxy)methyl)propyl l-isopropylpiperidine-4-carboxylate; 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z, 12Z)-octadeca-9, 12-di enoyloxy)methyl)propyl 1- (cyclopropylmethyl)piperidine-4-carboxylate; 3-((4,4-bis(octyl oxy)butanoyl)oxy)-2- (((9Z,12Z)-octadeca-9, 12-di enoyloxy)methyl)propyl l-methylpyrrolidine-3 -carboxylate; (9Z,9'Z,12Z,12Z)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((9Z,12Z)-Attorney Docket No.: 084177.0330 octadeca-9, 12-di enoy loxy)methyl)propane- 1,3 -diyl bis(octadeca-9, 12-dienoate); (2S)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl l-methylpyrrolidine-2-carboxylate; (2R)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9, 12-di enoyloxy)methyl)propyl l-methylpyrrolidine-2-carboxylate; 3 -((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z, 12Z)-octadeca-9, 12-di enoyloxy)methyl)propyl 4- methylmorpholine-2-carboxylate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9, 12-di enoyl oxy)methyl)propyl l,4-dimethylpiperidine-4-carboxylate; 3 -((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z, 12Z)-octadeca-9, 12-di enoyloxy)methyl)propyl 1- methylpiperidine-4-carboxylate; (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l- methylpyrrolidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z, 12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l-methylazetidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l- ethylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z, 12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpiperidin-4- yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l, 2,2,6, 6-pentamethylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate; (9Z, 12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propyl)carbamoyl)oxy)methyl)propyl octadeca-9, 12-dienoate; 3-((4,4-bis((2-propylpentyl)oxy)butanoyl)oxy)-2-(((9Z, 12Z)- octadeca-9, 12-di enoyloxy)methyl)propyl l,4-dimethylpiperidine-4-carboxylate; 3 -((6,6- bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((9Z,12Z)-octadeca-9, 12- di enoyloxy )methyl)propyl 1 ,4-dimethylpiperidine-4-carboxylate; 2-(((l - methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diyl bis(4,4- bis(octyloxy)butanoate); 2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3- diyl bis(6,6-bis(octyloxy)hexanoate); 2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propane- 1,3 -diyl bis(6,6-bis((2-propylpentyl)oxy)hexanoate); 2-(5- (3-(dodecanoyloxy)-2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propoxy)-5- oxopentyl)propane- 1,3 -diyl dioctanoate; 2-(5-(3-((l-methylpyrrolidine-3-carbonyl)oxy)-2- ((palmitoyloxy)methyl)propoxy)-5-oxopentyl)propane-l,3-diyl dioctanoate; 2-(S-(3-((l- methylpyrrolidine-3-carbonyl)oxy)-2-((tetradecanoyloxy)methyl)propoxy)-S- oxopentyl)propane- 1,3 -diyl dioctanoate; 3-((4,4-bis(octyl oxy)butanoyl)oxy)-2- ((dodecanoyloxy)methyl)propyl l-methylpyrrolidine-3 -carboxylate; 3 -((4,4-Attorney Docket No.: 084177.0330 bis(octyloxy)butanoyl)oxy)-2-((tetradecanoyloxy)methyl)propyl l-methylpyrrolidine-3-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((palmitoyloxy)methyl)propyl l-methylpyrrolidine-3- carboxylate; l-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((l-methylpyrrolidine-3- carbonyl)oxy)methyl)propyl) 8-methyl octanedioate; l-(3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propyl) 10- octyl decanedioate; l-(3-((6,6-bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4- dimethylpiperidine-4 carbonyl)oxy)methyl)propyl) 10-octyl decanedioate; l-(3-((6,6- bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4-dimethylpiperidine-4- carbonyl)oxy)methyl)propyl) 8-methyl octanedioate; and 8-dimethyl O'l,Ol-(2-(((l- methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diyl) dioctanedioate.In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: ((4,4'-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy))bis(propane- 2, 1,3-triyl) tetranonanoate; ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis (butanoyl))bis(oxy)) bis(propane-2, 1,3-triyl) tetraoctanoate; bis(l,3- bis(Nonanoyloxy)propan-2-yl) 5-((4-(dimethylamino)butanoyl)oxy)nonanedioate HCI salt; bis(l,3-bis(Octanoyloxy) propan-2-yl) 5-((4-(dimethylamino) butanoyl)thio)nonanedioate; bis(l,3-bis(N onanoyloxy) propan-2-yl) 4-((4-(dimethylamino) b utanoyl)oxy)heptanedioate; bis(l,3-bis(Octanoyloxy) propan-2-yl) 4-((4-(dimethylamino) butanoyl)thio)heptanedioate; ((2,2'-((((3-(Dimethyl-amino)propyl)thio) carbonyl)azanediyl)bis(acetyl))bis(oxy))bis(propane-2, 1,3-triyl) tetranon-anoate; bis(l,3- bis(N onanoyloxy)propan-2-yl) 4-((4-(dimethylamino) butanoyl)thio) heptanedioate; ((4,4'- ((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis (butanoyl)) bi s(oxy))bis(propane-2, 1,3-triyl) tetrakis(3-cyclohexylpropanoate); ((4,4'-((((3-(Dimethyl- amino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2, 1,3-triyl) tetrakis(4-cyclohexyl butanoate); ((6,6'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanedi-yl)bis(hexanoyl))bis(oxy))bis(propane-2,l,3-triyl)tetrakis(3- cyclohexylpropanoate); Nonanoic acid 2-(3-{(3-dimethylaminopropylsulfanylcarbonyl)- [2-(2-nonanoyloxy-l-nonanoyloxymethyl-ethoxycarbonyl)-ethyl]amino}-propionyloxy)- 3-octanoyloxy-propyl ester; ((4,4'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy))bis (propane-2, 1,3-triyl) tetrakis(2-(4-methylcyclohexyl) acetate); ((4,4 '-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2, 1,3-triyl) tetrakis(4-ethylcyclo hexane-1- carboxylate); ((4, 4'-((((3 -(Dimethylamino) propyl)thio)carbonyl) azanediyl)bis(butanoyl))Attorney Docket No.: 084177.0330 bis(oxy))bis(propane-2, 1 ,3 -triyl) tetrakis(3-cyclohexyl-2-methylpropanoate); ((4,4'-((((3 - (Dimethylamino)propyl)thio)carbonyl) azanediyl)bis(butanoyl)) bis(oxy))bis(propane-2.1.3-triyl) tetrakis(2-methyloctanoate); ((4,4 '-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(butanoyl)) bis(oxy))bis(propane-2.1.3-triyl)tetrakis(2,2-dimethylheptanoate); ((3,3'-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl) bis(propanoyl)) bis(oxy))bis (methylene))bis(2- methylpropane-2, 1,3 -triyl) tetrakis(3-(4-methyl cyclohexyl)propanoate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(propanoyl))bis(oxy)) bis (methylene)) bis(2-methyl propane-2, 1,3-triyl) tetrakis(2-(4-ethylcyclohexyl) acetate); ((3,3'-((((3-(Dimethylamino)propyl)thio)carbonyl) azanediyl) bis(propanoyl))bis(oxy)) bis(methylene)) bis(2-methyl propane-2, 1,3-triyl) tetrakis(2-(4-ethylcyclohexyl)acetate); ((3,3'-((((3- (Dimethylamino)propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methylene)) bis (2-methylpropane-2, l,3-triyl)tetrakis(3,3-dimethylheptanoate); ((3,3'-((((3- (Dimethylamino) propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methyl- ene))bis(2-methyl-propane-2, 1,3-triyl) tetrakis(octanoate); ((4,4'-((((3-(Dimethylamino) propyl)thio)carbonyl) azanediyl) bis(butanoyl))bis(oxy)) bis(methylene))bis(propane-2.1.3-triyl) tetranonanoate; ((3,3'-((((2-(Dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy)) bi s(methylene))bi s(propane-2, 1 , 3 -triyl)tetranonanoate; ((3 , 3 '-((((3 -(Dimethylamino)propyl) thio)carbonyl) azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis (propane-2, 1,3-triyl) tetrakis(3-(4- methylcyclohexyl)propanoate); ((4,4'-((((3-(Dimethyl-amino)propyl)thio) carbonyl)azanediyl) bis(butanoyl))bis (oxy )bis(propane-2, 1,3-triyl) tetrakis(octahydro-lH- indene); ((4,4 '-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl) bis(butanoyl))bis(oxy) ) bis(propane-2,l,3-triyl)tetrakis(octahydro-lH-indene-5- carboxylate); ((4,4'-(((3-(dimethyl-amino)propoxy)carbonyl) azanediyl)bis(butanoyl))bis(oxy)) bis(propane-2, 1,3-triyl) tetranonanoate; ((4,4'-(((3- (dimethylamino)propyl)carbamoyl) azanediyl)bis (butanoyl)) bis(oxy))bis(propane-2,l,3- triyl)tetranonanoate; ((4, 4'-((((3 -(dimethylamino) propyl)thio) carbonyl)azanediyl)bis(butanoyl)) bis(oxy)) bis(propane-2, 1,3-triyl) tetrakis(2-(p- tolyl)acetate); [2-[4-[3-(dimethylamino)propoxycarbonyl-[4-[2-(2-methyloctanoyloxy)-l- (2-methyloctanoyloxymethyl)ethoxy]-4-oxobutyl] amino]butanoyloxy]-3-(2- methyloctanoyl-oxy)propyl] 2-methyloctanoate; [2-[4-[3-Attorney Docket No.: 084177.0330(dimethylamino)propylcarbamoyl-[4-[2-(2-methyloctanoyl oxy)-l-(2- methyloctanoyloxymethyl)ethoxy ]-4-oxobutyl] amino ]butanoyl-oxy]-3-(2- methyloctanoyloxy)propyl] 2-methyloctanoate; [2-[4-[[3-(dimethylamino)propyl-methyl- carbamoyl][4-[2-(2-methyloctanoyloxy)-l-(2-methyloctanoyloxymethyl)ethoxy]-4- oxobutyl] amino] butanoyloxy]-3-(2-methyloctanoyloxy)propyl] 2-methyloctanoate; [2-[4- [5-(dimethylamino)pentanoyl-[4-[2-(2-methyloctanoyloxy)-l-(2-methyloctanoyloxym ethyl)ethoxy]-4-oxo-butyl] amino] butanoyloxy]-3-(2-methyloctanoyloxy)propyl] 2- methyloctanoate; [2-[4-[5-(dimethylamino)pentyl-[4-[2-(2-methyl octanoyloxy)-l-(2- methyl-octanoyloxym ethyl)ethoxy]-4-oxo-butyl] amino] butanoyloxy]-3-(2- methyloctanoyl-oxy)propyl] 2-methyloctanoate; bis [2-(2-methyloctanoyloxy)-l-(2- methyloctanoyl oxym ethyl)ethyl] 5-[ 4-(dimethylamino)butanoyloxy] nonanedioate; and ((4,4 '-((((3 -(dimethylamino) propyl)thio)carbonyl)azanediyl)bis(butanoyl)) bis(oxy)) bis(propane-2, 1,3 -triyl) tetrakis(2-methyl-4-(p-tolyl) butanoate).In certain embodiments, the cationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to: 6,6'- (Methylazanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-(Octylazanediyl)Bis(N,N-Dioctylhexan-amide); 6,6'-(Hexylazanediyl)Bis(N,N-Dioctylhexanamide); 10, 10'-(Methy 1 azanediy 1 )B i s (N,N -Di octyl decan ami de) ; 8,8' -(Methyl azanediy 1)B i s(N,N -Didecyloctanamide); 6,6'-(Methylazanediyl)Bis(N,N-Didecylhexanamide); 6,6'- (Methylazanediyl)Bis(N,N-Didodecyl-hexanamide); 6,6'-((2-Hydroxyethyl)Azanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctylhexanamide); 6,6'-((2-Hydroxyethyl)Azanediyl) Bis(N,N-Didecylhexanamide); 8,8'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctyloct-anamide); 10, 10'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Dioctyldecanamide); 10, 10'-((2-Hydroxy ethyl) Azanediy 1 )B i s(N,N -Didecyldecanamide); 8 , 8 ' - (( 5 -Hydroxypentyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8,8'-((4-Hydroxybutyl)Azanediyl)Bis(N,N-Didecyloct-anamide); 8,8'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8, 8'-((2 -Hydroxy - ethyl)Azanediyl)Bis(N,N-Didecyloctanamide); 10, 10'-((4-Hydroxybutyl)Azanediyl)Bis(N,N-Didecyldecanamide); N,N'-( (Methyl azanediy 1)Bis(Hexane-6, 1-Diyl) )Bis(N,2-Dihexyldecanamide); N,N'-((Methylazanediyl)Bis(Hexane-6, l-Diyl))Bis(N-Hexyl Palmitamide); (9z,9'z,12z,12'z)- N,N'-((Methylazanediyl)Bis(Hexane-6,L-Diyl))Bis(N-Hexyloctadeca-9,L2-Dienamide);Attorney Docket No.: 084177.0330N,N-Didecyl-8-((8-(Hexadecylamino)-8-Oxooctyl) (Methyl)Amino)Octanamide; 8,8'-( (8-(Decylamino )-8-Oxooctyl )Azanediyl )Bis(N,N-Didecyloctanamide); 8,8'-((6- (Dihexylamino)-6-Oxohexyl)Azanediyl)Bis(N,N-Didecyloctan-amide); 8,8'-((5-(Decylamino)-5-Oxopentyl)Azanediyl)Bis(N,N-Didecyloctanamide); 6,6'-( (8-(Decylamino )-8-Oxooctyl )Azanediyl )Bis(N,N-Didecylhexanamide); 6,6'-((2- (Dihexylamino)Ethyl)Azanediyl)Bis(N,N-Didecylhexanamide); 10,10'-((2-(Dimethyl- amino)Ethyl)Azanediyl)Bis(N,N-Didecyldecanamide); 2-Butyloctyl 6-(Bis(6-(Dioctylamino)-6-Oxohexyl)Amino)Hexanamide; 6,6'-((4-Hydroxybutyl)Azanediyl)Bis(N,N-Bis(2-Ethylhexyl)Hexanamide); 8,8'-((2-Hydroxyethyl)Azanediyl)Bis(N,N-Didodecyl-octanamide); 6,6'-((6-Hydroxyhexyl)Azanediyl)Bis(N,N-Didodecylhexanamide); N,N-Didecyl-8-((8-(Hexadecyl(Methyl)Amino)-8-Oxooctyl)(Methyl )Amino)Octanamide; 8,8'-(Methylazanediyl)Bis(N,N-Didodecyloctanamide); 8,8'-((3-Hydroxypropyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8,8'-( (2-(2-Hydroxyethoxy)Ethyl )Azanediyl )Bis(N,N- Di decyl octanami de); 8,8'-((5-Hydroxy-4,4-Dimethylpentyl)Azanediyl)Bis(N,N-Didecyl- octanamide); 8,8'-((3-(2-Methyl-Lh-Imidazol-L-Yl)Propyl)Azanediyl)Bis(N,N-Didecyloctan-amide); 8,8'-((7-Hydroxyheptyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-( (2-(2 -Methoxyethoxy )Ethyl)Azanediyl)Bis(N,N-Didecyloctanami de); 8,8'-((8-Hydroxyoctyl) Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((3-(Lh-Imidazol-L- Yl)Propyl)Azanediyl) Bis(N,N-Didecyloctanamide); 8, 8'-((2,2-Difluoro-3 -Hydroxypropyl) Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((3-((2-(Methylamino)-3,4-Dioxocyclobut- L-En-Lyl) Amino) Propyl)Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((2-Fluoro-3- Hydroxypropyl) Azanediyl)Bis(N,N-Didecyloctanamide); 8,8'-((3,3,3-Trifluoro-2- (Hydroxymethyl)Propyl) Azanediyl) Bis(N,Ndidecyloctanamide); 8,8'-((5- Methoxypentyl)Azanediyl)Bis(N,N-Didecyloctanamide); N,N-Didecyl-8-((8-(Dioctylamino)-8-Oxooctyl)(Methyl)Amino) Octanamide; Tert-Butyl (3-(Bis(10- (Didecylamino)-10-oxodecyl)Amino)Propyl)Carbamate; 10,10'-((3-(Lh-Imidazol-L- Yl)Propyl)Azanediyl)Bis(N,N-Didecyldecanamide); 8,8'-(Methylazanediyl)Bis(N,N- Dinonyloctanamide); Tert-Butyl (3-(Bis( 10-(Didecylamino )-10-oxodecyl )Amino )Propyl ) Carbamate; 10, 10'-((3-((2-(Methylamino)-3,4-Dioxocyclobut-L-En-Lyl)Amino)Propyl) Azanediyl) Bis(N,N-Didecyldecanamide); N.N'-((methylazanediyl) bis(octane-8, 1- diyl))bis(N-hexylhexanamide); N.N'-(((5-hydroxypentyl)azanediyl)bis (octane-8, 1- diyl))bis(N-hexylhexanamide); N.N'-((methylazanediyl)bis(octane-8, l-diyl))bis(N- octyloctanamide); N.N'-(((5-hydroxypentyl)azanediyl)bis(octane-8, l-diyl))bis(N-Attorney Docket No.: 084177.0330 octyloctanamide); N,N'-((methylazanediyl)bis(octane-8, l-diyl))bis(N-octyloctanamide); N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,l-diyl))bis(N-decyldecanamide); N,N'- ((methylazanediyl)bis(octane-8, 1 -diyl))bis(N-dodecyldodecanamide); N,N'-(((5- hydroxypentyl)azanediyl)bis( octane-8, l-diyl))bis(Ndodecyldodecanamide); N,N'- ((methylazanediyl)bis(octane-8, l-diyl))bis(2-hexyldecanamide); N,N'-((methylazanediyl)bis (octane-8, l-diyl))bis(2-hexyl-N-methyldecanamide); N,N'-(((5- hydroxypentyl)azanediyl)bis (octane-8, l-diyl))bis(2-hexyldecanamide); N,N'-(((5- hydroxypentyl)azanediyl)bis( octane-8, l-diyl))bis(2-hexyl-N methyldecanamide); N- decyl-N-(8-((8-(didecylamino)-8 oxooctyl) (methyl) amino) octyl)decanamide; N-decyl-N- (8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl)decanamide; 6,6'-((5- hydroxypentyl)azanediyl)bis(N,N-didecyl-hexanamide); 7, 7'-(methylazanediyl)bis(N,N- didecylheptanamide); 8,8'-((2-(dimethylamino) ethyl)azanediyl)bis(N,N- di decyl octanami de); 8,8'-((2-(pyrrolidin-l-yl)ethyl)azanediyl) bis(N,N-didecyloctanamide); N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(methyl) amino)octanamide; N,N- didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(2 -hydroxy ethyl) amino)octanamide; 8,8'- ((5-Hydroxypentyl)Azanediyl)Bis(N,N-Dinonyloctanamide); 8,8'-((5-Hydroxypentyl)Azanediyl)Bis(N,N-Didecyl-2-Fluorooctanamide); 8,8'-(Methyl- azanediyl)Bis(N,N-Didecyl-2-Fluorooctanamide); 2,2'-((5-Hydroxypentyl)Azanediyl) Bis(N,N-Didecylacetamide); and 4,4'-((5-Hydroxypentyl)Azanediyl)Bis(N,N- Di decy Ibutan-ami de) .In certain embodiments, the LNPs of the present disclosure comprise an ionizable lipid, e.g., those ionizable lipids disclosed in Table 14.Moreover, each of the following references, which disclose ionizable lipids, e.g., ionizable cationic lipids, and other lipid components that find use in connection with the compositions and methods disclosed herein, is hereby incorporated by reference in its entirety: international patent application publications W02015 / 095340, W02022 / 011156,WO2022 / 133344, WO2023 / 086514, WO2023 / 081776, W02023 / 010128WO2022 / 235972, WO2022 / 235935, WO2022 / 235923, WO2022 / 056413W02022 / 036170, W02020 / 191103, W02018 / 183901, WO201 8 / 119163W02015074085 Al, W02016081029A1, WO2017117530A1, W02019191780A,W02020 / 097548, W02020 / 097540, W02020 / 097520, W02020 / 097493,WO2016 / 197133, WO201 1 / 141705, WO2011 / 141704, WO20 11 / 000107,WO20 11 / 000106, W02010 / 144740, W02010 / 129709,W02010 / 088537,WO20 10 / 054406, WO2010 / 054405, WO20 10 / 054401, WO2010 / 054384,Attorney Docket No.: 084177.0330W02009 / 127060, W02008 / 042973, W02007 / 012191, W02006 / 074546WO2005 / 121348, W02005 / 120461, W02005 / 120152, W02005 / 026372W02005 / 007196, W02004 / 002453, W02002 / 087541, W02000 / 003683WO2023 / 114944, WO2023 / 114943, WO2023 / 114937, W02022 / 016070W02021 / 030701, W02020 / 146805, W02020 / 081938, W02020 / 061426WO20 19 / 089828, WO2019 / 036030, WO20 19 / 036028, W02019 / 036008 WO20 19 / 036000, WO2018 / 200943, WO2018 / 191719, WO2018 / 191657 W02018 / 081480, W02018 / 078053, WO2017 / 117528, WO2017 / 075531.WO20 17 / 004143, WO2015 / 199952, W02024 / 220807, WO2025 / 133951; US patent numbers US 10227302, US 10383952, US 11801306 and US 10526284; and US application publication US2020 / 0297634.In certain embodiments, the LNPs of the present disclosure comprise a non-cationic lipid, e.g., those non-cationic lipids disclosed in Table 14. In certain embodiments, the noncationic lipids that find use in the compositions and methods of the present disclosure include, but are not limited to lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoyl-phosphatidylglycerol (DPPG), dioleoyl phosphatidyl ethanol amine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl-phosphatidyl- ethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethyl-phosphatidyl ethanol amine, phosphatidyl-ethanolamine (DEPE), dimethyl-phosphatidylethanolamine, di el ai doy 1 stearoyl ol eoyl -phosphati dyl ethanol amine (S OPE) .In certain embodiments, the PEGylated lipids comprise a PEG molecule with a molecular weight from about 200Da to about 5000Da. In certain embodiments, the PEGylated lipids comprise a PEG molecule with a molecular weight of 2000Da (2kDa).In certain embodiments, the PEG-lipids can include, but are not limited to, those identified in Table 15. For example, but not by way of limitation, The PEG-lipid can comprise PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), methoxypolyethyleneglycol (PEG-DMG or PEG2000-DMG), PEG coupledAttorney Docket No.: 084177.0330 to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof. In certain embodiments, the PEG-lipid comprises detergent-like PEG lipids (e.g., PEG-DSPE).In certain embodiments, the PEG moiety is conjugated directly to the lipid. In certain embodiments, the PEG moiety is conjugated to the lipid via a linker moiety. Any linker moiety suitable for conjugating the PEG to a lipid can be used including, but not limited to, ester-containing linker moieties and / or non-ester-containing linker moieties. In certain embodiments, an ester-containing linker moiety is used to conjugate the PEG to the lipid. Exemplary ester-containing linker moieties include, e.g, carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof. Exemplary non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-0-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (- NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).In certain embodiments, phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the PEG-lipid conjugate. In certain embodiments, phosphatidylethanolamines comprising saturated or unsaturated fatty acids with carbon chain lengths in the range of CIO to C20 are employed in connection with the compositions and methods disclosed herein. In certain embodiments, phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. In certain embodiments, the phosphatidylethanolamines that find use in connection with the compositions and methods disclosed herein include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).In certain embodiments the PEG-DAA conjugate of the instant disclosure is a PEG- di decyl oxy propyl (CIO) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG- dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG-distearyloxypropyl (C18) conjugate. In certain of such embodiments, the PEG moiety has an average molecular weight of about 750 or about 2,000 daltons. In certain of such embodiments, the terminal hydroxyl group of the PEG moiety is substituted with a methyl group.Attorney Docket No.: 084177.0330In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxy propyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxy ethylcellulose.The lipids described herein can be combined in any number of molar ratios to produce an LNP. In certain embodiments, the LNP comprises a PEG-lipid where the PEG- lipid comprises at least 0.1 mol% of the total lipid. For example, but not by way of limitation, the PEG-lipid component can comprise from about 0.1 mol% to 5 mol% of the total lipid. In certain embodiments, the LNP comprises a cationic lipid where the cationic lipid component comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, the cationic lipid component can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid, the cationic lipid component can comprise from about 10 mol% to 20 mol% of the total lipid. In certain embodiments, the LNP comprises cholesterol where cholesterol comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, cholesterol can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the cholesterol can comprise from about 10 mol% to 30 mol% of the total lipid, the cholesterol can comprise from about 10 mol% to 20 mol% of the total lipid. In certain embodiments, the LNP comprises a non-cationic lipid where the non-cationic lipid comprises at least 10 mol% of the total lipid. For example, but not by way of limitation, the non-cationic lipid can comprise from about 10 mol% to 70 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 60 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 50 mol% of the total lipid. In certain embodiments, theAttorney Docket No.: 084177.0330 non-cationic lipid component can comprise from about 10 mol% to 40 mol% of the total lipid. In certain embodiments, the non-cationic lipid component can comprise from about 10 mol% to 30 mol% of the total lipid, the non-cationic lipid component can comprise from about 10 mol% to 20 mol% of the total lipid.In some embodiments, non-viral vectors include targeting modifications to improve uptake and / or selectively target certain cell types. These targeting modifications can include e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell penetrating peptides. Such vectors also optionally use fusogenic and endosome-destabilizing peptides / polymers, undergo acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo), and / or incorporate a stimuli-cleavable polymer, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.In certain embodiments, the targeting modification comprises N- acetylgalactosamine (GalNAc) or GalNAc derivatives. In some embodiments, the GalNAc targeting modification serves as a ligand that targets the vector, e.g., an LNP, to particular cells. GalNAc and GalNAc derivatives are capable of binding asialoglycoprotein receptor (ASGPR), also known as Ashwell-Morell receptor, a lectin predominantly expressed on liver hepatocytes. In some embodiments, the GalNAc targeting modification targets the vector, e.g., an LNP, to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes), via an LDLR-independent pathway. In some embodiments, the targeting modification comprises one or more GalNAc and / or GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent, trivalent, or tetravalent branched linker. Any suitable linker may be used. For example, linkers described in WO 2024 / 220807 A2, U.S. 11,801,306, and / or Kasiewicz et al. GalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy (Nat Commun. 2023 May 15; 14(1):2776) may be used. In some embodiments, an LNP comprises PEG, and the GalNAc moiety is attached to the PEG via a linker. In certain embodiments, one or more nucleic acid molecules (e.g, DNA molecules) other than the components of a genome editing system, e.g, the RNA-guided nuclease component (or a polynucleotide encoding the RNA-guided nuclease component) and / or the gRNA component described herein, are delivered. In certain embodiments, the nucleic acid molecule is delivered at the same time as one or more of the components of the genome editing system. In certain embodiments, the nucleic acid molecule is delivered before orAttorney Docket No.: 084177.0330 after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the genome editing system are delivered. In certain embodiments, the nucleic acid molecule is delivered by a different means than one or more of the components of the genome editing system, e.g., the RNA-guided nuclease component (or a polynucleotide encoding the RNA- guided nuclease component) and / or the gRNA component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an AAV or an integrationdeficient lentivirus, and the RNA-guided nuclease molecule component (or a polynucleotide encoding the RNA-guided nuclease component) and / or the gRNA component can be delivered by electroporation, e.g. , such that the toxicity caused by nucleic acids (e.g. , DNAs) can be reduced. In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In certain embodiments, the nucleic acid molecule comprises an RNA molecule, e.g., an RNA molecule described herein.7.1.2 Naked Nucleic AcidsNucleic acids encoding genome editing systems or components thereof can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., liver cells). Nucleic acid vectors, such as the vectors summarized in Table 12, can also be used. In some embodiments the genome editing system of the disclosure is delivered into cells by electroporation.7.1.3 Shuttle vectorsOne approach for cell therapy processes includes the direct delivery of active proteins into human cells. A protein delivery agent, the Feldan Shuttle, is a protein-based delivery agent, which is designed for cell therapy (Del’Guidice et al., PloSOne. 2018 Apr 4;13(4):e0195558; incorporated in its entirety herein by reference). In some embodiments the genome editing system of the disclosure are delivered into cells by the Feldan Shuttle.7.1.4 Nucleic acid vectorsNucleic acid vectors can comprise one or more sequences encoding genome editing system components, such as an RNA-guided nuclease, a gRNA and / or a donor template. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein. As one example, a nucleic acid vector can includeAttorney Docket No.: 084177.0330 an RNA-guided nuclease (e.g., Cas9 or Cast 2a) coding sequence that encodes one or more nuclear localization sequences (e.g., a nuclear localization sequence from SV40).The nucleic acid vector can also include any suitable number of regulatory / control elements, e.g., promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well known in the art and are described in Cotta-Ramusino.Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are set forth in Table 13, and additional suitable viral vectors and their use and production are described in Cotta-Ramusino. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver genome editing system components in nucleic acid and / or peptide form. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.7.2 Delivery of RNPs and / or RNA encoding genome editing system componentsRNPs (complexes of gRNAs and RNA-guided nucleases) and / or RNAs encoding RNA-guided nucleases and / or gRNAs, can be delivered into cells or administered to subjects by art-known methods, some of which are described in Cotta-Ramusino. In vitro, RNA-guided nuclease-encoding and / or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (see, e.g., Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc- or other conjugate-mediated delivery, and combinations thereof, can also be used for delivery in vitro and in vivo.In certain embodiments, the gRNA is conjugated to N-acetylgalactosamine (GalNAc) or GalNAc derivatives. GalNAc conjugates are described, for example, in U.S. Pat. No. 8,106,022 and U.S. Pat No. 11,801,306, both of which are incorporated herein by reference. In some embodiments, the conjugate targets the gRNA to a particular cell, e.g., a liver cell, e.g., a hepatocyte, via an LDLR-independent pathway. In some embodiments, the gRNA is conjugated to one or more GalNAc and / or GalNAc derivatives. The GalNAc and / or GalNAc derivatives may be attached via a linker, e.g., a bivalent, or trivalent branched linker. In some embodiments, the GalNAc or GalNAc derivative is conjugated to the 3' end or 5’ end of the gRNA via a linker. / / / vitro, delivery via electroporation comprises mixing the cells with the RNA encoding RNA-guided nucleases and / or gRNAs, with or without donor template nucleic acid molecules, in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. Systems andAttorney Docket No.: 084177.0330 protocols for electroporation are known in the art, and any suitable electroporation tool and / or protocol can be used in connection with the various embodiments of this disclosure.In certain embodiments, a genome editing system or ribonucleoprotein (RNP) complex, may comprise guide RNAs, Casl2a proteins, including modified Casl2a proteins (AsCasl2a variants). Non-limiting examples of Cast 2a (Cpfl) proteins are set forth in SEQ ID NOs: 66-70 and 72-75. In certain embodiments, a genome editing system or RNP complex may include a guide RNA (gRNA) complexed to a Cast 2a protein or a modified Casl2a protein. In certain embodiments a gRNA may comprise a sequence set forth in SEQ ID NOs: 35-45 or SEQ ID NOs: 46-56. In certain embodiments, a genome editing system or RNP complex may comprise an RNP complex set forth in Table 16. For example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 35-45, a modified Casl2a protein set forth in SEQ ID NO: 70 and target an ANGPTL3 gene at the sequence set forth in SEQ ID NO: 26. In another example, a genome editing system or RNP complex may include a gRNA comprising a sequence set forth in SEQ ID NOs: 35-45, a modified Casl2a protein set forth in SEQ ID NO: 70, and target an ANGPTL3 gene at the sequence set forth in SEQ ID NO: 31.In certain embodiments, a genome editing system comprises guide RNA and mRNA encoding the RNA-guided nuclease (Cast 2a proteins, including modified Cast 2a proteins e.g., AsCasl2a variants). Non-limiting examples of mRNAs encoding the RNA-guided nuclease are set forth in SEQ ID NOs: 80-84. Following delivery of the guide RNA and mRNA to the cell, the mRNA drives translation of the RNA-guided nucleases, leading to formation of the RNP complexes comprising a guide RNA (gRNA) and a Casl2a protein or a modified Casl2a protein. In certain embodiments, a gRNA can comprise a sequence set forth in SEQ ID NOs: 35-45 or SEQ ID NOs: 46-56. In certain embodiments, a genome editing system or RNP complex can comprise an RNP complex set forth in Table 16. For example, but not by way of limitation, a genome editing system or RNP complex can comprise a gRNA comprising a sequence set forth in SEQ ID NOs: 37, 48, or 58, a modified Casl2a protein set forth in SEQ ID NO: 70 and target an ANGPTL3 gene at the sequence set forth in SEQ ID NO: 26. Additionally or alternatively, a genome editing system or RNP complex can comprise a gRNA comprising a sequence set forth in SEQ ID NOs: 42, 53, or 103, a modified Casl2a protein set forth in SEQ ID NO: 70, and target an ANGPTL3 gene at the sequence set forth in SEQ ID NO: 31.Table 16: Exemplary ribonucleoprotein (RNP) ConfigurationAttorney Docket No.: 084177.03307.3 Editing efficiencyEditing the ANGPTL3 gene using the genome editing systems, the RNP complexes, or the LNP delivery systems described in this disclosure reduces ANGPTL3 expression by at least about 20% to about 100% when compared to control non-edited cells. In some embodiments, the ANGPTL3 expression level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least about 50% to at least about 95% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least about 90% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least about 95% compared to control cells.7.4 Target cellsThe genome editing systems, the RNP complexes, or the delivery systems described in this disclosure can be used to manipulate or alter a target cell, e.g., to edit or alter a target nucleic acid. In some embodiments, the genome editing system, the RNP complex, or the delivery system described in this disclosure, are used to edit target cells in a tissue, e.g., edit or alter a target nucleic acid in cells that make up the tissue. The manipulating can occur, in various embodiments, in vivo or ex vivo. In certain embodiments, the target cells are cells involved in metabolism. For example, in one embodiment, the target cells are liver cells (e.g., hepatocytes, hepatic stellate cells, a Kupffer cells, or liver stem cells). In one embodiment the target cell is a hepatocyte. In certain embodiments, the target cell comprises a genomic edit that results in loss of function of ANGPTL3. While not wishing to be bound by any particular theory, it is contemplated that reducing or disabling ANGPTL3 reduces the risk of major adverse cardiovascular events (MACE) including, heart attack, stroke, aortic stenosis, peripheral vascular disease, and renal dysfunction. In some embodiments, reducing or disabling ANGPTL3 expression reduces hyperlipidemia.Attorney Docket No.: 084177.0330In some embodiments, editing the ANGPTL3 gene in the liver cells using the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces ANGPTL3 expression levels by at least about 20% to about 100% when compared to a control, non-edited liver cells. In some embodiments, the ANGPTL3 expression level is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least 50% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least 80% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least 85% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least 90% compared to control cells. In some embodiments, the ANGPTL3 expression levels are reduced by at least about 95% compared to control cells.In some embodiments, editing the ANGPTL3 gene in a subject (e.g., in liver cells of the subject) by administering the genome editing system, the RNP complex, or the delivery system to the subject reduces an ANGPTL3 level in the subject, or in a cell, tissue, or fluid in the subject, by at least about 20% to about 100% relative to the ANGPTL3 level prior to administration, or relative to the ANGPTL3 level in a control (e.g., non-edted) subject, cell, tissue, or fluid. In some embodiments, the ANGPTL3 level (e.g., serum or plasma level) is reduced by about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In some embodiments, the ANGPTL3 level (e.g., serum or plasma level) is reduced by at least 50%. In some embodiments, the ANGPTL3 level (e.g., serum or plasma level) is reduced by at least 80%. In some embodiments, the ANGPTL3 level (e.g., serum or plasma level) is reduced by at least 85%. In some embodiments, the ANGPTL3 level (e.g., serum or plasma level) is reduced by at least 90%. In some embodiments, the ANGPTL3 level (e.g., serum or plasma level) is reduced by at least 95%.Provided herein are methods of administering the genome editing system, the RNP complex, or the delivery system described in this disclosure, to treat, prevent or reduce the risk of diseases, conditions, and disorders, including metabolic disorders. Non-limiting examples of such disease include hyperlipidemia, hypercholesterolemia atherosclerotic cardiovascular disease (ASCVD), heart attack, stroke, aortic stenosis, high blood pressure, peripheral vascular disease, diabetes, renal dysfunction, or a combination of these. In some embodiments, the genome editing system, the RNP complex, or the delivery system described in this disclosure is administered to a subject or patient having the particularAttorney Docket No.: 084177.0330 disease or condition to be treated. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition.In certain embodiments, reducing the ANGPTL3 expression level in a cell from the subject or reducing the serum or plasma ANGPTL3 level in the subject reduces the serum or plasma level of LDL and / or Lp(a).In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Lp(a) levels in the subject or patient to <150 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Lp(a) levels in the subject or patient to between about 5 mg / dL and about 140 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Lp(a) levels in the subject or patient to between about 60 mg / dL and about 100 mg / dL, between about 70 mg / dL and about 100 mg / dL, between about 80 mg / dL and about 100 mg / dL, between about 90 mg / dL and about 100 mg / dL, between about 50 mg / dL and about 80 mg / dL, between about 30 mg / dL and about 50 mg / dL, between about 40 mg / dL and about 55 mg / dL, between about 40 mg / dL and about 60 mg / dL, between about 50 mg / dL and about 60 mg / dL, or between about 60 mg / dL and about 75 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Lp(a) levels in the subject or patient to between about 5 mg / dL and about 10 mg / dL, between about 7 mg / dL and about 15 mg / dL, between about 20 mg / dL and about 30 mg / dL, between about 25 mg / dL and about 40 mg / dL, or between about 30 mg / dL and about 45 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Lp(a) levels in the subject or patient to less than 10 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Lp(a) levels in the subject or patient to between about 66 mg / dL and about 100 mg / dL. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system described in this disclosure reduces Lp(a) levels in the subject or patient to between about 30 mg / dL and about 50 mg / dL. In some embodiments, the Lp(a) level in the subject is measured at about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, or 20 weeks, about 1, 2, 3, 4, 5, or 6 months, or about 1, 2, 3, 4, or 5 years after administering the genome editing system, the RNP complex, or the delivery system described in this disclosure.Attorney Docket No.: 084177.0330In some embodiments, administering the genome editing system, the RNP complex, or the delivery system is complemented by providing to the subject or patient a standard of care (SOC) for treating the disease, condition, or disorder. Non-limiting examples of SOC include Apo(a) apheresis, one or more pharmacological agents, or a combination thereof. In some embodiments, the pharmacological agent is selected from statins, LY3473329, angiopoietin like 3 (ANGPTL3) inhibitors, and proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, or a combination thereof.The genome editing system, the RNP complex, or the delivery system can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous, or subcutaneous injections. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration. In some embodiments, it is administered by multiple bolus administrations, for example, over a period of no more than 3 days, or by continuous infusion administration.All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.8. Exemplary EmbodimentsA. A genome editing system comprising: (a) a gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene, and (b) an RNA-guided nuclease, or an RNA encoding the RNA-guided nuclease.Al. The genome editing system of A, wherein: (a) the target sequence of the ANGPTL3 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-34; and / or (b) the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.A2. The genome editing system of A or Al, wherein: (a) the target sequence of the ANGPTL3 gene comprises the nucleotide sequence set forth in SEQ ID NO: 26, or SEQ IDAttorney Docket No.: 084177.0330NO: 31; and / or (b) the targeting domain comprises the nucleotide sequence set forth in SEQ ID NO: 37, or SEQ ID NO: 42.A3. The genome editing system of any one of A-A2, wherein the RNA-guided nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Casl2h, Casl2i, CasX, CasY, and Cas .A4. The genome editing system of any one of A-A3, wherein the RNA-guided nuclease is a Casl2a protein.A5. The genome editing system of A4, wherein the Casl2a protein is a modified Cast 2a protein.A6. The genome editing system of A5, wherein the modified Casl2a protein is an activity enhanced Cast 2a protein.A7. The genome editing system of A5 or A6, wherein the modified Casl2a protein is a modified AsCasl2a protein.A8. The genome editing system of any one of A-A8, wherein the RNA-guided nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-70 and 72-75.A9. The genome editing system of any one of A-A8, wherein the RNA-guided nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 66-70 and 72-78.A10. The genome editing system of any one of A-A9, wherein the RNA encoding the RNA-guided nuclease comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 80-84.Al 1. The genome editing system of any one of A-A10, wherein the gRNA molecule further comprises a Casl2a stem loop.A12. The genome editing system of any one of A-Al 1, wherein the gRNA molecule further comprises a nucleotide extension, wherein the nucleotide extension is a 5' extension, a 3' extension, or a combination thereof.A13. The genome editing system of A12, wherein the nucleotide extension comprises one or more RNA bases, one or more DNA bases, or a combination thereof.A14. The genome editing system of any one of A-A13, wherein the gRNA molecule contains one or more modifications.A15. The genome editing system of any one of clauses A12-A14, wherein the nucleotide extension is a 5' extension comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-23.Attorney Docket No.: 084177.0330A16. The genome editing system of any one of A12-A15, wherein the nucleotide extension is a 5' extension comprising the nucleotide sequence set forth in SEQ ID NO: 7.Al 7. The genome editing system of any one of A-A16, wherein the gRNA molecule comprises a DNA / RNA oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 35-45.Al 8. The genome editing systems of any one of A-A17, wherein the gRNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 37, SEQ ID NO: 48, or SEQ ID NO: 58.Al 9. The genome editing systems of any one of A-Al 8, wherein the gRNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 42, SEQ ID NO: 53, or SEQ ID NO: 103.B. A ribonucleoprotein (RNP) complex comprising the genome editing system of any one of A- Al 9.Bl. The RNP complex of B, further comprising one or more N-acetylgalactosamine (GalNAc) and / or GalNAc derivatives.C. A delivery system for delivering the genome editing system of any one A-A19, wherein the delivery system comprises a DNA sequence encoding the gRNA molecule and / or RNA-guided nuclease, an RNA sequence encoding the gRNA molecule and / or RNA- guided nuclease, or combination thereof.Cl. The delivery system of C, wherein the delivery system comprises a lipid nanoparticle (LNP) encapsulating the gRNA molecule and an RNA sequence encoding the RNA-guided nuclease.C2. The delivery system of Cl, wherein the LNP comprises ionizable lipids, polyethylene glycol (PEG) lipids, helper lipids, sterols, or combinations thereof.C3. The delivery system of C2, wherein the ionizable lipid is selected from the group consisting of ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), and 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM- 102).C4. The delivery system of C2 or C3, wherein the PEG lipid is selected from the group consisting of dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG), distearoyl-sn-glycerol-3-methoxypolyethylene glycol (DSG-PEG), and di stearoyl -sn- glycero-3-phosphoethanolamine-N-methoxypolyethylene glycol (DSPE-PEG).Attorney Docket No.: 084177.0330C5. The delivery system of any one of C2-C4, wherein the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC) and 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine. (DOPE).C6. The delivery system of any one of C2-C5, wherein the sterol is selected from the group consisting of cholesterol and sitosterol.C7. The delivery system of Cl, wherein the LNP comprises ((4- Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315), DMG- PEG, a DSPC, and cholesterol.C8. The delivery system of any one of C1-C7, wherein the LNP comprises one or more N-acetylgalactosamine (GalNAc) and / or GalNAc derivatives.D. A method of editing an ANGPTL3 gene in a target cell comprising contacting the target cell with the genome editing system of any one of A-A19, the RNP complex of B or Bl, or the delivery system of any one of claims C-C8.DI. The method of D, wherein the target cell is in vivo.D2. The method of D or DI, wherein the target cell is a cell involved in metabolism.D3. The method of D-D2, wherein the target cell is a hepatocyte.E. A method of treating a disease or disorder comprising administering to a subject in need thereof the genome editing system of any one of A-A19, the RNP complex of B or Bl, or the delivery system of any one of C-C8.El. The method of E, wherein the disease or disorder is a hyperlipidemia or hypercholesterolemia.E2. The method of E or El, wherein the disease or disease is homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH).E3. The method of any one of E-E2, wherein the subject is suffering from an atherosclerotic cardiovascular disease (ASCVD).E4. The method of any one of E-E3, wherein the subject is identified to be at a high risk for a major adverse cardiovascular event (MACE) or has suffered from a MACE.E5. The method of any one of E-E4, wherein the subject has an Lp(a) level >150 mg / dL.E6. The method of any one of E-E5, further comprising administering to the subject a standard of care (SOC) for hyperlipidemia.E7. The method of E6, wherein the SOC is Apo(a) apheresis and / or at least one pharmacological agent.Attorney Docket No.: 084177.0330E8. The method of E7, wherein the pharmacological agent is selected from the group consisting of a statin, an angiopoietin like 3 (ANGPTL3) inhibitor, a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, and LY3473329, or a combination thereof.E9. The method of any one of E-E8, wherein administering the genome editing system, the RNP complex, or the delivery system reduces an ANGPTL3 protein level in the subject, or in a cell, tissue, or fluid of the subject, by at least about 50% to at least about 95%, relative to the ANGTPL3 protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject.E10. The method of E9, wherein administering the genome editing system, the RNP complex, or the delivery system reduces the ANGPTL3 protein level in the subject, or in a cell, tissue, or fluid of the subject, by at least 90%, relative to the ANGTPL3 protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject.El l. The method of any one of E-E8, wherein administering the genome editing system, the RNP complex, or the delivery system reduces an ANGPTL3 protein level in the subject, or in a cell, tissue, or fluid of the subject, to 10%, relative to the ANGTPL3 protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subj ectF. The genome editing system of A14, wherein the one or more modifications is selected from the group consisting of a 5' inverted thymidine (idT) modification, a 3' idT modification, a 2' fluoro modification, a 2' O-methyl modification, a phosphorothioate linkage, a 3' pseudoknot, a locked nucleic acid (LNA), and a combination thereof.Fl. The genome editing system of F, wherein the one or more modifications comprises the 5' inverted thymidine (idT) modification and the 3' idT modification.F2. The genome editing system of Fl, wherein the one or more modifications comprises the 2' fluoro modification.F3. The genome editing system of F, wherein the one or more modifications comprises one or more 2' fluoro modifications, and each of the 2' fluoro modifications modifies a nucleotide internal to the gRNA molecule.F4. The genome editing system of F3, wherein the gRNA molecule further comprises a 5' DNA extension.Attorney Docket No.: 084177.0330F5. The genome editing system of F4, wherein the 5' DNA extension comprises the sequence set forth in SEQ ID NO: 7.F6. The genome editing system of F5, wherein the gRNA molecule comprises the sequence set forth in SEQ ID NO: 91.G. A method of treating an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a formulation comprising: (i) a lipid nanoparticle (LNP); (ii) an mRNA encoding a Cast 2a nuclease; and (iii) a gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene of the subject; wherein the mRNA and the gRNA are encapsulated within the LNP.Gl. The method of G, wherein (a) the target sequence of the ANGPTL3 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24- 34; and / or (b) the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.G2. The method of G or Gl, wherein the gRNA molecule comprises the sequence set forth in SEQ ID NO: 37.G3. The method of any one of G-G2, wherein the gRNA molecule comprises the sequence and modifications set forth in SEQ ID NO: 91.G4. The method of any one of G-G3, wherein the subject has homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH).H. A gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene.Hl. The gRNA molecule of H, wherein the gRNA molecule comprises one or more modifications selected from the group consisting of a 5' inverted thymidine (idT) modification, a 3' idT modification, a 2' fluoro modification, a 2' O-methyl modification, a phosphorothioate linkage, a 3' pseudoknot, a locked nucleic acid (LNA), and a combination thereof.H2. The gRNA molecule of Hl, wherein the one or more modifications are on nucleotides positioned outside of the targeting domain.H3. The gRNA molecule of Hl or H2, wherein the gRNA comprises a 5' DNA extension.H4. The gRNA molecule of H3, wherein the 5' DNA extension comprises the sequence set forth in SEQ ID NO: 7.H5. The gRNA molecule of any one of H-H4, wherein the gRNA molecule comprises the sequence and modifications set forth in SEQ ID NO: 91.Attorney Docket No.: 084177.0330H6. The gRNA molecule of any one of H-H5, wherein the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 95); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.H7. The gRNA molecule of any one of H-H5, wherein the gRNA molecule comprises the following sequence: mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 96); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45, and wherein the 3' nucleotide of the targeting domain comprises a 2' O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.H8. The gRNA molecule of any one of H-H5, wherein the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTr UrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUx / 3InvdT / (SEQ ID NO: 97); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.H9. The gRNA molecule of any one of H-H5, wherein the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i 2FA / rUx / 3InvdT (SEQ ID NO: 98); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.H10. The gRNA molecule of any one of H-H5, wherein the gRNA molecule comprises the following sequence: mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 99); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45, and wherein the 3' nucleotide of the targeting domain comprises a 2' O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.Attorney Docket No.: 084177.0330Hl 1. The method of any one of clauses D-El 1 or G-G4, wherein administering the genome editing system, the RNP complex, or the delivery system reduces the Lp(a) level in the subject, or in a cell, tissue, or fluid of the subject, to <150 mg / dL9. ExamplesThe following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.General Methods9.1 Guide SynthesisGuide-RNAs (gRNAs) were synthesized via standard phosphoramidite chemistry. Purification was completed by ion-pair reversed-phase preparative HPLC, followed by desalting and sequence analysis.9.2 Lipid Nanoparticle (LNP) Formulation and Quality ControlLNPs were generally formulated with commercially available lipids using a NanoAssemblr® Ignite™ (Cytiva). Generally, LNP cargo was mRNA encoding engineered AsCasl2a nuclease and gRNA at a 1 : 1 ratio by weight. LNPs were evaluated for percent encapsulation greater than 80% by RiboGreen assay (ThermoFisher Scientific), poly dispersity index (PDI) <0.2, and average diameter size <105 nm by Zetasizer analysis (Malvern Panalytical, Model ZSU3205).9.3 Cell Culture TreatmentsCells were treated with LNPs at indicated concentrations of encapsulated AsCasl2a mRNA, and gDNA was isolated at 72 hours post transfection. Transfection of primary human hepatocytes (PHHs) included recombinant human Apolipoprotein E (ApoE). Amplicon based NGS was performed to determine the percentage of editing.9.4 In Vivo Editing in Mouse LiverLNPs were delivered via intravenous tail vein injection to C57B1 / 6 mice or humanized ANGPTL3 transgenic mice (Biocytogen C57BL / 6- Angptl3tm4^ANGPTL3^Bcgen / Bcgen; see biocytogen. com / products / humanized-cytokines_mice / b- hangptl3-mice-plus / ). One week to three weeks post-injection, the livers were dissected, gDNA was isolated, and amplicon based NGS was performed to determine the percentage of editing.9.5 In Vivo Editing in Nonhuman PrimatesNHPs were dosed intravenously with vehicle or two types of LNPs that each comprised a GalNAc targeting moiety but differed in the identity of the ionizable lipid (Attorney Docket No.: 084177.0330“LNP2” and “LNP3”). Dosing was over Ihr and monitored for 4 weeks. Genomic DNA was extracted from liver punches collected at the terminal endpoint and editing at ANGPTL3 was then assessed by NGS to determine the frequency of indels. ANGPTL3 reduction in serum was assessed by ELISA or other methodologies.Example 1: Screening ionizable lipids for LNP deliveryTo investigate the transfection efficiency of lipid nanoparticles (LNPs) formed with different ionizable lipids, LNPs were formulated with one of five different ionizable lipids, the structures of which are shown in Table 17 below: (1) MC3 (also called Dlin-MC3- DMA), (2) SM-102, (3) ALC-0315, (4) 5A2-SC8, and (5) BAMEA-O16B. LNPs (MC3, SM-102, ALC-0315, BAMEA-O16B) were formed with 50% ionizable lipid, 38.5% cholesterol, 10% distearoylphosphatidylcholine (DSPC), and 1.5% dimethylglycine (DMG)-PEG2k and encapsulated GFP mRNA. 5A2-SC8 LNP was formed with 25% ionizable lipid, 48.5% cholesterol, 25% DSPC, and 1.5% DMG-PEG2k and encapsulated GFP mRNA.Table 17. Exemplary Ionizable lipids used in LNPsAttorney Docket No.: 084177.0330Briefly, LNPs were formulated with an amine-to-RNA-phosphate (N:P) ratio of 4- 7. Unless otherwise specified, the N:P ratio = 6.88 was used. The lipid nanoparticle components (ionizable lipid, cholesterol, DSPC, and DMG-PEG2k) were dissolved in 100% ethanol and mixed in the indicated molar ratios. The RNA cargo (1 : 1 weight ratio 100% GFP mRNA) was dissolved in 50 mM citrate buffer (pH 4.5), resulting in a concentration of RNA cargo of approximately 0.12 mg / mL. LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems Ignite or Spark Instrument, in accordance with the manufacturer’s protocol. After mixing, the LNPs were collected and ethanol was removed by one of the following two methods: 1) LNPs were diluted in PBS or TBS (1 :40, vokvol) and loaded into Amicon™ Centrifugal Filter Units for ultrafiltration (MilliporeSigma, 30kD); or 2) LNPs were loaded into 10 kDa Slide-a-Lyzer G2 Dialysis Cassettes (ThermoFisher Scientific) for dialysis in TBS or PBS under gentle stirring (1 hour in room temperature and new buffer change, 3 hours in 4 °C and new buffer change, and overnight at 4 °C). The resultant mixture was concentrated to the target concentrations and then filtered using a 0.2- mm sterile filter. The filtrate was stored at 2 °C-8 °C for use within a week or stored at -80 °C for a longer time after addition of 10% sucrose.Attorney Docket No.: 084177.0330Example 2: RNP nucleofection screen for AsCasl2a gRNAs targeting ANGPTL3Using a bioinformatic approach, 11 potential AsCasl2a gRNA sequences were identified within the ANGPTL3 gene with no predicted off-target sites within the human genome and with zero or 1 mismatch with the corresponding cynomolgus macaque nonhuman primate (NHP) genomic location. The gRNAs utilize the canonical AsCasl2a PAM sequence TTTV (WT). These gRNAs target exon 1 within the ANGPTL3 gene, and were tested for their ability to generate insertion or deletion (indel) mutations and disrupt proper target protein production or function, thereby decreasing the levels of ANGPTL3 protein and potentially protecting against atherosclerosis and major adverse cardiac events.Table 18. ANGPTL3 Target Sequences And Mean Indel Fraction For Each gRNAThese gRNAs were screened for editing activity in the HepG2 hepatocellular carcinoma cell line. Briefly, AsCasl2a protein (SEQ ID NO: 70) was complexed with each gRNA at a 1 :2 molar ratio (RNP complex) and delivered to HepG2 cells via nucleofection. Three days after nucleofection, genomic DNA (gDNA) was extracted and the target sites were amplified by PCR and evaluated by next generation sequencing (NGS). FIG. 7; Table 18A combination of in silico and experimental methods were used to further analyze the potential for off-target sites in the human genome in connection with the development of gRNAs targeting target site 645 (SEQ ID No. 26) of Table 18. Using CALITAS (https: / / github.com / editasmedicine / calitas), an in silico prediction method, 359 potential off-target sites were identified using a gRNA having the sequence of SEQ ID NO: 91 when analyzed in the absence of a specific PAM sequence requirement. When using a gRNAAttorney Docket No.: 084177.0330 having the sequence of SEQ ID NO: 91 and requiring the AsCasl2a “TTTN” PAM sequence, only 9 potential off-target sites were identified. Experiments were also conducted using a gRNA having the sequence of SEQ ID NO: 91 formulated into LNPs with an engineered AsCasl2a mRNA as described in Example 3, below. For example, Digenome- seq was performed using Promega DNA with 1 nM, 10 nM, 100 nM, and 1000 nM of LNP. In these experiments, only one potential off-target site was identified, and it was identified only at the highest, 1000 nM, dose concentration. Guide-seq was also performed using a donor sample, and no off-targets were identified.Example 3: Efficient ANGPTL3 editing and ANGPTL3 knockdown achieved by gRNAs delivered by LNPs to primary human hepatocytes (PHHs) and HepG2 cells.Guide-RNAs were generated with the sequence SEQ ID NO: 91 and 92 for further analysis using lipid nanoparticle (LNP) delivery format. The gRNAs were formulated into LNPs with engineered AsCasl2a mRNA (SEQ ID NO: 81) at a 1 : 1 weight ratio between gRNA and mRNA using an ALC0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k) and incubated with PHH and HepG2 cells for 24 hours. At 3 days post treatment, genomic DNA was isolated from the cells and indel profiling was performed using next-generation sequencing (NGS). As shown in FIGs. 1A- 1B, robust editing was observed in LNP -treated PHHs and HepG2 cells.Table 19.Attorney Docket No.: 084177.0330Based on the above results, further experiments were performed using the gRNA of SEQ ID NO: 91 and PHHs from 3 different human donors. The gRNA was formulated into LNPs with engineered AsCasl2a mRNA (SEQ ID NO: 81) at a 1 : 1 weight ratio between gRNA and mRNA using an ALC0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k) and incubated with donor PHHs for 24 hours. Treated PHHs were analyzed for ANGPTL3 editing by NGS. ANGPTL3 protein knockdown was assessed using a commercially available ELISA (R&D Systems - Human Angiopoietin- like 3 ELISA Kit - Quantikine (Cat # DANL30)). As shown in FIGs. 1C-1E, robust editing and protein knockdown was observed in PHHs from various donors treated with LNPs containing the4M / 7J7 / .3-targeting gRNA of SEQ ID NO: 91 (ANGPTL3 496). In all cases, greater than 90% editing and greater than 80% protein knockdown was achieved.Example 4: In vivo editing of ANGPTL3 in WT mice.To assess ANGPTL3 editing in vivo, a surrogate gRNA was designed targeting a mouse ANGPTL3 sequence (SEQ ID NO: 94; Table 20) orthologous to the human ANGPTL3 target sequence of SEQ ID NO: 26 (Table 19) and incorporated along with AsCasl2a mRNA (SEQ ID NO: 84) into an ALC0315-based LNP formulation (50% ALC- 0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k) at a 1 : 1 weight ratio between gRNA and mRNA. The LNPs were delivered via tail vein injection into wild type C57B1 / 6 (WT) mice. After ten days, the mice were euthanized and liver tissue was collected for NGS editing analysis. Terminal serum samples were collected for mouse ANGPTL3 protein knockdown analysis. NGS analysis showed high levels of indel generation for all doses tested (FIG. 2).Table 20. Surrogate gRNA for targeting mouse ANGPTL3.Attorney Docket No.: 084177.0330A commercially available ELISA (R&D Systems - Mouse Angiopoietin-like 3 ELISA Kit - Quantikine (Cat #: MANL30)) was used for quantification of mouse ANGPTL3 protein from the terminal serum samples collected from the WT mice treated with the LNP formulation described above. Terminal samples from vehicle control or LNP treated mice were analyzed in triplicate as per the manufacturer's protocol. Concentrations of mouse ANGPTL3 protein within vehicle control or treated mouse serum are shown in FIG. 3A. The percentage of knockdown for each mouse was generated by comparing mouse ANGPTL3 protein concentration from each mouse to an average of the vehicle controls (FIG. 3B). Mouse ANGPTL3 protein was decreased by >70% in all treated serum samples (FIG. 3B).Example 5: In vivo editing of ANGPTL3 in humanized mice.To assess editing of a human ANGPTL3 gene in mice, LNPs were formulated with gRNA of SEQ ID NO: 91 and engineered AsCasl2a mRNA (SEQ ID NO: 84) at a 1 :1 weight ratio between gRNA and mRNA using an ALC0315-based LNP formulation (50% ALC-0315, 38.5% cholesterol, 10% DSPC, 1.5% DMG-PEG2k). LNPs were delivered via tail vein injection into humanized ANGPTL3 transgenic mice (Biocytogen C57BL / 6- Angptl3tm4(ANGPTL3)BcgenI c^, see https: / / biocytogen.com / products / humanized-cytokines_ mice / b-hangptl3-mice-plus / ). After 20 days, the mice were euthanized and liver tissue was collected for NGS editing analysis. Pre-dose and terminal serum samples were collected for ANGPTL3 protein knockdown analysis. NGS analysis demonstrated high editing for all samples at 0.1 mg / kg dose and higher (FIG. 4).Due to low levels of human ANGPTL3 protein in the plasma of humanized ANGPTL3 transgenic mice (FIG. 5A), a commercially available ELISA with a lower limit of quantification (LoQ) was used (Invitrogen - Human ANGPTL3 ELISA Kit (Cat #: EH29RB). Despite the improved sensitivity of this assay, levels of human ANGPTL3 within pre-dose samples were found to be close to or below the assay's limit of quantification (FIG. 5B). All ANGPTL3 values, including those below the assay's LoQ, were interpolated from obtained absorbance values using the four-parameter logistic regression equation generated by the standard curve.Terminal samples from vehicle control or LNP treated mice were analyzed in triplicate as per the manufacturer's protocol. All ANGPTL3 values, including those below the assay's LoQ, were interpolated from obtained absorbance values using the four- parameter logistic regression equation generated by the standard curve. The percentage ofAttorney Docket No.: 084177.0330 knockdown for each mouse was determined by comparing their average pre-dose and terminal human ANGPTL3 levels. Potent editing and protein knockdown was observed, with maximum effect observed at > 1 mg / kg (FIGs. 6A-6B).Example 6: In vivo editing of ANGPTL3 in nonhuman primates.To assess editing of a human ANGPTL3 gene sequence in nonhuman primates (“NHPs”), two specific LNP formulations comprising GalNAc and different ionizable lipids (“LNP2” and “LNP3”) were prepared with the gRNA of SEQ ID NO: 91 and engineered AsCasl2a mRNA (SEQ ID NO: 81) as cargo. NHPs were dosed intravenously with vehicle or LNP at the indicated doses over Ihr and monitored for 4 weeks. Genomic DNA was extracted from liver punches collected at the terminal endpoint and editing at ANGPTL3 was then assessed by NGS to determine the frequency of indels (FIG. 8A). ANGPTL3 reduction in serum was assessed by ELISA (FIG. 8A) or other methodologies (FIG. 8B). The percent change in ANGPTL3 levels was determined by comparing pre-dose samples to those collected each week. ANGPTL3 (FIG. 8A) baseline within serum was determined by taking the average of the screening and pre-dose samples. The maximum level of ANGPTL3 reduction over the 4-week study is shown, and potent editing and protein knockdown was observed (FIGs 8A-8B) Similar reduction was observed in ANGPTL3 mRNA levels (data not shown). Dotted line represents theoretical maximum hepatocyte editing based on the liver being composed of -70% hepatocytes.

Claims

Attorney Docket No.: 084177.0330WHAT IS CLAIMED IS:

1. A genome editing system comprising:(a) a gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene, and(b) an RNA-guided nuclease, or an RNA encoding the RNA-guided nuclease.

2. The genome editing system of claim 1, wherein:(a) the target sequence of the ANGPTL3 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-34; and / or(b) the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.

3. The genome editing system of claim 1 or 2, wherein:(a) the target sequence of the ANGPTL3 gene comprises the nucleotide sequence set forth in SEQ ID NO: 26, or SEQ ID NO: 31; and / or(b) the targeting domain comprises the nucleotide sequence set forth in SEQ ID NO: 37, or SEQ ID NO: 42.

4. The genome editing system of any one of claims 1-3, wherein the RNA-guided nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Casl2h, Casl2i, CasX, CasY, and Cas .

5. The genome editing system of any one of claims 1-4, wherein the RNA-guided nuclease is a Casl2a protein.

6. The genome editing system of claim 5, wherein the Casl2a protein is a modified Cast 2a protein.

7. The genome editing system of claim 6, wherein the modified Casl2a protein is an activity enhanced Cast 2a protein.

8. The genome editing system of claim 6 or claim 7, wherein the modified Cast 2a protein is a modified AsCasl2a protein.Attorney Docket No.: 084177.03309. The genome editing system of any one of claims 1-8, wherein the RNA-guided nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-70 and 72-75.

10. The genome editing system of any one of claims 1-9, wherein the RNA-guided nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 66-70 and 72-78.

11. The genome editing system of any one of claims 1-10, wherein the RNA encoding the RNA-guided nuclease comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 80-84.

12. The genome editing system of any one of claims 1-11, wherein the gRNA molecule further comprises a Cast 2a stem loop.

13. The genome editing system of any one of claims 1-12, wherein the gRNA molecule further comprises a nucleotide extension, wherein the nucleotide extension is a 5’ extension, a 3’ extension, or a combination thereof.

14. The genome editing system of claim 13, wherein the nucleotide extension comprises one or more RNA bases, one or more DNA bases, or a combination thereof.

15. The genome editing system of any one of claims 1-14, wherein the gRNA molecule contains one or more modifications.

16. The genome editing system of any one of claims 13-15, wherein the nucleotide extension is a 5’ extension comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-23.

17. The genome editing system of any one of claims 13-16, wherein the nucleotide extension is a 5’ extension comprising the nucleotide sequence set forth in SEQ ID NO: 7.Attorney Docket No.: 084177.033018. The genome editing system of any one of claims 1-17, wherein the gRNA molecule comprises a DNA / RNA oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 35-45.

19. The genome editing systems of any one of claims 1-18, wherein the gRNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 37, SEQ ID NO: 48, or SEQ ID NO: 58.

20. The genome editing systems of any one of claims 1-18, wherein the gRNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 42, SEQ ID NO: 53, or SEQ ID NO: 103.

21. A ribonucleoprotein (RNP) complex comprising the genome editing system of any one of claims 1-20.

22. The RNP complex of claim 21, further comprising one or more N- acetylgalactosamine (GalNAc) and / or GalNAc derivatives.

23. A delivery system for delivering the genome editing system of any one of claims 1- 20, wherein the delivery system comprises a DNA sequence encoding the gRNA molecule and / or RNA-guided nuclease, an RNA sequence encoding the gRNA molecule and / or RNA- guided nuclease, or combination thereof.

24. The delivery system of claim 23, wherein the delivery system comprises a lipid nanoparticle (LNP) encapsulating the gRNA molecule and an RNA sequence encoding the RNA-guided nuclease.

25. The delivery system of claim 24, wherein the LNP comprises ionizable lipids, polyethylene glycol (PEG) lipids, helper lipids, sterols, or combinations thereof.

26. The delivery system of claim 25, wherein the ionizable lipid is selected from the group consisting of ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2- hexyl decanoate) (ALC-0315), and 8-[(2 -hydroxy ethyl) [6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM- 102).Attorney Docket No.: 084177.033027. The delivery system of claim 25 or claim 26, wherein the PEG lipid is selected from the group consisting of dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG- PEG), distearoyl-sn-glycerol-3-methoxypolyethylene glycol (DSG-PEG), and distearoyl- sn-glycero-3-phosphoethanolamine-N-methoxypolyethylene glycol (DSPE-PEG).

28. The delivery system of any one of claims 25-27, wherein the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC) and 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine. (DOPE).

29. The delivery system of any one of claims 25-28, wherein the sterol is selected from the group consisting of cholesterol and sitosterol.

30. The delivery system of claim 24, wherein the LNP comprises ((4- Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315), DMG- PEG, a DSPC, and cholesterol.

31. The delivery system of any one of claims 24-30, wherein the LNP comprises one or more N-acetylgalactosamine (GalNAc) and / or GalNAc derivatives.

32. A method of editing an ANGPTL3 gene in a target cell comprising contacting the target cell with the genome editing system of any one of claims 1-20, the RNP complex of claim 21 or claim 22, or the delivery system of any one of claims 23-31.

33. The method of claim 32, wherein the target cell is in vivo.

34. The method of claim 32, wherein the target cell is a cell involved in metabolism.

35. The method of claim 32, wherein the target cell is a hepatocyte.

36. A method of treating a disease or disorder comprising administering to a subject in need thereof the genome editing system of any one of claims 1-20, the RNP complex of claim 21 or claim 22, or the delivery system of any one of claims 23-31.Attorney Docket No.: 084177.033037. The method of claim 36, wherein the disease or disorder is a hyperlipidemia or hypercholesterolemia.

38. The method of claim 36 or 37, wherein the disease or disease is homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH).

39. The method of any one of claims 36-38, wherein the subject is suffering from an atherosclerotic cardiovascular disease (ASCVD).

40. The method of any one of claims 36-39, wherein the subject is identified to be at a high risk for a major adverse cardiovascular event (MACE) or has suffered from a MACE.

41. The method of any one of claims 36-40, wherein the subject has an Lp(a) level >150 mg / dL.

42. The method of any one of claims 36-41, further comprising administering to the subject a standard of care (SOC) for hyperlipidemia.

43. The method of claim 42, wherein the SOC is Apo(a) apheresis and / or at least one pharmacological agent.

44. The method of claim 43, wherein the pharmacological agent is selected from the group consisting of a statin, an angiopoietin like 3 (ANGPTL3) inhibitor, a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor, and LY3473329, or a combination thereof.

45. The method of any one of claims 36-44, wherein administering the genome editing system, the RNP complex, or the delivery system reduces an ANGPTL3 protein level in the subject, or in a cell, tissue, or fluid of the subject, by at least about 50% to at least about 95%, relative to the ANGTPL3 protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject.

46. The method of claim 45, wherein administering the genome editing system, the RNP complex, or the delivery system reduces the ANGPTL3 protein level in the subject, or in aAttorney Docket No.: 084177.0330 cell, tissue, or fluid of the subject, by at least 90%, relative to the ANGTPL3 protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject.

47. The method of any one of claims 36-44, wherein administering the genome editing system, the RNP complex, or the delivery system reduces an ANGPTL3 protein level in the subject, or in a cell, tissue, or fluid of the subject, to 10%, relative to the ANGTPL3 protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject48. The genome editing system of claim 15, wherein the one or more modifications is selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LNA), and a combination thereof.

49. The genome editing system of claim 48, wherein the one or more modifications comprises the 5’ inverted thymidine (idT) modification and the 3’ idT modification.

50. The genome editing system of claim 49, wherein the one or more modifications comprises the 2’ fluoro modification.

51. The genome editing system of claim 48, wherein the one or more modifications comprises one or more 2’ fluoro modifications, and each of the 2’ fluoro modifications modifies a nucleotide internal to the gRNA molecule.

52. The genome editing system of claim 51, wherein the gRNA molecule further comprises a 5’ DNA extension.

53. The genome editing system of claim 52, wherein the 5’ DNA extension comprises the sequence set forth in SEQ ID NO: 7.

54. The genome editing system of claim 53, wherein the gRNA molecule comprises the sequence set forth in SEQ ID NO: 91.Attorney Docket No.: 084177.033055. A method of treating an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a formulation comprising:(i) a lipid nanoparticle (LNP);(ii) an mRNA encoding a Cast 2a nuclease; and(iii) a gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene of the subject; wherein the mRNA and the gRNA are encapsulated within the LNP.

56. The method of claim 55, wherein (a) the target sequence of the ANGPTL3 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24- 34; and / or(b) the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.

57. The method of claim 55 or claim 56, wherein the gRNA molecule comprises the sequence set forth in SEQ ID NO: 37.

58. The method of any one of claims 55-57, wherein the gRNA molecule comprises the sequence and modifications set forth in SEQ ID NO: 91.

59. The method of any one of claims 55-58, wherein the subject has homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH).

60. A gRNA molecule comprising a targeting domain that targets a sequence of an ANGPTL3 gene.

61. The gRNA molecule of claim 60, wherein the gRNA molecule comprises one or more modifications selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LNA), and a combination thereof.Attorney Docket No.: 084177.033062. The gRNA molecule of claim 61, wherein the one or more modifications are on nucleotides positioned outside of the targeting domain.

63. The gRNA molecule of claim 61 or claim 62, wherein the gRNA comprises a 5’ DNA extension.

64. The gRNA molecule of any one of claims 61-63, wherein the 5’ DNA extension comprises the sequence set forth in SEQ ID NO: 7.

65. The gRNA molecule of any one of claims 61-64, wherein the gRNA molecule comprises the sequence and modifications set forth in SEQ ID NO: 91.

66. The gRNA molecule of claim 61, wherein the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT / (SEQ ID NO: 95, SEQ ID NOs 111-120, respectively); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.

67. The gRNA molecule of claim 61, wherein the gRNA molecule comprises the following sequence:mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / / i2FC / / i2FU / / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 96, SEQ ID NOs 121-130, respectively); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45, and wherein the 3 ’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.

68. The gRNA molecule of claim 61, wherein the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTTrUrArArUrUrUrCrUrArCrUrCrUrUrGr UrArGrArUx / 3InvdT / (SEQ ID NO: 97, SEQ ID NOs 131-140, respectively);Attorney Docket No.: 084177.0330 wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.

69. The gRNA molecule of claim 61, wherein the gRNA molecule comprises the following sequence: / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i 2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx / 3InvdT (SEQ ID NO: 98, SEQ ID NOs 141-150, respectively); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45.

70. The gRNA molecule of claim 61, wherein the gRNA molecule comprises the following sequence:mA*TGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCrU / i2FA / / i2FC / r U / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUx (SEQ ID NO: 99, SEQ ID NOs 151- 160, respectively); wherein x consists of the targeting domain, and wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-45, and wherein the 3 ’ nucleotide of the targeting domain comprises a 2’ O-methyl modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.

71. The method of any one of claims 32-46 or 55-59, wherein administering the genome editing system, the RNP complex, or the delivery system reduces the Lp(a) level in the subject, or in a cell, tissue, or fluid of the subject, to <150 mg / dL.

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