Engineered crispr associated proteins

Abstract

The present disclosure provides compositions and methods for enhancing gene editing. In some aspects, the compositions and methods are used prevent the progression or treat diseases such as osteoarthritis and other joint disorders that are characterized by an inflammatory component.

Description

ENGINEERED CRISPR ASSOCIATED PROTEINSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 708,639, filed on October 17, 2024, the contents of which are hereby incorporated by reference herein, in its entirety, for all purposes.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML file, named 123994-5019-WO SequenceListing.xml, was created on October 17, 2025 and is 869 kilobytes in size.BACKGROUND

[0003] Gene therapy is a promising method for treating a wide range of diseases, including cancers (such as leukemias and lymphomas), genetic disorders (such as cystic fibrosis, hemophilia, and sickle cell disease), infectious diseases (such as AIDS), and progressive disorders (such as neurodegenerative diseases like Parkinson’s and Alzheimer’s or joint diseases such as arthritis and gout). Particular advantages of gene therapy include its ability to be tailored for specific medical conditions and its ability to treat the underlying causes of diseases rather than just symptoms, typically by reversing an abnormal condition or inducing a new trait.

[0004] Currently, the most common clinical use of gene therapy is treatment of cancers. See, Arabi, F., Mansouri, V, and Ahmadbeigi, N. (2022) Biomedicine & Pharmacotherapy 153: 113324. For example, one strategy used to treat hematological cancers is engineering chimeric receptors in T cells that can target specific cancer-related antigens and have enhanced T cell expansion abilities. A number of successful clinical trials have also targeted particular genetic disorders. For example, both sickle cell disease and beta-thalassemia have recently been treated with ex-vivo use of gene editing technology. Similarly, ATTR amyloidosis has been successfully treated by in vivo use of gene editing technology.

[0005] The particular advantages of gene therapy for the treatment of joint diseases stem from the fact that they are complex diseases marked by progressive joint failure with few options for either long term relief of symptoms or restoring joint function. The immune response plays a central role in progressive joint failure. For example, osteoarthritis is associated with acute chronic inflammation driven by increased levels of pro-inflammatory cytokines. See, Fearon, U., Hanlon, M., Wade, S., and Fletcher, J. (2018) The Journal of Translational Immunology 197(2):170-180. Cytokines are a broad group of small secreted signaling proteins that can bind to their cell surface receptors on target cells and are either pro-inflammatory or anti-inflammatory, sometimes with pleiotropic effects. Pro-inflammatory cytokines induce inflammation by the recruitment and proliferation of immune cells.

[0006] One of the most promising gene therapy techniques is a system using clustered regularly interspaced short palindromic repeats (CRISPR) and the Cas9 protein. This system uses a small guide RNA to direct Cas9 to specific sites in a cell’s genome for generating double strand breaks, which is followed by editing using either the cell’s non-homologous end joining (NHEJ) or homology-directed repair (HDR) DNA repair machinery. See, Li, T., Yang, Y., Qi, H., Zhang, L., Fu, X., He, X., Liu, M., Li, P. and Yu, T. (2023) Signal Transduction and Targeted Therapy 8:36. A template polynucleotide with homology to the region targeted by the guide RNA that also incorporates specific desired nucleotide changes is added to allow incorporation of the desired nucleotide changes into the genome during DNA repair. Nonetheless, the CRISPR Cas9 system is just one of a number of CRISPR-associated endonuclease systems that are capable of altering the expression of genes in mammalian cells with potential utility as a gene therapy agent. BRIEF SUMMARY

[0007] Increasing the efficiency by which a Cas endonuclease can be expressed and reach a target site within the nucleus of a cell therefore increases the editing efficiency in this system and is anticipated to have wide-reaching utility in treating a variety of diseases. This disclosure provides methods and compositions to address this and related needs. Advantageously, the present disclosure provides engineered Cas9 polypeptides fused to nuclear localization signals(NLS) and codon-optimized polynucleotides encoding the same that provide improved gene editing.

[0008] In accordance with some embodiments, the present disclosure provides fusion proteins comprising, from N- to C-terminal, a) a first nuclear localization signal (NLS); b) a CRISPR- associated endonuclease; c) a first linker polypeptide having a first length of from 15 to 17 amino acids; d) a second NLS; e) a second linker polypeptide having a second length of from 15 to 17 amino acids; and f) a third NLS.

[0009] In some embodiments, the first NLS is a first portion of a bipartite NLS. In some aspects, the second NLS is a second portion of the bipartite NLS. In some embodiments, the third NLS is a monopartite NLS. In some embodiments, the first NLS can bind to an importin alpha polypeptide. In some embodiments, the first NLS comprises a strong artificial bipartite NLS of the SV40 T antigen (bpSV40 T3 NLS). In some embodiments, the first NLS comprises an amino acid sequence of NLS1_AA (SEQ ID NO: 1). In some embodiments, the second NLS can bind to an importin alpha polypeptide. In some aspects, the second NLS comprises the bpSV40 T3 NLS. In some embodiments, the second NLS comprises an amino acid sequence of NLS2_AA (SEQ ID NO: 2). In some embodiments, the third NLS can bind to an importin alpha polypeptide. In some embodiments, the third NLS comprises the c-myc NLS. In some embodiments, the third NLS comprises NLS an amino acid sequence of NLS3_AA (SEQ ID NO: 3).

[0010] In some embodiments, the CRISPR-associated endonuclease comprises Cas3, Cas9, Cas12a, Cas13a, or Cas13b. In some embodiments, the CRISPR-associated endonuclease comprises a Cas protein devoid of nucleolytic activity (“dead Cas protein”).

[0011] In some embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease. In some aspects, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 95% identity to Cas9_AA (SEQ ID NO: 21). In some embodiments, the CRISPR-associated endonuclease comprises K848A, K1003A, and R1060A amino acid substitutions relative to SEQ ID NO: 21 (esCas91.1, Slaymaker et al. (2016) Science 351(6268):84-88). In some aspects, theCRISPR-associated endonuclease comprises a R691A amino acid substitution relative to SEQ ID NO: 21 (HiFi Cas9, Vakulskas et al. (2018) Nature 24:1216-1224). In some embodiments, the CRISPR-associated endonuclease comprises N497A, R661A, Q695A, and Q926A amino acid substitutions relative to SEQ ID NO: 21 (Cas9-HF1, Kleinstiver, B. et al. (2016) Nature 529(7587):490-495). In some embodiments, the CRISPR-associated endonuclease comprises N692A, M694A, Q695A, and H698A amino acid substitutions relative to SEQ ID NO: 21 (HypaCas9, Ikeda, A. et al. (2019) Commun Biol 2:371). In some aspects, the CRISPR- associated endonuclease comprises R63A and Q768A amino acid substitutions relative to SEQ ID NO: 21 (Cas9 R63A / Q768A, Bratovic et al. (2020) Nat Chem Biol 16(5):587-595). In some aspects, the CRISPR-associated endonuclease comprises K526E or K526N amino acid substitutions relative to SEQ ID NO: 21 or M495V, Y515N, K526E, and R661Q amino acid substitutions relative to SEQ ID NO: 21 (evoCas9, see, e.g., Casini et al. (2018) Nat Biotechnol 36(3):265-271). In some embodiments, the CRISPR-associated endonuclease comprises F539S, M763I, and K890N amino acid substitutions relative to SEQ ID NO: 21 (Sniper-Cas9, Kim et al. (2020) Nat Biotechnol 38:1328-1336). In some embodiments, the CRISPR-associated endonuclease comprises N690C, T769I, G915M and N980K amino acid substitutions relative to SEQ ID NO: 21 (LZ3 Cas9, Schmid-Burgk et al. (2020) Mol Cell 78(4):794-800.e8). In some embodiments, the CRISPR-associated endonuclease comprises an E1007L amino acid substitution relative to SEQ ID NO: 21.

[0012] In some embodiments , the CRISPR-associated endonuclease comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9_AA (SEQ ID NO: 4). In some aspects, the CRISPR-associated endonuclease comprises an amino acid sequence has 100% identity to Cas9_AA (SEQ ID NO: 21).

[0013] In some aspects, the fusion protein comprises an amino acid sequence having at least 95% identity to Cas9-FP-AA (SEQ ID NO: 23). In some embodiments, the fusion protein comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9-FP-AA (SEQ ID NO: 23). In some embodiments, the fusionprotein comprises an amino acid sequence having 100% identity to Cas9-FP-AA (SEQ ID NO: 23).

[0014] In some embodiments, the first linker polypeptide has a first length of 16 amino acids. In some embodiments, the first linker polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to Full_Linker_1 (SEQ ID NO: 4).

[0015] In some embodiments, the second linker polypeptide has a second length of 16 amino acids. In some embodiments, first linker polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to Full_Linker_2 (SEQ ID NO: 5).

[0016] In some embodiments, the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least 10, at least 11, at least 12, at least 13, or at least 14 of the amino acids in the amino acid sequence are selected from glycine, alanine, and serine. In some embodiments, the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least one amino acid is phenylalanine. In some embodiments, the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least one amino acid is glutamate.

[0017] Also provided herein is a polynucleotide encoding a Cas9 fusion protein comprising a nucleotide sequence having at least 95% identity to DNA (SEQ ID NO: 25).

[0018] Further provided is a polynucleotide encoding a HiFi-Cas9 fusion protein comprising a nucleotide sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9-CO-NA DNA (SEQ ID NO: 25) or its corresponding RNA. Further provided herein is a polynucleotide encoding a Cas9 endonuclease comprising a nucleotide sequence having 100% identity to Cas9-CO-NA DNA (SEQ ID NO: 25) or its corresponding RNA.

[0019] In some embodiments, any one of the polynucleotides as provided herein further comprises the nucleotide sequence for initiation site of T7 RNA polymerase-mediated transcription (Imburgio et al. (2000) Biochemistry 39(34):10419-30).

[0020] In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence encoding a recognition site for a translation initiation complex. In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence of synthetic 5’ UTR NeoUTR3 (Cao et al., (2021) Nat Commun 12, 4138).

[0021] In some embodiments, a polynucleotide as provided herein further comprises a nucleotide sequence of a synthetic eIF4G-recruiting aptamer (Tusup et al. (2018) World J Clin Oncol 9(3):42-55).

[0022] In some embodiments, a polynucleotide as provided herein further comprises a nucleotide sequence encoding an endogenous 5’ UTR of the human hemoglobin alpha-subunit 1 / 2 (HBA1 / 2). In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence encoding a minimalistic 5’ UTR. In some embodiments, any one of the polynucleotides provided herein further comprises nucleotide sequence encoding an endogenous 5’ UTR of hemoglobin beta-subunit (HBB). In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence encoding an a Kozak motif.

[0023] In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence of a polyadenylation signal. In some embodiments, a polynucleotide as provided herein further comprises the sequence for a polyadenylation tail. In some aspects, the polyadenylation tail is segmented by a linker sequence, wherein the linker sequence comprises a nucleotide sequence encoding restriction enzyme cleavage site (Trepotec et al. (2019) 25(4):507-518). In some embodiments, the restriction enzyme cleavage site is a NdeI restriction enzyme cleavage site.

[0024] In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence of a type IIS restriction enzyme site after the poly(A) tail togenerate a free 3’ poly(A) end rather than one extended with unrelated nucleotides (Holtkamp et al. (2006) Blood 108(13):4009-17). In some embodiments, the restriction enzyme cleavage site is a SapI restriction enzyme cleavage site.

[0025] In some embodiments, a polynucleotide as provided herein further comprises a single copy of the endogenous 3’ UTR of the human hemoglobin α-subunit 1 (HBA1).

[0026] In some embodiments, a polynucleotide as provided herein is a circular RNA. In some embodiments, a polynucleotide as provided herein further comprises a synthetic internal ribosome entry site containing a eukaryotic initiation factor 4G-recruiting aptamer. In some embodiments, a polynucleotide as provided herein further comprises 5% N6-methyladenosine. In some embodiments, a polynucleotide as provided herein further comprises the 3’ UTR of the human hemoglobin α-subunit 1 (HBA1).

[0027] In some embodiments, a polynucleotide as provided herein comprises a deoxyribonucleic acid (DNA).

[0028] Also provided herein is a plasmid construct comprising a polynucleotide as provided herein comprising a deoxyribonucleic acid (DNA).

[0029] Also provided herein is a composition comprising: (i) any one of the fusion proteins as provided herein, any one of the polynucleotides as provided herein, or any one of the plasmids as provided herein; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene.

[0030] In some embodiments, the composition is formulated for a non-therapeutic use. In some embodiments, the composition is formulated for the diagnosis of a disease or disorder in a subject.

[0031] In some embodiments, the composition is a pharmaceutical composition for the prevention or treatment of a disease or disorder in a subject. In some embodiments, the pharmaceutical composition is formulated for local, targeted, or regional administration in thesubject. In some embodiments, the pharmaceutical composition is formulated for systemic administration in the subject.

[0032] In some embodiments, the disease or disorder is a genetic disorder. In some embodiments, the disease or disorder is a cancer. In some embodiments, the disease or disorder is an inflammatory disorder.

[0033] In some embodiments, the disease or disorder is a joint disorder. In some embodiments, the joint disorder is arthritis. In some embodiments, the joint disorder is osteoarthritis. In some embodiments, the joint disorder is rheumatoid arthritis. In some embodiments, the joint disorder is post-traumatic arthritis. In some embodiments, the joint disorder is gout. In some embodiments, the joint disorder is pseudogout. In some embodiments, the disease or disorder is tendinopathy in any mammalian species. In some embodiments, the joint disorder occurs in dogs, cats, or horses.

[0034] In some embodiments, the at least one guide RNA targets the coding region for: (i) a transmembrane receptor, (ii) a cytokine, or (iii) a gene associated with the production, blocking, or removal of reactive oxygen species (ROS). In some embodiments, the transmembrane receptor is a cytokine receptor. In some embodiments, the cytokine receptor is an interleukin-1 receptor, an interleukin-6 receptor, or a co-receptor necessary for signal transduction. In some embodiments, the at least one guide RNA targets a gene selected from the group consisting of IL1R1, IL1RAP, IL1R5, IL1R6, IL1R7, and IL1R9. In some embodiments, the at least one guide RNA targets an ILR6 gene. In some embodiments, the cytokine is IL-1 alpha, IL-1 beta, or IL-6. In some embodiments, the at least one guide RNA targets the IL1A or IL1B gene. In some embodiments the at least one guide RNA targets the TNF-alpha or TGF-beta gene. In some embodiments, the at least one guide RNA targets the IL6 gene. In some embodiments, the gene associated with the production, blocking, or removal of reactive oxygen species (ROS) is an NF- kappa B1 or NF-kappa B2 gene. In some embodiments, the at least one guide RNA is a single guide RNA (sgRNA). In some embodiments, the at least one guide RNA targets a human gene. In some embodiments, the at least one guide RNA targets a canine gene. In some embodiments, the at least one guide RNA targets an equine gene. In some embodiments, the at least one guideRNA targets a feline gene. In some embodiments, the at least one guide RNA targets a mammalian gene.

[0035] In some embodiments, the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a viral vector. In some embodiments, the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a lipid nanoparticle (LNP) or a polymeric nanoparticle. In some embodiments, the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a liposome. In some embodiments, the fusion protein and at least one guide RNA or a nucleic acid are encapsulated in a virus-like particle.

[0036] Also provided is a method for altering the genome of a cell comprising contacting the cell with a composition as provided herein. In some embodiments, the cell is synovial cell. In some embodiments, the cell is a synovial fibroblast, a synoviocyte, a chondrocyte, or a synovial macrophage. In some embodiments, the method further comprises administering a therapeutically effective amount of a composition as provided herein to an animal in need thereof. In some embodiments, the method further comprises administering a therapeutically effective amount of a composition as provided herein by injection into a joint. In some embodiments, the composition as provided herein is administered locally to the site of a treatment selected from the group consisting of a surgery, an application of topical ointment, and a combination thereof. DESCRIPTION OF THE DRAWINGS

[0037] The presently disclosed embodiments will be further explained with reference to the attached drawings. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the presently disclosed embodiments.

[0038] FIG.1A illustrates a map of an example coding sequence for an engineered Cas9 protein, in accordance with some embodiments of the present disclosure. FIGS.1B and 1C collectively depict the sequences of N- and C-terminal NLS1_BPSV40 (SEQ ID NOs: 1-2), the C-terminal NLS3_C-Myc (SEQ ID NO: 3), the full linkers 1 (SEQ ID NO: 4) and 2 (SEQ ID NO: 5), the t7 promoter sequence (SEQ ID NO: 6), the Kozak consensus sequence (SEQ ID NO:7), the polyadenylation tail RNA sequence (SEQ ID NO: 8 , the synthetic 5’-UTR NeoUTR3, the eIF4G-recuiting aptamer (SEQ ID NO: 10), and the human hemoglobin subunit alpha endogenous 5’UTR (SEQ ID NO: 11) (FIG.1B); various minimalistic 5’-UTR UTR1 to UTR-7 (SEQ ID NO: 12-18), human hemoglobin subunit beta endogenous 5’UTR (SEQ ID NO: 19), and the human hemoglobin subunit alpha endogenous 3’UTR (SEQ ID NO: 20) (FIG.1C), comprised in the exemplary engineered Cas9 system of FIG.1A.

[0039] FIGs.2A, B, and C collectively show use of 3D structure predictions by AlphaFold2 multimer v3 to optimize binding of NLS to importin-α1 protein.

[0040] FIGs.3A and 3B show wild-type Cas9 (SEQ ID NO: 21) and the HiFi Cas9 (SEQ ID NO: 22) amino acid sequences (FIG.3A); and the Cas9-FP (SEQ ID NO: 23) amino acid sequence (FIG.3B)

[0041] FIGs.4A, 4B, 4C, 4D, and 4E show the nucleic acid sequences for the Cas9-CO-NA construct DNA sequence (SEQ ID NO: 24, FIG.4A, 4B), the human codon-optimized Cas9-CO- NA DNA sequence (SEQ ID NO: 25, FIG.4C), and the Cas9-CO-NA RNA sequence (SEQ ID NO: 26, FIG.4D, 4E).

[0042] FIG.5 shows a gel electrophoresis image of a linearised DNA plasmid encoding an engineered Cas9 protein, in accordance with some embodiments of the present disclosure.

[0043] FIGs.6A and 6B depict examples of human codon-optimized Cas9 mRNA constructs used in the studies described herein.

[0044] FIG.7 illustrates the components used to create an engineered Cas9 mRNA construct for in vivo studies in mice, in accordance with some embodiments of the present disclosure.

[0045] FIG.8 illustrates the results of duplicate experiments of in vitro editing using various Cas9 proteins and 5’ mRNA caps, as described in Example 3.

[0046] FIG.9 depicts dose-dependent induction of frameshift edits of the IL1B gene in canine monocytes following co-electroporation of increasing doses an mRNA encoding an engineeredCas9 protein, in accordance with some embodiments of the present disclosure, and the single guide RNA (sgRNA) OCB02, targeting the canine IL1B gene, as described in Example 4.

[0047] FIG.10A depicts the frameshift efficacy of engineered Cas9 mRNA variants and commercially available Cas9 protein variants in canine monocytes DH82 when delivered together with modified sgRNA OCB02 above their dose-saturation level.

[0048] FIG.10B depicts the frameshift efficacy of engineered Cas9 mRNA variants in canine monocytes DH82 when delivered together with highly modified sgRNA at suboptimal dose level.

[0049] FIGs.11A and 11B illustrate results of in -vivo editing of the IL1B and IL1R1 genes following administration of LNP CRISPR formulations comprising either OCB02 (OBT1, SEQ ID NO: 341) or OCR10 (OBT2, SEQ ID NO: 359) guide RNAs. Figure 11A depicts the analysis of the OBT1-mediated on-target and off-target editing in the synovium, the main source of the inflammatory mediator IL1B, and various off-tissues. For genotyping reconstructed Amp- Seq fragments require at least 80% homology with reference sequence and any detected indels must be within 2 bp from the cut site. Boxplot (all datapoints shown) with corrected two-way ANOVA test: ***, adj. p-value <0.001; ****, adj. p-value, <0.0001. Figure 11B depicts the detection of CRISPR-related on-target editing of IL1R1 in synovial membrane. For genotyping reconstructed Amp-Seq fragments require at least 80% homology with reference sequence and any detected indels must be within 2 bp from the cut site. OBT2-5, injected with OBT2 containing 5% sucrose; OBT2-10, injected with OBT2 containing 10% sucrose. Boxplot (all datapoints shown) with corrected two-way ANOVA test: **, adj. p-value <0.01; ****, adj. p- value, <0.0001.

[0050] FIGs.11C and 11D collectively show the nucleotide sequence for HiFi-Cas9 OBTv3 mRNA (SEQ ID NO: 27) used in the OBT1 and OBT2 formulations.

[0051] FIGs.12A and 12B show the Cas9-OBTv3 complete mRNA sequence (SEQ ID NO: 28). FIG 12B depicts the Cas9-OBTv3 complete mRNA sequence (SEQ ID NO: 28, continued) and the Cas9-OBTv3 coding mRNA sequence (SEQ ID NO: 29). FIG 12C depicts the Cas9-OBTv3 Coding mRNA sequence (SEQ ID NO: 29, continued) and the Cas9-OBTv3 nuclease mRNA sequence (SEQ ID NO: 30). FIG.12D depicts the OBTv3 nuclease mRNA sequence (SEQ ID NO: 30, continued).

[0052] FIG.13A depicts the HiFiCas9-OBTv1 Complete mRNA sequence (SEQ ID NO: 31). FIG.13B depicts the HiFiCas9-OBTv1 Complete mRNA sequence (SEQ ID NO: 31, continued) and the HiFiCas9-OBTv1 Coding mRNA sequence (SEQ ID NO: 32). FIG.13C depicts the - HiFiCas9-OBTv1 Coding mRNA sequence (SEQ ID NO: 32, continued) and the HiFiCas9- OBTv1 nuclease mRNA sequence (SEQ ID NO: 33). FIG.13D depicts the HiFiCas9-OBTv1 nuclease mRNA sequence (SEQ ID NO: 33, continued).

[0053] FIG.14A depicts the HiFiCas9-OBTv2 complete mRNA sequence (SEQ ID NO: 34). FIG.14B depicts the HiFiCas9-OBTv2 complete mRNA sequence (SEQ ID NO: 34, continued) and the HiFiCas9-OBTv2 coding mRNA sequence (SEQ ID NO: 35). FIG.14C depicts the HiFiCas9-OBTv2 coding mRNA sequence (SEQ ID NO: 35, continued) and the HiFiCas9- OBTv2 nuclease mRNA sequence (SEQ ID NO: 36). FIG.14D depicts the Cap1-HiFiCas9- OBTv2 nuclease mRNA sequence (SEQ ID NO: 36, continued).

[0054] FIG.15A depicts the HiFiCas9-OBTv3 complete mRNA sequence (SEQ ID NO: 37). FIG.15B depicts the HiFiCas9-OBTv3 complete mRNA sequence (SEQ ID NO: 37, continued) and the HiFiCas9-OBTv3 coding mRNA sequence (SEQ ID NO: 38). FIG.15C depicts the HiFiCas9-OBTv3 coding mRNA sequence (SEQ ID NO: 38, continued) and the HiFiCas9- OBTv3 nuclease mRNA sequence (SEQ ID NO: 39). FIG.15D depicts the HiFiCas9-OBTv3 nuclease mRNA sequence (SEQ ID NO: 39, continued).

[0055] FIG.16A depicts the HiFiCas9-OBTv4 complete mRNA sequence (SEQ ID NO: 40). FIG.16B depicts the HiFiCas9-OBTv4 complete mRNA sequence (SEQ ID NO: 40, continued) and the HiFiCas9-OBTv4 coding mRNA sequence (SEQ ID NO: 41). FIG.16C depicts the HiFiCas9-OBTv4 coding mRNA sequence (SEQ ID NO: 41, continued) and the HiFiCas9- OBTv4 nuclease mRNA sequence (SEQ ID NO: 42). FIG.16D depicts the HiFiCas9-OBTv4 nuclease mRNA sequence (SEQ ID NO: 42, continued).

[0056] FIG 17A depicts the HiFiCas9-OBTv5 complete mRNA sequence (SEQ ID NO: 43). FIG.17B depicts the HiFiCas9-OBTv5 complete mRNA sequence (SEQ ID NO: 43, continued) and the HiFiCas9-OBTv5 coding mRNA sequence (SEQ ID NO :44). FIG.17C depicts the HiFiCas9-OBTv5 coding mRNA sequence (SEQ ID NO: 44, continued) and the HiFiCas9- OBTv5 nuclease mRNA sequence (SEQ ID NO: 45). FIG.17D depicts the HiFiCas9-OBTv5 nuclease mRNA sequence (SEQ ID NO:45, continued).

[0057] FIG.18A depicts Cas9-LD-OBTv3 complete mRNA (SEQ ID NO: 46). FIG.18B Cas9-LD-OBTv3 complete mRNA (SEQ ID NO: 46, continued) and Cas9-LD-OBTv3 Coding mRNA (SEQ ID NO: 47). FIG.18C depict the Cas9-LD-OBTv3 coding mRNA (SEQ ID NO: 47, continued) and the Cas9-LD-OBTv3 nuclease mRNA (SEQ ID NO: 48). FIG.18D depicts the Cas9-LD-OBTv3 nuclease mRNA (SEQ ID NO: 48, continued).

[0058] FIG.19A depicts the HiFiCas9-LD-OBTv3 complete mRNA sequence (SEQ ID NO: 49). FIG.19B depicts the HiFiCas9-LD-OBTv3 complete mRNA sequence (SEQ ID NO: 49, continued) and the HiFiCas9-LD-OBTv3 Coding mRNA sequence (SEQ ID NO: 50). FIG.19C depicts the HiFiCas9-LD-OBTv3 Coding mRNA sequence (SEQ ID NO: 50, continued) and the HiFiCas9-LD-OBTv3 nuclease mRNA sequence (SEQ ID NO: 51). FIG.19D depicts the HiFiCas9-LD-OBTv3 nuclease mRNA sequence (SEQ ID NO: 51, continued).

[0059] FIG.20A depicts the HiFiCas9-OBTv1 Complete mRNA sequence (SEQ ID NO: 52). FIG.20B depicts the HiFiCas9-OBTv1 Coding mRNA sequence (SEQ ID NO: 52, continued), and the HiFiCas9-OBTv1 Coding mRNA sequence (SEQ ID NO: 53). FIG.20C depicts the HiFiCas9-OBTv1 Coding mRNA sequence (SEQ ID NO: 53, continued) and the HiFiCas9- OBTv1 Nuclease mRNA sequence (SEQ ID NO: 54). FIG.20D depicts the HiFiCas9-OBTv1 Nuclease mRNA sequence (SEQ ID NO: 54, continued).

[0060] FIG.21A depicts the ultraHiFiCas9-LD-OBTv3 complete mRNA sequence (SEQ ID NO: 55). FIG.21B depicts the ultraHiFiCas9-LD-OBTv3 complete mRNA sequence (SEQ ID NO: 55, continued) and the ultraHiFiCas9-LD-OBTv3 coding mRNA sequence (SEQ ID NO: 56). FIG.21C depict the ultraHiFiCas9-LD-OBTv3 coding mRNA sequence (SEQ ID NO: 56,continued) and the ultraHiFiCas9-LD-OBTv3 nuclease mRNA sequence (SEQ ID NO: 57). FIG. 21D depict the ultraHiFiCas9-LD-OBTv3 nuclease mRNA sequence (SEQ ID NO: 57, continued).

[0061] FIG.22A depicts the GA_Cas9v3_pmRVac complete DNA sequence (SEQ ID NO: 58). FIG.22B GA_Cas9v3_pmRVac complete DNA sequence (SEQ ID NO: 58, continued) and GA_Cas9v3_pmRVac coding DNA sequence (SEQ ID NO: 59). FIG.22C depicts the GA_Cas9v3_pmRVac coding DNA sequence (SEQ ID NO: 59, continued) and the GA_Cas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 60). FIG.22D depicts the GA_Cas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 60, continued).

[0062] FIG.23A GA_HiFiCas9v1_pmRVac complete DNA sequence (SEQ ID NO: 61). FIG. 23B GA_HiFiCas9v1_pmRVac complete DNA sequence (SEQ ID NO: 61, continued) and GA_HiFiCas9v1_pmRVac coding DNA sequence (SEQ ID NO: 62). FIG.23C depicts the GA_HiFiCas9v1_pmRVac coding DNA sequence (SEQ ID NO: 62, continued) and the GA_HiFiCas9v1_pmRVac nuclease DNA sequence (SEQ ID NO: 63). FIG.23D depicts the GA_HiFiCas9v1_pmRVac nuclease DNA sequence (SEQ ID NO: 63, continued).

[0063] FIG.24A depicts the GA_HiFiCas9v2_pmRVac complete DNA sequence (SEQ ID NO :64). FIG.24B depicts the GA_HiFiCas9v2_pmRVac complete DNA sequence (SEQ ID NO: 64, continued) and the GA_HiFiCas9v2_pmRVac coding DNA sequence (SEQ ID NO: 65). FIG. 24C depicts the GA_HiFiCas9v2_pmRVac coding DNA sequence (SEQ ID NO: 65, continued) and GA_HiFiCas9v2_pmRVac nuclease DNA sequence (SEQ ID NO: 66). FIG.24D depicts the GA_HiFiCas9v2_pmRVac nuclease DNA sequence (SEQ ID NO: 66, continued).

[0064] FIG.25A depicts the GA_HiFiCas9v3_pmRVAc complete DNA sequence (SEQ ID NO: 67). FIG.25B depicts the GA_HiFiCas9v3_pmRVAc complete DNA sequence (SEQ ID NO: 67, continued) and the GA_HiFiCas9v3_pmRVAc coding DNA sequence (SEQ ID NO: 68). FIG.25C depicts the GA_HiFiCas9v3_pmRVAc coding DNA sequence (SEQ ID NO: 68, continued) and the GA_HiFiCas9v3_pmRVAc nuclease DNA sequence (SEQ ID NO: 69). FIG.25D depicts the GA_HiFiCas9v3_pmRVAc nuclease DNA sequence (SEQ ID NO: 69, continued).

[0065] FIG.26A depicts the GA_HiFiCas9v4_pmRVAc complete DNA sequence (SEQ ID NO: 70). FIG.26B depicts the GA_HiFiCas9v4_pmRVAc complete DNA sequence (SEQ ID NO: 70, continued) and the GA_HiFiCas9v4_pmRVAc coding DNA sequence (SEQ ID NO: 71). FIG.26C depicts the GA_HiFiCas9v4_pmRVAc coding DNA sequence (SEQ ID NO: 71, continued) and the GA_HiFiCas9v4_pmRVAc nuclease DNA sequence (SEQ ID NO: 72). FIG. 26D depicts the GA_HiFiCas9v4_pmRVAc nuclease DNA sequence (SEQ ID NO: 72, continued). FIG.26E depicts the GA_HiFiCas9v4_pmRVAc nuclease DNA sequence (SEQ ID NO: 72 continued).

[0066] FIG.27A depicts the GA_HiFiCas9v5_pmRVAc complete DNA sequence (SEQ ID NO: 73). FIG.27B depicts the GA_HiFiCas9v5_pmRVAc complete DNA sequence (SEQ ID NO: 73, continued) and the GA_HiFiCas9v5_pmRVAc coding DNA sequence (SEQ ID NO: 74). FIG.27C depicts the GA_HiFiCas9v5_pmRVAc coding DNA sequence (SEQ ID NO: 74, continued) and the GA_HiFiCas9v5_pmRVAc nuclease DNA sequence (SEQ ID NO: 75). FIG. 27D depicts the GA_HiFiCas9v5_pmRVAc nuclease DNA sequence (SEQ ID NO: 75, continued). FIG.27E depicts the GA_HiFiCas9v5_pmRVAc nuclease DNA sequence (SEQ ID NO: 75, continued).

[0067] FIG.28A depicts the LD_Cas9v3_pmRVac complete DNA sequence (SEQ ID NO: 76). FIG.28B depicts the LD_Cas9v3_pmRVac complete DNA sequence (SEQ ID NO: 76, continued) and the LD_Cas9v3_pmRVac coding DNA sequence (SEQ ID NO: 77). FIG.28C depicts the LD_Cas9v3_pmRVac coding DNA sequence (SEQ ID NO: 77, continued) and the LD_Cas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 78). FIG.28D depicts the LD_Cas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 78 continued). FIG.28E depicts the LD_Cas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 78, continued).

[0068] FIG.29A depicts the LD_HiFiCas9v3_pmRVac complete DNA sequence (SEQ ID NO: 79). FIG.29B depicts the LD_HiFiCas9v3_pmRVac complete DNA sequence (SEQ IDNO: 79, continued) and the LD_HiFiCas9v3_pmRVac coding DNA sequence (SEQ ID NO: 80). FIG.29C depicts the LD_HiFiCas9v3_pmRVac coding DNA sequence (SEQ ID NO: 80, continued) and the LD_HiFiCas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 81). FIG. 29D depicts the LD_HiFiCas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 81, continued).

[0069] FIG.30A depicts the LD_ultraHiFiCas9v3_pmRVac complete DNA sequence (SEQ ID NO: 82). FIG.30B depicts the LD_ultraHiFiCas9v3_pmRVac complete DNA sequence (SEQ ID NO: 82, continued) and the LD_ultraHiFiCas9v3_pmRVac coding DNA sequence (SEQ ID NO: 83). FIG.30C depicts the LD_ultraHiFiCas9v3_pmRVac coding DNA sequence (SEQ ID NO: 83, continued) and the LD_ultraHiFiCas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 84). FIG.30D depicts the LD_ultraHiFiCas9v3_pmRVac nuclease DNA sequence (SEQ ID NO: 84, continued).

[0070] FIG.31 depicts the C-terminally tagged HiFi-Cas9v3 with the stabilising peptides Exin21 (Stabilon #1), or human STABILON (Stabilon #2), or both Stabilon #1 and Stabilon #2 (HiFi-Cas9v3a, HiFi-Cas9v3b, and HiFi-Cas9v3c, respectively).

[0071] FIGs.32A, and 32B depict the HiFi-Cas9v3a amino acid sequence (FIG.32A, SEQ ID NO: 85), HiFi-Cas9v3b amino acid sequence (FIG.32A, SEQ ID NO: 86), HiFi-Cas9v3c amino acid sequence (FIG.32B, SEQ ID NO: 87), and HiFi-Cas9-FP (FIG.32B, SEQ ID NO: 611) amino acid sequences.

[0072] FIGs.33A, 33B, 33C, and 33D depict the HiFi-Cas9v3a mRNA sequence (FIGs.33A- 33B, SEQ ID NO: 88), the HiFi-Cas9v3b mRNA sequence (FIGs.33B-33C, SEQ ID NO: 89), HiFi-Cas9v3c mRNA sequence (FIGs.33C-33D, SEQ ID NO: 90).

[0073] FIGs.34A, 34B, 34C, 34D, and 34E depict the HiFi-Cas9v3a plasmid DNA sequence (FIGs.34A-34B, SEQ ID NO: 91), HiFi-Cas9v3b plasmid DNA sequence (FIGs.34B-34D, SEQ ID NO: 92), HiFi-Cas9v3c plasmid sequence (FIGs.34D-34E, SEQ ID NO: 93).

[0074] FIG.35A illustrates editing efficiency of the human IL1β gene following electroporation of U-937 monocytic cell line with 80 pmol highly modified sgRNAs against different regions and exons of the human gene coding for IL-1β (OHB series) designed to target the gene and 500 ng OBT WT Cas9 V3 mRNA (SEQ ID NO:94). Frameshift edits were detected using Sanger Sequencing. Error bars are S.E.M with N=1 where there are no error bars and N=2 or N=3 where there are error bars.

[0075] FIGs.35B and 35C collectively show the nucleotide sequence for Cas9 OBTv3 mRNA (SEQ ID NO: 94).

[0076] FIG.36 illustrates editing of the IL1B gene across different dosages of HiFi-Cas9 mRNA v3 and hmOCB02 sgRNA in canine monocytes. Error bars, standard deviation (n=2).

[0077] FIG.37A depicts the architecture of the HiFi-Cas9 mRNA constructs. FIG.37B shows the in vitro performance of the optimized HiFi-Cas9 mRNA constructs in comparison to commercially available wild-type Cas9 protein. Indels were quantified by Amp-Seq (n=2). *, adj. p-value <0.1 according to Tukey test.

[0078] FIGs.38A, 38B, 38C, and 38D depict the ultraHiFi-Cas9v3 complete coding DNA sequence (FIGs.38A-38B, SEQ ID NO: 95), ultraHiFi-Cas9v3 amino acid sequence (FIG.38C, SEQ ID NO: 96) and the ultraHiFi-Cas9v3 mRNA sequence (FIGs.38C-38D, SEQ ID NO: 97), as described in Example 10.

[0079] FIGs.39A, 39B, 39C, and 39D depict the ultra2HiFi-Cas9v3 complete coding DNA sequence (FIGs.39A-39B, SEQ ID NO: 98), ultra2HiFi-Cas9v3 amino acid sequence (FIG. 36C, SEQ ID NO: 99) and the ultra2HiFi-Cas9v3 mRNA sequence (FIGs.36C-36D, SEQ ID NO: 100), as described in Example 10.

[0080] FIG.40A details the experimental groups of the in vivo study on the effects of OBT1 pre- treatment on MSU-induced synovitis in Beagle dogs of Examples 11 and 12. FIG.40B shows the Dunning scale used to score the lameness of treated subjects during the in vivo study.

[0081] FIGs.40C and 40D collectively depict the pre-clinical efficacy of OBT1 in vivo treatment against MSU-induced synovitis, through reduction of lameness score in OBT1-treated dogs (FIG.40C), and preservation of peak vertical force in OBT1-treated dogs (FIG.40D) from Examples 11-12.

[0082] FIG.41A illustrates the synovial fluid sampling schedule throughout the in vivo study of examples 11 and 12.

[0083] FIGs.42A, 42B, 42C, 42D, 42E, 42F, 42G, 42H, 42I, 42J, 42K, 42L, 42M, 42N, 42O, 42P, 42Q, 42R, 42S, 42T, and 42U collectively illustrate spacer sequences for sgRNA targeting different regions and exons of the murine IL1-RAP gene (FIG.42A, SEQ ID NOs: 101-106), the human IL1α gene (SEQ ID Nos: 107-132), the human IL1β gene (FIG.42B, SEQ ID NOs: 133- 150), the human IL1-RAP gene (FIG.42C, SEQ ID NOs: 151-176), the human IL1-R1 gene (FIG.42D, SEQ ID NOs: 177-199), the human IL6-ST gene (FIG.42F, SEQ ID NOs: 200-233), the human TGFβ gene (FIG.42G, SEQ ID NOs: 234-262), the human TGFβIR gene (FIG.42H, SEQ ID NOs: 263-278), the human TGFβIIR gene (FIG.42I, SEQ ID NOs: 279-292), the human TNFα gene (FIG.42J, SEQ ID NOs: 293-318), the canine IL1α gene (FIG.42K, SEQ ID NOs: 319-339), the canine IL1β (FIG.42L, SEQ ID NOs: 340-347), the canine IL1-R1 gene (FIG.42M, SEQ ID NOs: 348-381), the canine IL1-RAP gene (FIG.42N, SEQ ID NOs: 382- 402), the canine IL6-ST gene (FIG.42O, SEQ ID NOs: 403-413), the canine TGFβ gene (FIG. 42P, SEQ ID NOs: 414-441), the canine TNFα gene (FIG.42Q, SEQ ID NOs: 442-471), the equine IL1α gene (FIG.42R, SEQ ID NOs: 472-495), the equine IL1β gene (FIG.42S, SEQ ID NOs: 496-521), the equine IL1-R1 gene (FIG.42T, SEQ ID NOs: 522-550), and the equine IL1- RAP gene (FIG.42U, SEQ ID NOs: 551-580), in accordance with some embodiments of the present disclosure.

[0084] FIG.43 illustrates genotyping results of the dose escalation study described in Examples 11-12. *, p-value of Welch t-test <0.005.

[0085] FIGs.44A and 44B illustrate levels of local monocyte infiltration (FIG.44A) and monocyte reactivity (FIG.44B) in MSU-challenged synovial tissue with and without prophylactic OBT1 treatment.

[0086] While the above-identified drawing sets forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments. DETAILED DESCRIPTION I. Introduction

[0087] Gene therapies such as gene editing hold great promise for treating a wide variety of ailments, from cancers to genetic abnormalities to progressive diseases. However, even the most widely used gene editing methods currently in use that incorporate CRISPR Cas endonucleases have not been optimized for the stability and expression of its components in vitro and in vivo. The present disclosure relates to the development of new Cas endonuclease fusion proteins that can be targeted more efficiently to the nucleus and contain one of a variety of Cas endonuclease variants previously reported to have enhanced activity but not previously tested within this context. In some embodiments further described below, these are expressed from an mRNA polynucleotide that has enhanced stability and is optimized for efficient translation. Compared to other delivery formats such as viral DNA, RNA offers several advantages such as transient expression, no risk of chromosomal integration, low immunogenicity, and scalable production. The CRISPR Cas system requires both a guide RNA, which is a small RNA sequence (sometimes only 100 nucleotides long) that can be chemically synthesized without a template, and a CRISPR Cas protein that cleaves DNA near or within a polynucleotide sequence targeted by the guide RNA. Unlike the guide RNA, the mRNA encoding the CRISPR Cas protein is relatively large (> 4000 nucleotides long) and so cannot be synthesized without a DNA template.In some embodiments further described below, the CRISPR Cas endonuclease mRNA is transcribed from a linearized DNA plasmid. This DNA polynucleotide in turn contains a polyadenylation signal as well as other non-coding nucleotide sequences that enhance the efficiency of starting and stopping transcription.

[0088] The increased expression and stability of these new fusion proteins and polynucleotides results in enhanced efficacy of CRISPR Cas system activity in vitro and in vivo. Examples of such enhancement are described below using a CRISPR Cas9 system optimized for targeting cytokines, which are important for the inflammatory immune response associated with joint diseases. II. Definitions

[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

[0090] The term “in vivo” refers to an event that takes place in a subject’s body.

[0091] The term “in vitro” refers to an event that takes places outside of a subject’s body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0092] The term “reactive oxygen species” or “ROS” can refer to superoxide and its reduced oxygen derivatives and / or peroxynitrite (product of nitric oxide and superoxide interaction).

[0093] The term “effective amount” or “therapeutically effective amount” refers to that amount of a composition or combination of compositions as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration. The term also applies to a dosethat will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compositions chosen, the dosing regimen to be followed, whether the composition is administered in combination with other compositions or compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the composition is carried.

[0094] The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. For example, a composition, method, or system of the present disclosure may be administered as a prophylactic treatment to a subject that has a predisposition for a given condition (e.g., arthritis). “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, canine, feline, or equine, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine. It is understood that compositions and methods of the present disclosure are applicable to treat all mammals, including, but not limited to human, canine, feline, equine, and bovine subjects.

[0095] The terms “polynucleotide,” “nucleotide,” and “nucleic acid” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA, lncRNA, RNA antagomirs, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), aptamers, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA).Polynucleotides also include non-coding RNA, which include for example, but are not limited to, RNAi, miRNAs, lncRNAs, RNA antagomirs, aptamers, and any other non-coding RNAs known to those of skill in the art. Polynucleotides include naturally occurring, synthetic, and intentionally altered or modified polynucleotides as well as analogues and derivatives. The term “polynucleotide” also refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof, and is synonymous with nucleic acid sequence. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment as described herein encompassing a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. Polynucleotides can be single, double, or triplex, linear or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5’ to 3’ direction.

[0096] The term “gene” or “nucleotide sequence encoding a polypeptide” refers to the segment of DNA involved in producing a polypeptide chain. The DNA segment may include regions preceding and following the coding region (leader and trailer) involved in the transcription / translation of the gene product and the regulation of the transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons). For example, a gene includes a polynucleotide containing at least one open reading frame capable of encoding a particular protein or polypeptide after being transcribed and translated.

[0097] The terms “sequence identity,” “percent identity,” and “sequence percent identity” (or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government’s National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. ClustalW and ClustalX may be used to produce alignments, Larkin et al., Bioinformatics 23:2947-2948 (2007); Goujon et al., Nucleic Acids Research, 38 Suppl :W 695-9 (2010); and, Me William et al., Nucleic Acids Research 41(Web Server issue):W 597-600 (2013). One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0098] As used herein, the term “variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0099] Examples of cancers that may be treated using the methods of the invention include vascularized tumors, melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), mesothelioma, squamous cell cancer, lung cancer, small-cell lung cancer, non-smallcell lung cancer, neuroendocrine lung cancer (including pleural mesothelioma, neuroendocrine lung carcinoma), NSCL (large cell), NSCLC large cell adenocarcinoma, non-small cell lung carcinoma (NSCLC), NSCLC squamous cell, soft-tissue sarcoma, Kaposi’s sarcoma, adenocarcinoma of the lung, squamous carcinoma of the lung, NSCLC with PDL1 >=50% TPS, neuroendocrine lung carcinoma, atypical carcinoid lung cancer, cancer of the peritoneum, esophageal cancer, hepatocellular cancer, liver cancer (including HCC), gastric cancer, stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, urothelial cancer, bladder cancer, hepatoma, glioma, brain cancer (as well as edema, such as that associated with brain tumors), breast cancer (including, for example, triple negative breast cancer), testis cancer, testicular germ cell tumors, colon cancer, colorectal cancer (CRC), colorectal cancer MSS (MSS-CRC); refractory MSS colorectal; MSS (microsatellite stable status), primary peritoneal cancer, primary peritoneal ovarian carcinoma, microsatellite stable primary peritoneal cancer, platinum resistant microsatellite stable primary peritoneal cancer, CRC (MSS unknown), rectal cancer, endometrial cancer (including endometrial carcinoma), uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell cancer (RCC), renal cell carcinoma (RCC), gastro-esophageal junction cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, carcinoid carcinoma, head and neck cancer, B-cell lymphoma (including non-Hodgkin’s lymphoma, as well as low grade / follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, Diffuse Large B cell lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström’s Macroglobulinemia, Hodgkin’s lymphoma (HD), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), T cell Acute Lymphoblastic Leukemia (T-ALL), Acute myeloid leukemia (AML), Hairy cell leukemia, chronic myeloblastic leukemia, multiple myeloma, post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, Meigs’ syndrome, Merkel Cell cancer, MSI‐high cancer, KRAS mutant tumors, adult T‐cell leukemia / lymphoma, adenoid cystic cancer (including adenoid cystic carcinoma), melanoma, malignant melanoma, metastatic melanoma, pancreatic cancer, pancreatic adenocarcinoma,ovarian cancer (including ovarian carcinoma), pleural mesothelioma, cervical squamous cell carcinoma (cervical SCC), anal squamous cell carcinoma (anal SCC), carcinoma of unknown primary, gallbladder cancer, pleural mesothelioma, chordoma, endometrial sarcoma, chondrosarcoma, uterine sarcoma, uveal melanoma, amyloidosis, AL-amyloidosis, astrocytoma, and Myelodysplastic syndromes (MDS), renal clear cell carcinoma (RCC), lung cancer, NSCLC, lung adenocarcinoma, lung squamous cell carcinoma, gastric adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer (TNBC), head and neck tumor, colorectal adenocarcinoma, melanoma, and metastatic melanoma.

[0100] “Joint disease” is defined as measurable abnormalities in the cells or tissues of the joint that could lead to illness, for example, metabolic and molecular derangements triggering anatomical and / or physiological changes in the joint. These include, but are not limited to, radiographic detection of joint space narrowing, subchondral sclerosis, subchondral cysts, and osteophyte formation.

[0101] As used herein, a sgRNA (single guide RNA) is a RNA, preferably a synthetic RNA, composed of a targeting sequence and scaffold. It is used to guide Cas9 to a specific genomic locus in genome engineering experiments. The sgRNA can be administered or formulated, e.g., as a synthetic RNA, or as a nucleic acid comprising a sequence encoding the gRNA, which is then expressed in the target cells. As would be evident to one of ordinary skill in the art, various tools may be used to design and / or optimize the sequence of a sgRNA, for example to increase the specificity and / or precision of genomic editing. In general, candidate sgRNAs may be designed by identifying a sequence within the target region that has a high predicted on-target efficiency and low off-target efficiency based on any of the available web-based tools. Candidate sgRNAs may be further assessed by manual inspection and / or experimental screening. Examples of web-based tools include, without limitation, CRISPR seek, CRISPR Design Tool, Cas- OFFinder, E-CRISP, ChopChop, CasOT, CRISPR direct, CRISPOR, BREAKING-CAS, CrispRGold, and CCTop. See, e.g., Safari, et al. Current Pharma. Biotechol. (2017) 18(13), which is incorporated by reference herein in its entirety for all purposes. Such tools are also described, for example, in PCT Publication No. WO2014093701A1 and Liu, et al.,“Computational approached for effective CRISPR guide RNA design and evaluation”, Comput Struct Biotechnol J., 2020; 18: 35–44, each of which is incorporated by reference herein in its entirety for all purposes.

[0102] As used herein, “Cas9” refers to CRISPR Associated Protein; the Cas9 nuclease is the active enzyme for the Type II CRISPR system. “nCas9” refers to a Cas9 that has one of the two nuclease domains inactivated, i.e., either the RuvC or HNH domain. nCas9 is capable of cleaving only one strand of target DNA (a “nickase”). The term “Cas9” refers to an RNA-guided double- stranded DNA-binding nuclease protein or nickase protein, or a variant thereof. Herein, “Cas9” refers to both naturally-occurring and recombinant Cas9s. Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. Cas9 enzymes described herein can comprise a HNH or HNH-like nuclease domain and / or a RuvC or RuvC- like nuclease domain. Cas9 can induce double-strand breaks in genomic DNA (target locus) when both functional domains are active. The Cas9 enzyme can comprise one or more catalytic domains of a Cas9 protein derived from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the two catalytic domains are derived from different bacteria species.

[0103] As used herein, “PAM” refers to a Protospacer Adjacent Motif and is necessary for Cas9 to bind target DNA, and immediately follows the target sequence. The Cas9 can be administered or formulated, e.g., as a protein (e.g., a recombinant protein), or as a nucleic acid comprising a sequence encoding the Cas9 protein, which is then expressed in the target cells. Naturally occurring Cas9 molecules recognize specific PAM sequences (e.g., the PAM recognition sequences for S. pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis). In an embodiment, a Cas9 molecule has the same PAM specificities as a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule has a PAM specificity not associated with a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule’sPAM specificity is not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered such that the PAM sequence recognition is altered to decrease off target sites, improve specificity, or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule may be altered (e.g., to lengthen a PAM recognition sequence, improve Cas9 specificity to high level of identity, to decrease off target sites, and / or increase specificity). In an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. In some embodiments, a Cas9 molecule may be altered to ablate PAM recognition.

[0104] The term “promoter” is used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Other elements that may be present in an expression vector include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators), as well as those that confer certain binding affinity or antigenicity to the recombinant protein produced from the expression vector.

[0105] The term “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0106] The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0107] As used herein, the term “a”, “an”, or “the” generally is construed to cover both the singular and the plural forms.

[0108] The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that compositions, amounts, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0109] The transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed methods and compositions. All compositions, methods, and kits described herein that embody the present disclosure can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

[0110] A subject treated by any of the methods or compositions described herein can be of any age and can be an adult, infant or child. In some cases, the subject is 0, 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, or 99 years old, or within a range therein (e.g., without limitation, between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). The subject can be a human or non-human subject. A particular class of subjects that can benefit from the compositions and methods of the present disclosure include subjects over the age of 40, 50, or 60 years. Another class of subjects that can benefit from the compositions and methods of the present disclosure are subjects that have arthritis (e.g., osteoarthritis).

[0111] Any of the compositions disclosed herein can be administered to a non-human subject, such as a laboratory or farm animal. Non-limiting examples of a non-human subject include laboratory or research animals, pets, wild or domestic animals, farm animals, etc., e.g., a dog, a goat, a guinea pig, a hamster, a mouse, a pig, a non-human primate (e.g., a gorilla, an ape, an orangutan, a lemur, a baboon, etc.), a rat, a sheep, a horse, a cow, or the like. III. CRISPR / Cas systems – minimum requirements

[0112] In one aspect, clustered regularly interspaced short palindromic repeats and CRISPR- associated RNA-guided nuclease-related methods, components and compositions of the disclosure (hereafter, CRISPR / Cas systems) minimally require at least one isolated or non- naturally-occurring protein component (e.g., a Cas protein) and at least one isolated or non- naturally-occurring nucleic acid component (e.g., a guide RNA (gRNA)) to effectuate augmentation of a `nucleic acid sequence (e.g., genomic DNA).

[0113] In some embodiments, a CRISPR / Cas system effectuates the alteration of a targeted gene or locus in a eukaryotic cell by effecting an alteration of the sequence at a target position, e.g., by creating an insertion or deletion (collectively, an indel) resulting in loss-of-function of (i.e., knocking out) the affected gene or allele. For example, a CRISPR / Cas system mayintroduce a nucleotide substitution resulting in a truncation, nonsense mutation, or other type of loss-of-function of an encoded gene product of a target gene, a deletion of one or more nucleotides resulting in a truncation, nonsense mutation, or other type of loss-of-function of a gene product, or an insertion resulting in a truncation, nonsense mutation, or other type of loss- of-function of an encoded gene product.

[0114] CRISPR / Cas systems effectuate changes to the sequence of a nucleic acid through nuclease activity. For example, a CRISPR associated (Cas) protein, e.g., Cas9, is guided by a nucleic acid guide that hybridizes to a target position within a targeted gene or locus through sequence complementarity with a CRISPR RNA (crRNA) sequence, e.g., in a synthetic single guide RNA (sgRNA). The Cas protein then cleaves the genomic DNAupon recognition of a, nuclease-specific motif called the protospacer adjacent motif (PAM). See generally, Collias, D., & Beisel, C. L. (2021). Nature Communications, 12(1), 1-12, the disclosure of which is incorporated herein by reference in its entirety.

[0115] The nuclease activity of the Cas protein induces a double-strand break (DSB). Endogenous cellular mechanisms of DSB repair, namely non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination, result in erroneous repair at a given target position with some calculable frequency as a result of interference from said components of the CRISPR / Cas system, thereby introducing substitutions or indels into the genomic DNA. See generally Scully, R., et al. (2019). Nature Reviews Molecular Cell Biology, 20(11), 698-714, the disclosure of which is incorporated herein by reference in its entirety. At some frequency, these indels and / or substitutions may result in frameshifts, nonsense mutations (i.e., early stop codons) or truncations that impact the availability of gene products, such as mRNA and / or protein. In certain embodiments, the CRISPR / Cas system may induce a homology-directed repair (HDR) mechanism leading to insertions of non-random sequences at a target position through the use of templates (e.g., an HDR template) provided to the cell as part of the system along with the nuclease and gRNA. See Bloh, K., & Rivera-Torres, N. (2021). International Journal of Molecular Sciences, 22(8), 3834, the disclosure of which is incorporated herein by reference in its entirety.

[0116] In general, the minimum requirements of a CRISPR / Cas system are dependent upon the nuclease, e.g., the Cas protein, used in the system.. To this extent, these bacterially-derived nucleases have been functionally divided into Types I, III, and V, which all fall into Class 1 and Types II, IV, and VI that are grouped into Class 2. A. Class 1 CRISPR / Cas systems

[0117] The exact components, compositions, and methods for effectuating a change in a targeted nucleic acid sequence using a Class 1 CRISPR / Cas system will vary, but should minimally include: a nuclease (selected from at least Types I, and III), at least one guide RNA selected from 1) sgRNA or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems have been categorized together as Class 1 CRISPR / Cas systems due to their similarities in requirements and mode of action within a eukaryotic cell. To this end, compositions, components, and methods among Class 1 constituents may be considered functionally interchangeable, and the following details, provided merely for exemplary purposes, do not represent an exhaustive list of class members:

[0118] Cas3 (see Table 1) is the prototypical Type I DNA nuclease that functions as the effector protein as part of a larger complex (the Cascade complex comprising Cse1, Cse2,), that is capable of genome editing. See generally He, L., et al. (2020). Genes, 11(2), 208. Unlike other CRISPR / Cas systems, Type I systems localize to the DNA target without the Cas3 nuclease via the Cascade complex, which then recruits Cas3 to cleave DNA upon binding and locating the 3’ PAM. The Cascade complex is also responsible for processing crRNAs such that they can be used to guide it to the target position. Because of this functionality, Cascade has the ability to process multiple arrayed crRNAs from a single molecule. See . Luo, M. (2015). Nucleic Acids Research, 43(1), 674-681. As such, Type I system may be used to edit multiple targeted genes or loci from a single molecule.

[0119] Because the natural Cas3 substrate is ssDNA, its function in genomic editing is thought to be as a nickase; however, when targeted in tandem, the resulting edit is a result of blunt endcuts to opposing strands to approximate a blunt-cutting endonuclease, such as Cas9. See Pickar- Oliver, A., & Gersbach, C. A. (2019). Nature Reviews Molecular Cell Biology, 20(8), 490-507.

[0120] Like Type I nucleases, the Type III system relies upon an complex of proteins to effect nucleic acid cleavage. Particularly, CaslO possesses the nuclease activity to cleave ssDNA in prokaryotes. See Tamulaitis, G. Trends in Microbiology, 25(1), 49-61. Interestingly, this CRISPR / Cas system, native to archea, exhibits dual specificity and targets both ssDNA and ssRNA. Aside from this change, the system functions much like Type I in that the crRNA targets an effector complex (similar to Cascade) in a sequence-dependent manner. Similarly, the effector complex processes crRNAs prior to association. The dual nature of this nuclease makes its applications to genomic editing potentially more powerful, as both genomic DNA and, in some cases, mRNAs with the same sequence may be targeted to silence particular targeted genes.B, Class 2 CRISPR / Cas systems

[0121] The exact components, compositions, and methods for effectuating a change in a targeted nucleic acid sequence using a Class 2 CRISPR / Cas system will vary, but should minimally include: a nuclease (selected from at least Types II, and V), at least one guide RNA selected from 1) sgRNA or 2) a combination of crRNA and tracrRNA. These CRISPR / Cas systems have been categorized together as Class 2 CRISPR / Cas systems due to their similarities in requirements and mode of action within a eukaryotic cell. To this end, compositions, components, and methods among Class 2 constituents may be considered functionally interchangeable, and the following details, provided merely for illustrative purposes, do not represent an exhaustive list of class members:

[0122] Type II nucleases are the best-characterized CRISPR / Cas systems, particularly the canonical genomic editing nuclease Cas9 (see Table 1). Multiple Cas9 proteins, derived from various bacterial species, have been isolated. The primary distinction between these nucleases is the PAM, a required recognition site within the targeted dsDNA. After association with a gRNA molecule, the crRNA (or targeting domain of a sgRNA) orients the nuclease at the properposition, but the protein’s recognition of the PAM is what induces a cleavage event near that site, resulting in a blunt DSB.

[0123] In addition to the naturally-derived Cas9 proteins, several engineered variants have similarly been reported. These range from Cas9 with enhanced specific (i.e., less off-target activity), such as eSpCas9 v1.1. Others have been catalytically modified via point mutations in the RuvC (e.g., D10A) and HNH (e.g., H840A) domains such that they induce only single-strand breaks (i.e., Cas9 nickases). See Frock, R. et al. (2015). Nature Biotechnology, 33(2), 179-186. These have also been shown to be less error-prone in editing. Such mitigation of off-target effects becomes paramount when selecting for a desired insertion (i.e., a knock in mutation, in which a desired nucleotide sequence is introduced into a target nucleic acid molecule) rather than a deletion. Indeed, less off-target effects may aid in the preferred DNA repair mechanism (HDR, in most instances for knock in mutations). See generally Naeem, M., et al. (2020). Cells, 9(7), 1608.

[0124] Additional examples of engineered Cas variants are found, for example, in WO2015035162A2, WO2019126716A1, WO2019126774A1, WO2014093694A1, and WO2014150624A1, the disclosures of which are incorporated herein by reference in their entireties.

[0125] Like the canonical Cas9 systems, Type V nucleases only require a modified sgRNA with a targeting domain complementary to a genomic sequence to carry out genomic editing. These nucleases contain a RuvC domain but lack the HNH domain of Type II nucleases. Further, Cas12, for example, leaves a staggered cut in the dsDNA substrate distal to the PAM, as compared to Cas9’s blunt cut next to the PAM. Both Cas12a, also known as Cpf1, and Cas12b, also known as C2c1 (see Table 1), act as part of larger complex of two gRNA-associated nucleases that ) acts on dsDNA as quarternary structure nicking each strand simultaneously. See Zetsche B, , et al. Cell.2015;163(3):759-771.; see also Liu L, Chen P, Wang M, et al. Mol Cell. 2017;65(2):310-322. Additionally, Cas12b (C2c1) is a highly accurate nuclease with little tolerance for mismatches. See Yang H, et al. Cell.2016;167(7):1814-1828.e12.Table 1. Exemplary list of Cas nucleases and their requirements

[0126] See generally Wang, J., Zhang, C., & Feng, B. (2020). Journal of Cellular and Molecular Medicine, 24(6), 3256-3270, the disclosure of which is incorporated herein by reference in its entirety. In one aspect, the CRISPR / Cas system of the present disclosure comprises at least one Cas protein derived from one or more of the following selected bacterialgenera: Corynebacterium, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavobacterium, Spirochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Nitratifractor, Campylobacter, Pseudomonas, Streptomyces, Staphylococcus, Francisella, Acidaminococcus, Lachnospiraceae, Leptotrichia, and Prevotella. In some embodiments, the Cas protein is derived from Deltaproteobacteria or Planctomycetes bacterial species. C. “Dead Cas protein” systems

[0127] In some embodiments, the CRISPR-associated endonuclease comprises a Cas protein devoid of nucleolytic activity (“dead Cas protein”). Dead Cas proteins can be used, for example, for investigating chromatin structure if tethered to enzymes that are able to mark chromatin factors, base editing if coupled to base editing enzymes such as cytidine or adenine deaminases, epigenetic remodeling, and transcriptional regulation if tethered to transcriptional suppressors or activators, as described in Brezgin et al. (2019) Int. J. Mol. Sci.20(23):6041. One example of an effective CRISPR-based repressor is the fusion of the repressor KRAB-MeCP2 domain to a nuclease-deficient Cas9 domain, as described in Yeo et al. (2018) Nat. Methods 15(8):611-616. D. CRISPR guide RNAs

[0128] Some aspects of the present disclosure provide strategies, methods, compositions, and treatment modalities for altering a targeted sequence within a gene locus (e.g., altering the sequence of a wild type and / or of a mutant in a cell or in a patient having or experiencing effects of rheumatoid arthritis, osteoarthritis, or other inflammatory diseases of the joint or cells therein) by insertion or deletion of one or more nucleotides mediated by an RNA-guided nuclease and one or more guide RNAs (gRNAs), resulting in loss of function of the targeted gene product. Such an alteration is alternatively referred to as “knocking out” the gene of interest (i.e., generation of a “knock out”).

[0129] In one aspect, the CRISPR / Cas system of the present disclosure further provides a gRNA molecule (e.g., an isolated or non-naturally occurring RNA molecule) that interacts with the Cas protein. In certain embodiments, the gRNA is an sgRNA, in which the targeting (i.e.,complementary) domain, comprising a nucleotide sequence which is complementary with a target domain from a targeted gene, is incorporated into a single RNA molecule with the protein- interacting domain. In certain embodiments, the targeting domain is a crRNA that is provided to eukaryotic cells with tracrRNA, which acts as a scaffold through interactions with both the crRNA and the nuclease. In some embodiments, the system is further, optionally, comprised of an oligonucleotide—an HDR template with homology to either side of the target position. See, for example, Bloh, K., & Rivera-Torres, N. (2021). International Journal of Molecular Sciences, 22(8):3834, doi.org:10.3390 / ijms22083834, the disclosure of which is incorporated herein by reference in its entirety..

[0130] In an embodiment, the targeting domain of the gRNA molecule is configured to orient an associated nuclease such that a cleavage event, (e.g., a double strand break or a single strand break) occurs sufficiently close to a target position, in the targeted gene or locus, thereby facilitating an alteration in the nucleic acid sequence. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0131] In some embodiments, the targeting domain orients the nuclease such that a cleavage event occurs within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 nucleotides of a target position. The double-strand or single-strand break, may be positioned upstream or downstream of a target position, and either within or upstream a functional domain cluster within the targeted gene. IV. Fusion Protein

[0132] In accordance with some embodiments, the present disclosure provides fusion proteins comprising, from N- to C-terminal, a) a first nuclear localization signal (NLS); b) a CRISPR- associated endonuclease; c) a first linker polypeptide having a first length of from 15 to 17 amino acids; d) a second NLS; e) a second linker polypeptide having a second length of from 15 to 17 amino acids; and f) a third NLS.

[0133] In some aspects, the fusion protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9-FP- AA (SEQ ID NO: 23). In some embodiments, the fusion protein comprises an amino acid sequence having 100% identity to Cas9-FP-AA (SEQ ID NO: 23). In some aspects, the fusion protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to HiFi-Cas9 v3a (SEQ ID NO: 85).

[0134] In some embodiments, the fusion protein comprises an amino acid sequence having 100% identity to HiFi-Cas9 v3a (SEQ ID NO: 85). In some aspects, the fusion protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to HiFi-Cas9 v3b (SEQ ID NO: 86). In some embodiments, the fusion protein comprises an amino acid sequence having 100% identity to HiFi-Cas9 v3b (SEQ ID NO: 86). In some aspects, the fusion protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to HiFi-Cas9 v3c (SEQ ID NO: 87). In some embodiments, the fusion protein comprises an amino acid sequence having 100% identity to HiFi-Cas9 v3c (SEQ ID NO: 87).

[0135] In some aspects, the fusion protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to ultraHiFi-Cas9 v3 (SEQ ID NO: 96). In some embodiments, the fusion protein comprises an amino acid sequence having 100% identity to ultraHiFi-Cas9 v3 (SEQ ID NO: 96). In some aspects, the fusion protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to ultra2HiFi-Cas9 v3 (SEQ ID NO: 99). In some embodiments, the fusion protein comprises an amino acid sequence having 100% identity to ultra2HiFi-Cas9 v3 (SEQ ID NO: 99).

[0136] A. NLS

[0137] Nuclear localization signals are necessary for the transport of proteins from the cytoplasm into the nucleus through the nuclear pore complex. See, Lu, J., Zhang, B., Liu, S.,Song, W., Qiao, J., and Ruan, H. (2021) Cell Commun Signal 19:60, the disclosure of which is incorporated herein by reference in its entirety. An NLS can be monopartite or bipartite. Monopartite NLS typically have a single cluster of 4-8 basic amino acids with 4 or more positively charged amino acids. A consensus motif for a monopartite NLS is K (K / R) X (K / R), where X can be any amino acid. In contrast, bipartite NLS typically contain two clusters of 2-3 positively charged amino acids separated by an intervening sequence. A consensus sequence for bipartite NLS is R / K (X)10-12KRXK. Even though the intervening sequence was originally defined as 10-12 amino acids long, an artificial bipartite NLS derived from SV40 NLS (bpSV40 T3 NLS) has been found to have at least partial import activity when engineered with longer intervening sequences. See, Lange, A., McLane, L., Mills, R., Devine, S., and Corbett, A. (2010) Traffic 11(3):311-323, the disclosure of which is incorporated herein by reference in its entirety.

[0138] Classical NLS are recognized by an importin alpha (also known as karyopherin alpha) adapter protein, which in turn associates with an importin beta (also known as kayopherin beta), which is the transport factor that carries proteins through the nuclear pore complex. A Ras GTPase provides the energy for transport. The mammalian importin alpha superfamily contains six members: alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha 7.

[0139] In some embodiments, the first NLS is a first portion of a bipartite NLS. In some aspects, the second NLS is a second portion of the bipartite NLS. In some embodiments, the third NLS is a monopartite NLS. In some embodiments, the first NLS can bind to an importin- alpha polypeptide. In some embodiments, the first NLS comprises bpSV40 T3 NLS. In some embodiments, the first NLS comprises an amino acid sequence of NLS1_AA (SEQ ID NO: 1). In some embodiments, the second NLS can bind to an importin-alpha polypeptide. In some aspects, the second NLS comprises the bipartite SV40 T3 NLS. In some embodiments, the second NLS comprises an amino acid sequence of NLS2_AA (SEQ ID NO: 2). In some embodiments, the third NLS can bind to an importin-alpha polypeptide. In some embodiments, the third NLS comprises the c-myc NLS. In some embodiments, the third NLS comprises NLS an amino acid sequence of NLS3_AA (SEQ ID NO: 3).B. CRISPR-Cas

[0140] In some embodiments, the CRISPR-associated endonuclease comprises a Cas3, Cas9, Cas12a, Cas13a, or Cas13b polypeptide.

[0141] In some embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease. In some aspects, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 95% identity to Cas9_AA (SEQ ID NO: 21). In some embodiments, the CRISPR-associated endonuclease comprises K848A, K1003A, and R1060A amino acid substitutions relative to SEQ ID NO: 21. In some aspects, the CRISPR-associated endonuclease comprises a R691A amino acid substitution relative to SEQ ID NO: 21 and comprise an amino acid sequence having at least 95% identity to HiFi_Cas9 (SEQ ID NO: 22). In some embodiments, the CRISPR-associated endonuclease comprises N497A, R661A, Q695A, and Q926A amino acid substitutions relative to SEQ ID NO: 21. In some embodiments, the CRISPR-associated endonuclease comprises N692A, M694A, Q695A, and H698A amino acid substitutions relative to SEQ ID NO: 21. In some aspects, the CRISPR-associated endonuclease comprises R63A and Q768A amino acid substitutions relative to SEQ ID NO: 21. In some aspects, the CRISPR-associated endonuclease comprises M495V, Y515N, K526E and R661Q amino acid substitutions relative to SEQ ID NO: 21. In some aspects, the CRISPR-associated endonuclease comprises K526E or K526N amino acid substitutions relative to SEQ ID NO: 21. In some embodiments, the CRISPR-associated endonuclease comprises F539S, M763I, and K890N amino acid substitutions relative to SEQ ID NO: 21. In some embodiments, the CRISPR-associated endonuclease comprises a E1007L amino acid substitution relative to SEQ ID NO: 21. In some embodiments, the CRISPR-associated endonuclease comprises N690C, T769I, G915M and N980K amino acid substitutions relative to SEQ ID NO: 21. In some embodiments, the CRISPR-associated endonuclease comprises an E1007L amino acid substitution relative to SEQ ID NO: 21.

[0142] In some embodiments , the CRISPR-associated endonuclease comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9_AA (SEQ ID NO: 21). In some aspects, the CRISPR-associated endonuclease comprisesan amino acid sequence has 100% identity to Cas9_AA (SEQ ID NO: 21). In some embodiments , the CRISPR-associated endonuclease comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to HiFi_Cas9_AA (SEQ ID NO: 22). In some aspects, the CRISPR-associated endonuclease comprises an amino acid sequence has 100% identity to Cas9_AA (SEQ ID NO: 22). C. Linker Peptides

[0143] In some embodiments, the first linker polypeptide has a first length of 16 amino acids. In some embodiments, the first linker polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to Full_Linker_1 (SEQ ID NO: 4).

[0144] In some embodiments, the second linker polypeptide has a second length of 16 amino acids. In some embodiments, second linker polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to Full_Linker_2 (SEQ ID NO: 5).

[0145] In some embodiments, the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least 10, at least 11, at least 12, at least 13, or at least 14 of the amino acids in the amino acid sequence are selected from glycine, alanine, and serine. In some embodiments, the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least one amino acid is phenylalanine. In some embodiments, the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least one amino acid is glutamate. V. Nucleic acids encoding an engineered Cas9 polypeptide

[0146] Since amino acids can be encoded by more than one codon, it is also possible to engineer codon optimized gene coding sequences. One consideration with codon optimization is avoiding rare codons for a particular host organism that would otherwise reduce protein expression in that organism. Another consideration is the avoidance of genetic elements thatcould interfere with cloning or expression of a gene. Such elements include extra restriction sites that may interfere with desired cloning strategies. Also potentially problematic are elements that could serve as promoter sequences, internal ribosome entry sites, putative splice donor and acceptor sites, alternative start or stop codons in different reading frames, intragenic polyadenylation sites, or sites that initiate RNAse cleavage or methylation-dependent gene silencing. See, Mauro, V. and Chappell, S. (2014) Trends Mol Med 20(11):604-613. Several algorithms have been developed to design codon optimized gene sequences used for cloning, each taking into account different considerations that can then be tested empirically.

[0147] In some embodiments, the engineered Cas9 protein is encoded by an mRNA that comprises at least one modified nucleoside triphosphate, defined herein as nucleotide analogs / modifications such as backbone modifications, sugar modifications or base modifications that can enhance the expression or stability of the mRNA. A backbone involves modification the phosphates of the backbone of chemically modified nucleotides. In this context, a sugar modification is a chemical modification of the sugar of the nucleotides, and a base modification is a chemical modification of the base moiety of the nucleotides. Such modifications can enhance the expression and / or stability of an mRNA molecule. See, e.g., Li et al. (2016) Bioconjugate Chem 27:849-53.

[0148] Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.

[0149] For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.

[0150] In some embodiments of nucleotide analogs / modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5 ‘- triphosphate, 2-Aminopurine-riboside-5’- triphosphate; 2-aminoadenosine-5 ‘-triphosphate, 2’- Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5 ‘-triphosphate, 2-thiouridine-5’-triphosphate, 2’-Fluorothymidine-5’- triphosphate, 2’-0-Methyl inosine-5’-triphosphate 4- thiouridine-5’-triphosphate, 5- aminoallylcytidine-5’-triphosphate, 5-aminoallyluridine-5’- triphosphate, 5-bromocytidine- 5’-triphosphate, 5-bromouridine-5’-triphosphate, 5-Bromo-2’- deoxycytidine-5’- triphosphate, 5-Bromo-2’-deoxyuridine-5’-triphosphate, 5 -iodocytidine-5 ‘- triphosphate, 5- Iodo-2’-deoxycytidine-5’-triphosphate, 5-iodouridine-5’-triphosphate, 5-Iodo- 2’- deoxyuridine-5’-triphosphate, 5-methylcytidine-5’-triphosphate, 5-methyluridine-5’- triphosphate, 5-Propynyl-2’-deoxycytidine-5’-triphosphate, 5-Propynyl-2’-deoxyuridine-5’- triphosphate, 6-azacytidine-5’-triphosphate, 6-azauridine-5 ‘-triphosphate, 6- chloropurineriboside-5’-triphosphate, 7-deazaadenosine-5’-triphosphate, 7-deazaguanosine- 5 ‘- triphosphate, 8-azaadenosine-5’-triphosphate, 8-azidoadenosine-5’-triphosphate, benzimidazole- riboside-5’-triphosphate, Nl-methyladenosine-5’-triphosphate, Nl- methylguanosine-5’- triphosphate, N6-methyladenosine-5’-triphosphate, 06- methylguanosine-5’-triphosphate, pseudouridine-5’-triphosphate, or puromycin-5’- triphosphate, xanthosine-5’-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5- methylcytidine-5 ‘-triphosphate, 7-deazaguanosine- 5’-triphosphate, 5-bromocytidine-5’- triphosphate, and pseudouridine-5’-triphosphate.

[0151] In some embodiments, the modified nucleosides comprise 4-pyridine-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl- pseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio- uridine, 5- methyl-uridine, 1 -methyl-pseudouridine, 4-thio- 1 -methyl-pseudouridine, 2-thio- 1-methyl- pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza- pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2- methoxyuridine, 5-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4- methoxy-2-thio-pseudouridine.

[0152] In some embodiments, the modified nucleosides comprise 5-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine,5- hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4- thio- 1 -methyl -pseudoisocytidine, 4-thio-l -methyl- 1-deaza-pseudoisocytidine, 1 -methyl- 1- deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4- methoxypseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine .

[0153] In some embodiments, the modified nucleosides comprise 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza- 2- aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine.

[0154] In other embodiments, modified nucleosides comprise inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-m ethylinosine, 6-m ethoxy -guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, I-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine. In some embodiments, the nucleotide can be modified on the major groove face.

[0155] Provided is a polynucleotide encoding a Cas9 endonuclease comprising a nucleotide sequence having at least 80%, at least 85%, or at least 90% identity to Cas9-CO-NA DNA (SEQ ID NO: 25) or its corresponding RNA (SEQ ID NO: 26).

[0156] Provided herein is a polynucleotide encoding a Cas9 endonuclease comprising a nucleotide sequence having at least 95% identity to Cas9-CO-NADNA (SEQ ID NO: 25) or its corresponding RNA (SEQ ID NO:26).

[0157] Further provided is a polynucleotide encoding a Cas9 endonuclease comprising a nucleotide sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9-CO-NA DNA (SEQ ID NO: 25) or its corresponding RNA (SEQ ID NO:26).

[0158] Further provided herein is a polynucleotide encoding a Cas9 endonuclease comprising a nucleotide sequence having 100% identity to Cas9-CO-NA DNA (SEQ ID NO: 25) or its corresponding RNA (SEQ ID NO:26). A. RNA synthesis promoter

[0159] In some embodiments, any one of the polynucleotides as provided herein further comprises the coding sequence for an initiation site for T7 RNA polymerase-mediated transcription. T7 RNA polymerase is an RNA polymerase derived from T7 bacteriophage that is able to synthesize RNA from DNA in the 5’ to 3’ direction and therefore is often used for in vitro transcription of RNA from a DNA template. B. Translation initiation and 5’ UTR sequences

[0160] In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence encoding a recognition site for a translation initiation complex. Translation is initiated in mammalian cells by the formation of a pre-initiation complex comprised of a 40S ribosomal subunit, an initiator tRNA, and initiation factors. The mRNA is subsequently attached at the capped 5’ end and poly(A)-binding protein (PABP). The pre- initiation complex then scans the mRNA 5’ untranslated region (UTR) for an AUG start codon. Even though translation is frequently initiated at the first AUG during the scanning, in some instances the first AUG is flanked by unfavorable sequences and is skipped during the scanning. A favorable context for the start codon is within a Kozak sequence, for which the consensus is 5’ (A / G)CCAUGG 3’ (SEQ ID NO:7, see FIG.1B).

[0161] The length and sequence of the 5’ UTR can vary due to the presence of different sequences such as promoter sequences, regulatory elements such as CpG sites and untranslatedORFS, alternative splicing mechanisms, and secondary structures such as hairpin loops. These in turn affect the translational efficiency at the start codon. Both naturally occurring and synthetic 5’ UTRs are available for altering gene expression. An example of a synthetic 5’ UTR is NeoUTR3 described in Cao, J., Novia, E., Zhang, Z., Chen, W., Liu, D., Choi, G., Wong, A., Wehrspaun, C., Kellis, M., and Lu, T. (2021) Nature Communications 12(1):4138.

[0162] In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence of a synthetic 5’ UTR (NeoUTR3) (SEQ ID NO: 9; see FIG. 1B).

[0163] In some embodiments, a polynucleotide as provided herein further comprises a nucleotide sequence of a synthetic eIF4G-recruiting aptamer (SEQ ID NO: 10; see FIG.1B).

[0164] In some embodiments, a polynucleotide as provided herein further comprises a nucleotide sequence of an endogenous 5’ UTR of the human hemoglobin α-subunit 1 / 2 (HBA1 / 2) (SEQ ID NO: 11; see FIG.1B). In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence of a minimalistic 5’ UTR (selected from SEQ ID NOs: 12-18; see FIG.1C). In some embodiments, any one of the polynucleotides provided herein further comprises nucleotide sequence of an endogenous 5’ UTR of hemoglobin β-subunit (HBB) (SEQ ID NO:19; see FIG.1C). In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence encoding a Kozak motif. C. Polyadenylation and the 3’UTR

[0165] In mammalian cells, polyadenylation at the 3’ end of mRNAs requires cleavage of the mRNA before addition of adenine nucleotides by a poly(A) polymerase enzyme to form a polyadenylation tail. Cleavage occurs between a polyadenylation signal (typically AAUAA in eukaryotes) and a variable GU-rich region. Nonetheless, polyadenylation tails can be engineered to incorporate specific sequences using specific DNA templates, such as segmented poly(A) tails which contain stretches of polyadenylation sequences separated by spacers, sometimes with restriction enzyme cleave sites. Segmented poly(A) tails can be useful for controlling the lengthand content of polyadenylation tails on synthetic mRNAs as well as reducing recombination of plasmids in E. coli hosts without negatively impacting mRNA translation or half-life (Trepotec et al. (2019) 25(4):507-518).

[0166] In some embodiments, any one of the polynucleotides provided herein further comprises a nucleotide sequence encoding a polyadenylation signal. In some embodiments, a polynucleotide as provided herein further comprises the coding region for a polyadenylation tail. In some embodiments, the coding region for a polyadenylation tail comprises nucleotide sequence in SEQ ID NO: 8 (see FIG.1B). In some embodiments, other polyadenylation tails are used. In some aspects, the polyadenylation tail comprises a linker sequence, wherein the linker sequence comprises a nucleotide sequence encoding at least one type IIS restriction enzyme cleavage site. In some embodiments, the type IIS restriction enzyme cleavage site is a Sap1 restriction enzyme cleavage site.

[0167] The 3’ UTR is located within the mRNA immediately preceding the translation termination codon and can affect mRNA stability and interact with regulatory elements such as miRNAs. In some embodiments, a polynucleotide as provided herein further comprises a single copy of the endogenous 3’ UTR of the human hemoglobin α-subunit 1 (HBA1) (SEQ ID NO:20; see FIG.1C). D.5’ terminal mRNA cap

[0168] In some embodiments, a polynucleotide as provided herein further comprises comprises a ribonucleic acid (RNA). In some embodiments, the RNA is an mRNA comprising a 5’ terminal cap. Adding a 5’ cap comprising an inverted 7-methylguanosine connected to the rest of the eukaryotic RNA via a 5’-5’ triphosphate bridge is required for interaction with a cap binding complex in the nucleus and enables nuclear export. The 5’ cap is also required for interaction between the RNA and the eukaryotic translation initiation factor (eIF4E) in the cytoplasm which is required for cap-dependent translation of the RNA. A variety of 5’ terminal cap structures are known. In some embodiments, the 5’ terminal cap comprises m7G(5’)ppp(5’)(2’OmeA)pG, m7(3'OMeG)(5’)ppp(5’)(2’OmeA)pG or m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG.

[0169] In some contexts, use of circular RNA enhances gene expression, particularly when optimized for vector topology, 5’ UTR and 3’ UTR sequences, and internal ribosome entry sites, and with the incorporation of synthetic aptamers such as the eukaryotic initiation factor 4G- recruiting aptamer (which increases mRNA translation) and N6-methyladenosine incorporation (which helps with resistance to nucleases). See, Chen, R., Wang, S., Belk, J., Amaya, L., Li, Z., Cardenas, A., Abe, B., Chen, C., Wender, P., and Chang, H. (2022) Nature Biotechnology 41:262-272. In some embodiments, a polynucleotide as provided herein further comprises a ribonucleic acid (RNA). In some embodiments, the RNA is an mRNA comprising a 5’ terminal cap. In some embodiments, the 5’ terminal cap comprises m7G(5’)ppp(5’)(2’OmeA)pG, m7(3'OMeG)(5’)ppp(5’)(2’OmeA)pG or m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG. E. Circular RNA

[0170] In some embodiments, a polynucleotide as provided herein is a circular RNA. In some embodiments, the polynucleotide as provided herein is a circular RNA comprising a synthetic internal ribosome entry site. Nonlimiting examples of such circular RNAs are described in U.S. Patent No.11,560,567. In some embodiments, a polynucleotide as provided herein further comprises a synthetic internal ribosome entry site containing a eukaryotic initiation factor 4G- recruiting aptamer. In some embodiments, a polynucleotide as provided herein further comprises 5% N6-methyladenosine. In some embodiments, a polynucleotide as provided herein further comprises comprises the endogenous 3’UTR of the human hemoglobin α-subunit 1 (HBA1) (SEQ ID NO:20). F. DNA template

[0171] In some embodiments, a polynucleotide as provided herein comprises a deoxyribonucleic acid (DNA).

[0172] Also provided herein is a plasmid construct comprising a polynucleotide as provided herein comprising a deoxyribonucleic acid (DNA).VI. Compositions

[0173] Also provided herein is a composition comprising: (i) any one of the fusion proteins as provided herein, any one of the polynucleotides as provided herein, or any one of the plasmids as provided herein; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene.A, Non-therapeutic Uses and Therapeutic Uses

[0174] In some embodiments, the composition is formulated for a non-therapeutic use. In some embodiments, the composition is formulated for the diagnosis of a disease or disorder in a subject. Diagnostic uses of CR1SPR Cas endonuclease technology are based on the ability of this system to precisely target polynucleotide sequences for binding and cleavage, including abnormal or disease-related genomic or extrachromosomal DNA alterations. Broadly, these diagnostic methods can be categorized as based on the detection of sequence-specific target binding, detection of sequence-specific target cleavage, and detection of target-specific transcleavage. See, Kim, S., Ji, S., and Koh, H. (2021) Biomolecules 11 : 1162.B, Pharmaceutical Compositions and Methods of Administration

[0175] In some embodiments, the composition is a pharmaceutical composition for the prevention or treatment of a disease or disorder in a subject. In some embodiments, the pharmaceutical composition is formulated for local, targeted, or regional administration in the subject. In some embodiments, the pharmaceutical composition is formulated for systemic administration in the subject.

[0176] Depending on the method / route of administration, pharmaceutical dosage forms come in several types. These include many kinds of liquid, solid, and semisolid dosage forms. Common pharmaceutical dosage forms include pill, tablet, or capsule, drink or syrup, and natural or herbal form such as plant or food of sorts, among many others. Notably, the route of administration (ROA) for drug delivery is dependent on the dosage form of the substance inquestion. A liquid pharmaceutical dosage form is the liquid form of a dose of a chemical compound used as a drug or medication intended for administration or consumption.

[0177] In one embodiment, a composition of the present disclosure can be delivered to a subject subcutaneously (e.g., intra-articular injection), dermally (e.g., transdermally via patch), and / or via implant. Exemplary pharmaceutical dosage forms include, e.g., pills, osmotic delivery systems, elixirs, emulsions, hydrogels, suspensions, syrups, capsules, tablets, orally dissolving tablets (ODTs), gel capsules, thin fdms, adhesive topical patches, lollipops, lozenges, chewing gum, dry powder inhalers (DPIs), vaporizers, nebulizers, metered dose inhalers (MDIs), ointments, transdermal patches, intradermal implants, subcutaneous implants, and transdermal implants.

[0178] As used herein, “dermal delivery” or “dermal administration” can refer to a route of administration wherein the pharmaceutical dosage form is taken to, or through, the dermis (i.e., layer of skin between the epidermis (with which it makes up the cutis) and subcutaneous tissues). “Subcutaneous delivery” can refer to a route of administration wherein the pharmaceutical dosage form is to or beneath the subcutaneous tissue layer.

[0179] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21sted., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0180] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0181] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0182] Therapeutic compounds that are or include nucleic acids can be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No.6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Pat. No.6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Pat. No.6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).

[0183] Therapeutic compounds can be prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3- bis(carboxyphenoxy)propane-co-sebacic-acid] (PCPP-SA) matrix, fatty acid dimer-sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyorthoesters, polyethyleneglycol-coated liposomes, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811. Semisolid, gelling, soft-gel, or other formulations (including controlled release) can be used, e.g., when administration to a surgical site is desired. Methods of making such formulations are known in the art and can include the use of biodegradable, biocompatible polymers. See, e.g., Sawyer et al., Yale J Biol Med.2006 December; 79(3-4): 141-152.

[0184] The pharmaceutical compositions can be included in a container, kit, pack, or dispenser together with instructions for administration.1. Non-inflammatory disorders

[0185] In some embodiments, the disease or disorder is a genetic disorder. In some instances, the genetic disorder can be caused by a mutation in one gene. In some instances, the genetic disorder is caused by mutations in multiple genes (i.e., a multifactorial inheritance disorder). In some instances, the genetic disorder is caused by a combination of gene mutations and environmental factors. In some instances, the genetic disorder is caused by damage to chromosomes such as a change in the number of chromosomes or a change in the structure of a chromosome).

[0186] In some embodiments, the disease or disorder is a cancer. Over 120 types of cancer are currently recognized and have the shared hallmark of uncontrolled cell division of abnormal cell types. In some embodiments, the disease or disorder is heart disease. In some instances the heart disease is a blood vessel disease such as coronary artery disease. In some instances, the heart disease is arrhythmia, or irregular heartbeats. In some embodiments, the disease or disorder is a cardiovascular disease. In some instances, the cardiovascular disease is coronary heart disease, cerebrovascular disease, or rheumatic heart disease.2, Inflammatory disorders

[0187] In some embodiments, the disease or disorder is an inflammatory disorder. In some embodiments, the disease or disorder is a joint disorder.

[0188] In some embodiments, the joint disorder is arthritis. In some embodiments, the present disclosure includes a method for the treatment or prevention of an arthritis. Non-limiting examples of arthritis the can be treated using the compositions and methods described herein include post-traumatic arthritis, osteoarthritis (a degenerative condition that affects the joints, most commonly the hips, knees, and hands), rheumatoid arthritis (an autoimmune disorder that causes inflammation in the joints and surrounding tissue), psoriatic arthritis (a type of arthritis that occurs in people with psoriasis, a skin condition characterized by scaly red patches), gout (a type of arthritis caused by the buildup of uric acid crystals in the joints), lupus (a chronic autoimmune disorder that can cause inflammation and damage to the joints, as well as otherorgans), ankylosing spondylitis (a type of arthritis that primarily affects the spine, causing inflammation and stiffness), reactive arthritis (a type of arthritis that occurs as a reaction to an infection in the body), septic arthritis (a type of arthritis caused by an infection in the joint), juvenile idiopathic arthritis (a form of arthritis that affects children under the age of 16), and fibromyalgia (a chronic pain disorder that can cause widespread pain and stiffness, including in the joints). In some embodiments, the disease or disorder is degenerative disc disease.

[0189] In some embodiments, the joint disorder is osteoarthritis. In some embodiments, the joint disorder is rheumatoid arthritis. In some embodiments, the joint disorder is post-traumatic arthritis.

[0190] In some embodiments, the joint disorder is gout. Gout is a chronic inflammatory condition that affects joints. The underlying cause is monosodium urate (MSU) crystal deposition and the resultant host response, particularly in joint structures (as well as subcutaneous tissues and other sites). See, Dalbeth, N., & Stamp, L. (2014). Annals of the Rheumatic Diseases, 73(9):1598-1600. The clinical manifestations include recurrent acute flares of severe inflammatory arthritis and tendinobursitis. IL-1 and other pro-inflammatory mediators are a major contributor to this host response. See, Dinarello, C. A. (2014). Molecular Medicine, 20(1):S43-S58. To this end, effective blockade of these signaling pathways may provide relief to gout patients.

[0191] In some embodiments, the joint disorder is pseudogout. Pseudogout is caused by the accumulation of calcium pyrophosphate dihydrate (CPPD) crystals and can cause symptoms analogous to inflammatory arthritis. Currently, there is no effective agent to reduce the crystals that accumulate, so therapies are targeted towards reducing inflammation. See, MacMullan, P. and McCarthy, G. (2012) Ther Adv Musculoskelet Dev 4(2):121-131.

[0192] In some embodiments, the joint disorder is tendinopathy in any mammalian species. Tendinopathy occurs when tiny tears and degeneration of collagen protein in the tendon typically due to overuse injuries.

[0193] All mammals are susceptible to joint disorders. In some embodiments, the joint disorder occurs in dogs, cats, or horses. VII. Target Polynucleotides

[0194] In some embodiments, the at least one guide RNA targets the coding region for: (i) a transmembrane receptor, (ii) a cytokine, or (iii) a gene associated with the production, blocking, or removal of reactive oxygen species (ROS). In some embodiments, the transmembrane receptor is a cytokine receptor. In some embodiments, the cytokine receptor is an interleukin-1 receptor, an interleukin-6 receptor, or a co-receptor necessary for signal transduction. In some embodiments, the at least one guide RNA targets a gene selected from the group consisting of IL1R1, IL1RAP, IL1R5, IL1R6, IL1R7, IL1R9, TGFβ, TNFα, TGFβR1, and TGFβRII. In some embodiments, the at least one guide RNA targets an IL6R or IL6ST gene. In some embodiments, the cytokine is IL-1 alpha, IL-1 beta, or IL-6. In some embodiments, the at least one guide RNA targets the IL1A or IL1B gene. In some embodiments, the at least one guide RNA targets the IL6 gene. In some embodiments, the gene associated with the production, blocking, or removal of reactive oxygen species (ROS) is an NF-kappa B1 or NF-kappa B2 gene. In some embodiments, the at least one guide RNA is a single guide RNA (sgRNA). In some embodiments, the at least one guide RNA targets a human gene. In some embodiments, the at least one guide RNA targets a canine gene. In some embodiments, the at least one guide RNA targets an equine gene. In some embodiments, the at least one guide RNA targets a feline gene. In some embodiments, the at least one guide RNA targets a mammalian gene. VIII. Vectors And Delivery

[0195] A variety of vectors have been used to deliver biological drugs to intracellular targets. A. Viral vectors

[0196] In one aspect, the present disclosure encompasses methods of delivery of a CRISPR gene-editing system targeting a gene, and combinations thereof using one or more recombinantviral particle. In some embodiments, the one of more viral vectors comprise a recombinant virus selected from a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, and a herpes simplex virus-1. In some embodiments, the one of more viral vectors comprise a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant AAV is of serotype 5 (AAV5). In some embodiments, the recombinant AAV is of serotype 6 (AAV6). In some embodiments, the one or more viral vectors comprise: a first viral vector comprising a first nucleic acid, in the one or more nucleic acids, encoding the Cas protein; and a second viral vector comprising a second nucleic acid, in the one or more nucleic acids, encoding the at least one guide RNA. In some embodiments, the one or more viral vectors comprise a viral vector comprising a single nucleic acid, wherein the single nucleic acid encodes the Cas9 protein and the at least one guide RNA.1. Adeno-associated virus (AAV)

[0197] A viral vector system useful for delivery of nucleic acids is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. For a review see Muzyczka et al., Curr. Topics in Micro. And Immunol. 158:97-129 (1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62: 1963-1973 (1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81 :6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51 :611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). The identification of Staphylococcus aureus (SaCas9) and other smaller Cas9 enzymes that can be packaged into adeno-associated viral (AAV) vectorsthat are highly stable and effective in vivo, easily produced, approved by FDA, and tested in multiple clinical trials, paves new avenues for therapeutic gene editing.

[0198] According to particular embodiments, a CRISPR gene-editing system targeting a gene, and combinations thereof further comprise a recombinant AAV vector. In some embodiments, the CRISPR gene-editing system is encoded by a nucleic acid, wherein the nucleic acid is a recombinant AAV genome. In some embodiments, the AAV vector is selected from an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, and an AAV10 vector.

[0199] In some aspects, the AAV vector comprises a serotype selected from the group consisting of: AAV1, AAV1(Y705+731F+T492V), AAV2(Y444+500+730F+T491V), AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6 (VP3 variant Y705F / Y731F / T492V), AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9 (VP3 variant Y731F), AAV10(Y733F), AAV-ShH10, and AAV-DJ / 8. In some aspects, the AAV vector comprises a serotype selected from the group consisting of: AAV1, AAV5, AAV6, AAV6 (Y705F / Y731F / T492V), AAV8, AAV9, and AAV9 (Y731F).

[0200] In some embodiments, use of the CRISPR gene-editing system further comprising one or more LNPs to target a gene, and combinations thereof is therapeutic. 2. Lentivirus

[0201] In some aspects, the viral vector is a lentivirus. In an aspect, the lentivirus is selected from the group consisting of: human immunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).

[0202] Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat'l Acad. Sci.2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol.1997, 15,871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Patent No.6,627,442, the disclosures of each of which are incorporated by reference herein. 4. Lipid Nanoparticles (LNP)

[0203] In some embodiments, a CRISPR gene-editing system is delivered by a nanoparticle. Without wishing to be bound by any particular theory, in certain embodiments, nucleic acids, when present in the nanoparticle, are resistant in aqueous solution to degradation with a nuclease. In other embodiments, proteins are protected from protease degradation. In some embodiments, proteins and nucleic acids encapsulated by nanoparticles are capable of penetrating the cellular plasma membrane. B. Lipid Nanoparticles

[0204] Lipid nanoparticles comprising nucleic acids and their method of preparation is disclosed in at least WO2017 / 019935, WO2017 / 049074, WO2017 / 201346, WO2017 / 218704,WO2018 / 006052,WO2018 / 013525,WO2018 / 089540,WO2018 / 119115,WO20 18 / 126084,WO2018 / 157009,WO2018 / 170336,WO2018 / 222890,WO2019 / 046809,WO2019 / 089 828,WO2020 / 061284,WO2020 / 061317,WO2020 / 081938,WO2020 / 097511,WO2020 / 097520,W O2020 / 097540,WO2020 / 097548,WO2020 / 214946,WO2020 / 219941,WO2020 / 232276,WO2020 / 227615,WO2020 / 061295,WO2021 / 007278,WO2021 / 016430,WO2021 / 021988, EP Patent No. EP 2972360, US20200155691, US20200237671, U.S. Patent Nos.8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,404,127, 9,504,651, 9,593,077, 9,738,593, 9,868,691, 9,868,692, 9,950,068, 10,138,213, 10,166,298, 10,221,127, 10,238,754, 10,266,485, 10,383,952, 10,730,924, 10,766,852, 11,079,379, 11,141,378 and 11,246,933, which are incorporated herein by reference in their entirety for all purposes.

[0205] In some embodiments, the largest dimension of a nanoparticle composition is 1 micrometer or shorter (e.g., 1 micrometer, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter), e.g., when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Nanoparticle compositions include, for example, lipidnanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and / or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels. In various embodiments , lipid nanoparticles described herein have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic.

[0206] In certain embodiments, the lipid nanoparticles described herein comprise one or more components, including a lipid component„ and (optionally) a structural component. The lipid component comprises lipids selected from ionizable and / or cationic lipids (i.e., lipids that may have a positive or partial positive charge at physiological pH), neutral lipids (e.g., phospholipids, or sphingolipids), and polymer-conjugated lipids (e.g., PEGylated lipids). In some embodiments, the lipid component comprises a single ionizable lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 ionizable lipids. In some embodiments, the lipid component comprises a single neutral lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 neutral lipids. In some embodiments, the lipid com- ponent comprises a single polymer-conjugated lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 polymer-conjugated lipids. In some embodiments, the structural component comprises a single structural lipid. In other embodiments, the structural component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 structural lipids. In some embodiments, the lipid component comprises at least one cationic lipid, at least one neutral lipid, and at leastone polymer-conjugated lipid. The present disclosure contemplates that the lipid component may comprise any combination of the foregoing constituents.

[0207] In some embodiments, the lipid component comprises an ionizable lipid. In some embodiments, the ionizable lipid is anionic. In other embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the lipid component comprises cationic lipids including, but not limited to, a cationic lipid selected from the group consisting of 3-(didodecylamino)- N1,N1,4-tridodecy1-1-piperazineethanamine (KL 10), N1-[2-(didodecylarnino)ethyl]- N1,N4,N4-tridodecy1-1,4-piperazinediethanamine (KL22), 14,25-ditridecy1-15,18,21,24- tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (Dlin-DMA), 2,2-dilinoley1-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA), heptatriaconta- 6,9,28,31-tetraen-19-y14-(dimethylamino)butanoate (Dlin-MC3-DMA), 2,2-dilinoley1-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (Dlin-KC2-DMA), 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octylloxy)-N,N- dimethy1-3-[(9Z,12Z)- -octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), (2R)-2-( { 8- [(3. Beta. )-cholest-5-en-3 -yloxy] octyl } oxy)-N,N-dimethy1-3-[(9Z- ,12Z)-octadeca-9,12- dien-1-yloxy] propan-1-amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[(3.beta.)-cholest-5-en-3 - yloxy] octyl } oxy)-N,N-dimethy1-3-[(9Z- ,12Z)-octadeca-9,12-dien-1-yloxy] propan-1-amine (Octyl-CLinDMA (2S)), a lipid including a cyclic amine group, and mixtures thereof.

[0208] In some embodiments, the lipid component further comprises neutral lipids including, but not limited to, a phospholipid selected from the group consisting of 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoy1-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn-glycero-3- phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin (SM), and mixtures thereof.

[0209] In some embodiments, the lipid component further comprises polymer-conjugated lipids, including, but not limited to, a PEGylated lipid selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEGr000-c-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA or a PEG-DSPE lipid.

[0210] In some embodiments, the LNP further comprises a structural component. See generally Patel, S., et al. (2020). Nature Communications, 11(1), 1-13. In some embodiments, the structural component comprises a sterol including, but not limited to, a sterol selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmastanol, sitosterol, r3-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9, 11-dehydroergosterol, tomatidine, tomatine, a-tocopherol, and mixtures thereof. In other embodiments, the structural lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0211] Nanoparticle compositions may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic. A nanoparticle composition may be designed for one or more specific applications or targets. The elements of a nanoparticle composition may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a nanoparticle composition may be selected for aparticular application or target according to, for example, the efficacy and toxicity of particular combinations of elements.

[0212] The lipid component of a nanoparticle composition may include, for example, a cationic lipid, a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC), a PEG lipid, and a structural lipid. The elements of the lipid component may be provided in specific fractions.

[0213] In some embodiments, the lipid component of a nanoparticle composition includes an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid com-ponent of the nanoparticle composition includes about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 0 mol % to about 10 mol % of PEG lipid, and about 17.5 mol % to about 50 mol % structural lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % compound of ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 0 mol % to about 10 mol % of PEG lipid, and about 30 mol % to about 40 mol % structural lipid. In a particular embodiment, the lipid component includes about 50 mol % said compound, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another embodiment, the lipid component includes about 40 mol % said compound, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and / or the structural lipid may be cholesterol.

[0214] In some embodiments, the ionizable lipids comprise between about 20 and about 60 mol % of the lipid component. In other embodiments, the ionizable lipids comprise between about 35 and about 55 mol % of the lipid component. In various embodiments, the ionizable lipids comprise about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60 mol % of the lipid component.

[0215] In some embodiments, the neutral lipids comprise between about 0 and about 30 mol % of the lipid component. In other embodiments, the neutral lipids comprise between about 5 andabout 25 mol % of the lipid component. In various embodiments, the neutral lipids comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mol % of the lipid component.

[0216] In some embodiments, the polymer-conjugated lipids comprise between about 0 and about 15 mol % of the lipid component. In other embodiments, the polymer-conjugated lipids comprise between about 0.5 and about 10 mol % of the lipid component. In various embodiments, the polymer-conjugated lipids comprise about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.59, 9.5, 10, or 15 mol % of the lipid component.

[0217] In some embodiments, the structural component comprises about 17.5 mol % to about 50 mol % of the lipid component. In other embodiments, the structural component comprises about 30 to about 40 mol % of the lipid component. In various embodiments, the structural component comprises about 17.5, 20, 22.5, 25, 27.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mol % of the lipid component.

[0218] The structural component may alternatively be expressed as a ratio relative to the lipid component. In some embodiments, the structural component is in a ratio of about 1:1 with the lipid component (sterol:lipids). In other embodiments, the structural component is in a ratio of about 1:5 with the lipid component (sterol:lipids). In various embodiments, the structural component is in a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, or 1:25 with the lipid component (sterol:lipids).

[0219] Nanoparticle compositions may be designed for one or more specific applications or targets. For example, a nanoparticle composition may be designed to deliver a therapeutic and / or prophylactic such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal’s body. Physiochemical properties of nanoparticle compositions may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. The therapeutic and / or prophylactic included in a nanoparticle composition may also be selected based on the desired delivery target or targets. For example, a therapeutic and / or prophylactic may be selected for a particularindication, condition, disease, or disorder and / or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery). In certain embodiments, a nanoparticle composition may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition may be designed to be specifically delivered to a particular organ. In some embodiments, a composition may be de-signed to be specifically delivered to a mammalian joint.

[0220] The amount of a therapeutic and / or prophylactic in a nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic and / or prophylactic. For example, the amount of an RNA useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic and / or prophylactic and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic in a nanoparticle composition may be from about 5: 1 to about 60: 1, such as 5:1, 6: 1, 7:1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15:1, 16:1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35:1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic may be from about 10: 1 to about 40:1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of a therapeutic and / or prophylactic in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0221] In some embodiments, the therapeutic and / or prophylactic comprises a nucleic acid component. In some embodiments, the nucleic acid component comprises RNA including, but not limited to, RNA selected from the group consisting of messenger RNA (mRNA), CRISPR RNA (crRNA), tracrRNA, single-guide RNA (sgRNA), short interfering RNA (siRNA), antisense oligonucleotides (ASO), and mixtures thereof. In other embodiments, the nucleic acid component comprises DNA including, but not limited to, DNA selected from the group consisting of linear DNA, plasmid DNA, antisense oligonucleotide, and mixtures thereof.

[0222] In some embodiments, a nanoparticle composition includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:Pratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio may be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio may be about 5.67:1.

[0223] In some embodiments, the nucleic acid component is comprised of a modified nucleic acid. For example, an RNA may be a modified RNA. That is, an RNA may include one or more nucleobases, nucleosides, nucleotides, or linkers that are non-naturally occurring. A “modified” species may also be referred to herein as an “altered” species. Species may be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase species may include one or more substitutions that are not naturally occurring.

[0224] The characteristics of a nanoparticle composition may depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid may have different characteristics than a nanoparticle composition that includes a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0225] Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) may also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0226] The mean size of a nanoparticle composition may be between 10 nm and 1 micrometer, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.

[0227] A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition may be from about 0.10 to about 0.20.

[0228] The zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species mayinteract undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0229] The efficiency of encapsulation of a therapeutic and / or prophylactic describes the amount of therapeutic and / or prophylactic that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic (e.g., RNA) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0230] A nanoparticle composition may optionally comprise one or more coatings. For example, a nanoparticle composition may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0231] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosurebelongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0232] In some embodiments, the CRISPR gene-editing system comprises one or more RNA- containing compositions. In some embodiments, the CRISPR gene-editing system further comprises one or more nanoparticles. In some embodiments, said one or more RNA-containing compositions comprises a guide RNA. In some embodiments, said one or more RNA-containing compositions comprises an mRNA. In some embodiments, said one or more RNA-containing compositions comprises an RNP (e.g., Cas9 and a guide RNA). In some embodiments, said one or more nanoparticles are lipid nanoparticles (LNP).

[0233] In some embodiments, the CRISPR gene-editing system comprises one or more LNPs collectively encapsulating (i) the RNA-guided nuclease or the nucleic acid encoding the RNA- guided nuclease and (ii) the at least one guide RNA or the nucleic acid encoding the at least one guide RNA. In some embodiments, the one or more LNPs comprises a first plurality of LNP encapsulating the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and a second plurality of LNP encapsulating the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0234] In some embodiments, the one or more LNP comprises a component selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecy1-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecy1-1,4-piperazinediethanamine (KL22), 14,25-ditridecy1-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (Dlin-DMA), 2,2-dilinoley1-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-y14-(dimethylamino)butanoate (Dlin-MC3- DMA), 2,2-dilinoley1-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (Dlin-KC2-DMA), 1,2- dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-( I 8- [(3.beta.)-chol est-5 -en-3-yloxy] octyl oxy)-N,N-dimethy1-3-[(9Z,12Z)- -octadeca-9,12-dien-1-yloxy] propan- 1 -amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy] octyl oxy)-N,N-dimethy1-3-[(9Z- ,12Z)-octadeca-9,12-dien-1-yloxy]propan- 1 -amine (Octyl-CLinDMA (2R)), (2S)-2-({8- [(3.beta.)-cholest-5-en-3-yloxy] octyll oxy)-N,N-dimethy1-3-[(9Z- ,12Z)-octadeca-9,12-dien-l- yloxy]propan-l-amine (Octyl-CLinDMA (2S)), a lipid including a cyclic amine group, and a mixture thereof.

[0235] In some embodiments, the one or more LNP comprises a component selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn- glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoy1-2-oleoyl-sn- glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoy1-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin (SM), and a mixture thereof.

[0236] In some embodiments, the one or more LNP comprises a component selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA, a PEG-DSPE lipid, and a mixture thereof.

[0237] In some embodiments, the one or more LNP comprises a component selected from the group consisting of a cholesterol, fecosterol, stigmasterol, stigmastanol, sitosterol, r3-sitosterol,lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9, 11-dehydroergosterol, tomatidine, tomatine, a-tocopherol, and a mixture thereof. Example LP01 LNP Formulations

[0238] In some embodiments, a composition is provided comprising lipid nanoparticles (LNP) comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene, where the LNP comprises LP01.

[0239] In some embodiments, the LNP is composed of the following structural component: an LP01 ionizable lipid, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component. In some such embodiments of the LP01-based LNPs, the neutral / helper lipid component comprises DSPC. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0240] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): LP01 (40- 50), neutral / helper lipid component (6-12), steroid system component (39-49), and a pegylated lipid component (1-3). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNPs, the steroid systemcomponent comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0241] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): LP01 (41- 49), neutral / helper lipid component (6.5-11.5), steroid system component (40-48), and a pegylated lipid component (1.25-2.75). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the LP01 -based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the LPOl-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the LPOl-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0242] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): LP01 (42- 48), neutral / helper lipid component (7-11), steroid system component (41-47), and a pegylated lipid component (1.5-2.5). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the LPOl-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the LPOl-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the LPOl-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0243] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): LP01 (43- 47), neutral / helper lipid component (7.5-10.5), steroid system component (42-46), and a pegylated lipid component (1.5-2.5). In some such embodiments, the neutral / helper lipidcomponent comprises DSPC. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0244] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): LP01 (44- 46), neutral / helper lipid component (8-10), steroid system component (43-45), and a pegylated lipid component (1.5-2.5). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0245] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): LP01 (45), neutral / helper lipid component (9), steroid system component (44), and a pegylated lipid component (2). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the LP01-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the LP01-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2-Dimyristoyl-sn- glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG- PEG2000. In some such embodiments of the LP01-based LNPs, the steroid system componentcomprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.Example SM-102 LNP Formulations

[0246] In some embodiments, a composition is provided comprising lipid nanoparticles (LNP) comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene, where the LNP comprises SM-102.

[0247] In some embodiments, the LNP is composed of the following structural component: an SM-102 ionizable lipid, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component. In some such embodiments of the SM- 102-based LNPs, the neutral / helper lipid component comprises DSPC. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; l,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the SM- 102-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0248] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): SM-102 (45-55), neutral / helper lipid component (7-13), steroid system component (33.5-43.5), and a pegylated lipid component (0.5-2.5). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the SM-102- based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG- PEG; l,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNPs, thesteroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0249] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): SM-102 (46-54), neutral / helper lipid component (7.5-12.5), steroid system component (34.5-42.5), and a pegylated lipid component (0.75-2.25). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the SM-102- based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG- PEG; l,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0250] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): SM-102 (47-53), neutral / helper lipid component (8-12), steroid system component (35.5-41.5), and a pegylated lipid component (1-2). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the SM-102 -based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0251] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): SM-102 (48-52), neutral / helper lipid component (8.5-11.5), steroid system component (36.5-40.5), and a pegylated lipid component (1-2). In some such embodiments, the neutral / helper lipid componentcomprises DSPC. In some such embodiments of the SM-102 -based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0252] In some embodiments, the stmctural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): SM-102 (49-51), neutral / helper lipid component (9-11), steroid system component (37.5-39.5), and a pegylated lipid component (1-2). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the SM-102-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0253] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): SM-102 (50), neutral / helper lipid component (10), steroid system component (38.5), and a pegylated lipid component (1.5). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the SM- 102-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the SM-102-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2-Dimyristoyl-sn- glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG- PEG2000. In some such embodiments of the SM- 102-based LNPs, the steroid systemcomponent comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone. Example ALC0315 LNP Formulations

[0254] In some embodiments, a composition is provided comprising lipid nanoparticles (LNP) comprising (i) an RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene, where the LNP comprises ALC0315.

[0255] In some embodiments, the LNP is composed of the following structural component: an ALC0315 ionizable lipid, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component. In some such embodiments of the ALC0315- based LNPs, the neutral / helper lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2-Dimyristoyl-sn-glycero-3- methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0256] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): ALC0315 (45-55), neutral / helper lipid component (7-13), steroid system component (33.5-43.5), and a pegylated lipid component (0.5-2.5). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315- based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG- PEG; 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNPs, thesteroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0257] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): ALC0315 (46-54), neutral / helper lipid component (7.5-12.5), steroid system component (34.5-42.5), and a pegylated lipid component (0.75-2.25). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315- based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG- PEG; 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0258] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): ALC0315 (47-53), neutral / helper lipid component (8-12), steroid system component (35.5-41.5), and a pegylated lipid component (1-2). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0259] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): ALC0315 (48-52), neutral / helper lipid component (8.5-11.5), steroid system component (36.5-40.5), and a pegylated lipid component (1-2). In some such embodiments, the neutral / helper lipid componentcomprises DSPC. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0260] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): ALC0315 (49-51), neutral / helper lipid component (9-11), steroid system component (37.5-39.5), and a pegylated lipid component (1-2). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG-PEG2000. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.

[0261] In some embodiments, the structural components of the LNP are present in the following relative molar amounts (provided in parenthesis following the component): ALC0315 (50), neutral / helper lipid component (10), steroid system component (38.5), and a pegylated lipid component (1.5). In some such embodiments, the neutral / helper lipid component comprises DSPC. In some such embodiments of the ALC0315-based LNPs, the steroid system component comprises cholesterol. In some such embodiments of the ALC0315-based LNPs, the pegylated lipid component comprises a pegylated myristoyl diglyceride (DMG-PEG; 1,2-Dimyristoyl-sn- glycero-3-methoxypolyethylene glycol). In some embodiments, the DMG-PEG is DMG- PEG2000. In some such embodiments of the ALC0315-based LNPs, the steroid systemcomponent comprises cholesterol and a glucocorticoid steroid. In some embodiments, the glucocorticoid steroid is dexamethasone.Example LNP Systems

[0262] In some embodiments, an LNP system selected from those listed in the table below is used to formulate pharmaceutical compositions described herein:TABLE 5: LNP Systems (all values are a molar ratio relative to a 100 total)

[0263] In some embodiments of any one LNP system LNP001 to LNP240, the LNP system comprises the steroid component in a molar ratio from about 36 to about 46, or from about 38 to about 42. In some embodiments of any one LNP system LNP001 to LNP240, the LNP system comprises the steroid component in a molar ratio of about 36±0.5, about 37±0.5, about 38±0.5, about 39±0.5, about 40±0.5, about 41±0.5, about 42±0.5, about 43±0.5, about 44±0.5, about 45±0.5, or about ±0.5. In some embodiments, the LNP system comprises the steroid componentin a molar ratio of about 37, about 38, about 39, about 40, about 41, or about 42. In some embodiments of any one LNP system LNP001 to LNP240, the steroid component comprises cholesterol. In some embodiments of any one LNP system LNP001 to LNP240, the steroid component comprises cholesterol and one or more additional steroids, wherein cholesterol is about 60%(w / w) or more, about 65%(w / w) or more, about 70%(w / w) or more, about 75%(w / w) or more, or about 80%(w / w) or more of the steroid component. In some embodiments, the steroid component comprises dexamethasone. In some embodiments, the steroid component comprises a modified cholesterol, e.g., a hydroxy- or alkyl modification. In some embodiments, the steroid component comprises any other steroid disclosed herein. In some embodiments, the LNP system comprises the PEGylated Lipid Component in a molar ratio from about 1.5 to about 2.5. In some embodiments, the PEGylated Lipid Component comprises a PEG2000 lipid. In some embodiments, the PEGylated Lipid Component comprises one or more of DMG-PEG and / or DMG-C-PEG. In some embodiments, the PEGylated Lipid Component comprises one or more of DMG-PEG2000, DMG-C-PEG2000, DSG-PEG2000, C14-PEG2000, C16-PEG2000, and / or C18-PEG2000.

[0264] LNPs described herein typically include a Biodegradable Ionizable Lipid, a Helper phospholipid, Cholesterol (with the potentially addition of dexamethasone or other steroids), and a PEG2000 component. Molar Lipid Ratio described herein are ratio of ionizable lipid : helper lipid : cholesterol [: Dexamethasone] : PEG.

[0265] Modifying molar lipid ratios: Without wishing to be bound by any particular theory, when altering the molar lipid ratio of ionizable lipids, the amount of cholesterol or helper lipid would either be supplemented or decreased. Typically, ionizable lipids are kept at a set ratio, while helper lipids, cholesterol and PEG content are varied. If you were to alter the amount of PEG, you would subsequently adjust the amount of primarily cholesterol and sometimes the helper lipid. If altering the helper lipid, the amount of cholesterol in combination with this is adjusted. Dexamethasone, or another similar steroid in the LNP structure directly replaces some of the cholesterol due to similar chemical structures / function in the LNP.

[0266] Typical ionizable lipids are: Dlin-KC2-DMA: molar ratios between 42-50, e.g., and without limitation, 44; Dlin-MC3-DMA: molar ratios between 44-50, e.g., and without limitation, 50; SM-102 (Lipid H): molar ratios between 44-50, e.g., and without limitation, 50; ALC-0315: molar ratios between 44-50, e.g., and without limitation, 50; LP01 (LP000001): molar ratios between 42-50, e.g., and without limitation, 45. Helper Lipids can have molar ratios between 9-11, e.g., and without limitation, 10. Helper lipids include DSPC, DOPE, DOTMA, and DPPC. PEG2000 lipids have typical molar ratios between about 1.5 and about 2.5. These include, without limitation, DMG-PEG, DMG-C-PEG, DMG-PEG2000, DMG-C-PEG2000, DSG-PEG2000, and C14,16,18-PEG2000. Cholesterol molar lipid ratios are typically between about 36 and about 46. Dexamethasone or similar steroids supplement a portion of the molar lipid ratio of cholesterol, for example, and without limitation, a ratio of cholesterol : dexamethasone (C:D) of 9:1. Molar lipid ratio ranges of Cholesterol can be, without limitation, between about 8 and about 10 and Dexamethasone, without limitation, between about 0.1 and about 2. Without wishing to be bound by any particular theory, modifications or replacements for cholesterol can be considered, for example, and without limitation, hydroxy- or alkyl modification (e.g., to improve mRNA delivery) or substitution with potentially therapeutic moieties such as anti-inflammatory steroids. In some embodiments, Molar N / P ratios cab be between 1-8. Without wishing to be bound by any particular, it is believed that to reduce inflammatory response of LNPs in vivo, the ideal range is 1-5 for N / P, however, N / P ratios of 6-8 are tested.

[0267] In some embodiments, LNP formulations described herein include LP01:DSPC:Cholesterol:DMG-PEG2000, LP01:DSPC:Cholesterol:Dexamethasone:DMG- PEG2000, MC3:DSPC:Cholesterol:DMG-PEG2000, MC3:DSPC:Cholesterol:Dexamethasone:DMG-PEG2000, MC3:DSPC:Cholesterol:DMG-C- PEG2000, MC3:DSPC:Cholesterol:Dexamethasone:DMG-C-PEG2000, SM- 102:DSPC:Cholesterol:DMG-PEG2000, SM-102:DSPC:Cholesterol:Dexamethasone:DMG- PEG2000, ALC-0315:DSPC:Cholesterol:DMG-PEG2000, ALC-0315:DSPC:Cholesterol:Dexamethasone:DMG-PEG2000. In some embodiments, molar ratios of formulations described herein include 50:10:38.5:1.5; 45:9:44:2.

[0268] Biophysical Assays: LNPs efficiency to encapsulate nucleic acids, meet sizing criteria and are homogeneity, stability after freeze-thaw cycles. Payloads encapsulated: GFP mRNA, Luciferase mRNA, and our CRISPR / cas9 therapeutic (sgRNA and cas9 mRNA).

[0269] Table 6: LNPs of various formulations created and screened.Additional notes on LNP components:

[0270] Ionizable lipid (titratable charge): Contains a tertiary amine, making this positively charged at an acidic pH, neutrally charged at a physiological pH. Become protonated after endosomal uptake into the cytosol. See below for specifications on different types of ionizable lipids.

[0271] Helper phospholipid: anionic endosomal phospholipids that interact with protonated ionizable lipids to form cone shaped ion pairs that enhance cell membrane fusion and disruption, endosomal escape, and cargo release into cell cytosol.

[0272] Cholesterol: maintains nanoparticle membrane integrity, assists encapsulation of nucleic acids, and enhances circulation by reducing surface bound proteins.

[0273] Dexamethasone or similar steroids: provide an anti-inflammatory component to the LNPs, which decrease immunogenicity and increase transfection rates, particularly in vivo.

[0274] PEGylated lipid (PEG2000): improves circulation half-life, reduces aggregation of LNPs, and reduces interactions with serum proteins such as opsonins, enhancing stability in vivo. Additionally assists particle stability, particle sizing, and biodistribution.

[0275] Ionizable lipids:LP01(pKa = 6.41)

[0276] Cationic lipids: such as DODMA, DOTMA, and DOTAP have a quaternary amine group, which leaves them permanently positively charged. Use of this alone was in the past proven to have poor circulation and increased toxicity in vivo. Led to the development of ionizable lipids.

[0277] Ionizable lipids are characterised by a replacing quaternary amine to be tertiary, which enables them to be pH dependent, i.e., is neutrally charged at physiological pH and becomes positively charged (protonated) at acidic pH.[00278J This increases circulation half-life and reduces toxicity in vivo.

[0279] pKa of ionizable lipid drives performance in vivo. pKas between 6-6.7 have been shown to be optimal for delivery of RNA therapeutics. However, the relative pKa of combination of all components in LNP influences LNP transfection - tertiary amine, quaternary amino and hydroxyl group from ionizable lipid, helper lipids and cholesterol all alter relative pKa due to proximity between headgroups. This subsequently affects overall surface charge.

[0280] PH dependency enables efficient encapsulation of RNAs in acidic buffer as well as assists RNA release once uptaken by cells.

[0281] Further, these lipid pairs form an inverted hexagonal HII phase, which assists membrane disruption, endosomal escape, and subsequent payload release into cytosol of cells.

[0282] Albertson et al., 2022, Lipid packing theory showing relationship between amphipathic compounds and geometry once self-assembled. Proposed mechanism by which ionizable lipids mediate endosomal disruption (also known as molecular shape hypothesis). https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9250827 /

[0283] MC3, ALC-0135, SM-102 all have tertiary amines (making them ionizable), no stereo centres, and ester linkers. ALC-0135 and SM-102 have been improved upon MC3 to incorporate additional ester bonds (to assist with biodegradability and reduce bioaccumulation).

[0284] Unsaturation of linear tails increases delivery efficiency and fluidity in ionizable lipids, i.e., this enables bilayer lipids to form a non-bilayer phase that will increase membrane disruption and payload release. Additional research explored branching the tails of ionizable lipids, which further increased potency when delivering mRNA therapeutics due to an increase in protonation of ionizable lipids at endosomal pH and cross-section of lipid tails. This modification also led to an increase in cone-shape structure, which facilitates membrane disruption, endosomal escape, and payload release into the cytosol (Albertson et al., 2022).

[0285] LP01 incorporates increase in ester bonds as well as branching of tails, which has been shown to be an efficient and safe delivery system for in-vivo gene editing in animal models. LP01 has less liver bioaccumulation and fewer safety risks.

[0286] In some embodiments, use of the CRISPR gene-editing system further comprising one or more LNPs to target a gene, and combinations thereof is therapeutic. C. Virus-like particles

[0287] In one aspect, the present disclosure encompasses means for delivering a CRISPR gene-editing system to a mammalian cell via a virus-like particle (VLP). In some embodiments, a CRISPR gene-editing system is delivered by a VLP. Without wishing to be bound by any particular theory, in certain embodiments, nucleic acids, when present in the particle, are resistant in aqueous solution to degradation with a nuclease. In other embodiments, proteins are protected from protease degradation while present in the particle. In some embodiments, proteins and nucleic acids encapsulated by VLPs are capable of penetrating the cellular plasma membrane.

[0288] In some embodiments, the CRISPR gene-editing system comprises one or more RNA- containing compositions. In some embodiments, the CRISPR gene-editing system further comprises one or more VLPs. In some embodiments, said one or more RNA-containingcompositions comprises a guide RNA. In some embodiments, said one or more RNA-containing compositions comprises an mRNA. In some embodiments, said one or more RNA-containing compositions comprises an RNP (e.g., Cas9 and a guide RNA).

[0289] In some embodiments, the CRISPR gene-editing system comprises one or more viruslike particles collectively encapsulating (i) the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease and (ii) the at least one guide RNA or the nucleic acid encoding the at least one guide RNA. In some embodiments, the one or more virus-like particles comprises a first plurality of virus-like particles encapsulating the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and a second plurality of virus-like particles encapsulating the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0290] In some embodiments, use of the CRISPR gene-editing system further comprising one or more LNPs to target a gene, and combinations thereof is therapeutic.

[0291] In one embodiment, the disclosure provides an LNP comprising a 50 : 10 : 38.5 : 1 .5 molar ratio of the ionizable lipid SM-102 : helper lipid DSPC : cholesterol : DMG-PEG2000, a codon-optimized mRNA encoding an engineered high-fidelity SpCas9 protein fused to bipartite and monopartite nuclear localization signals (NLS) with rationally designed linkers having the sequence shown in Figures 9C-9D, and an sgRNA having a spacer sequence of ACUCUUGUUACAGAGCUGGU.

[0292] In one embodiment, the disclosure provides an LNP comprising a 50 : 10 : 38.5 : 1.5 molar ratio of the ionizable lipid SM-102 : helper lipid DSPC : cholesterol : DMG-PEG2000, a codon-optimized mRNA encoding an engineered high-fidelity SpCas9 protein fused to bipartite and monopartite nuclear localization signals (NLS) with rationally designed linkers having the sequence shown in Figures 9C-9D, and an sgRNA having a spacer sequence of AAAGCAAUAUCGUAUUACCC.D. Liposomes

[0293] In some embodiments, nucleic acids encoding a CRISPR gene-editing system targeting a gene, and combinations thereof (e.g., Cas9 or gRNA) are entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins), which can be tagged with antibodies against cell surface antigens of the target cells. These delivery vehicles can also be used to deliver Cas9 protein / gRNA complexes.

[0294] In some embodiments, the CRISPR gene-editing system comprises one or more RNA- containing compositions. In some embodiments, the CRISPR gene-editing system further comprises one or more liposomes. In some embodiments, said one or more RNA-containing compositions comprises a guide RNA. In some embodiments, said one or more RNA-containing compositions comprises an mRNA. In some embodiments, said one or more RNA-containing compositions comprises an RNP (e.g., Cas9 and a guide RNA).

[0295] In some embodiments, wherein the composition comprises one or more liposomes collectively encapsulating (i) the RNA-guided nuclease or the nucleic acid encoding the RNA- guided nuclease and (ii) the at least one guide RNA or the nucleic acid encoding the at least one guide RNA. In some embodiments, the one or more liposomes comprises a first plurality of liposomes encapsulating the RNA-guided nuclease or a nucleic acid encoding an RNA-guided nuclease and a second plurality of liposomes encapsulating the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

[0296] In some embodiments, use of the CRISPR gene-editing system further comprising one or more LNPs to target a gene, and combinations thereof is therapeutic.

[0297] In some embodiments, the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a viral vector. In some embodiments, the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a lipid nanoparticle (LNP). In some embodiments, the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a liposome. In some embodiments, the fusion protein and at least one guide RNA or a nucleic acid are encapsulated in a virus-like particle.E. Example Nanoparticle Compositions

[0298] IL1a - In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1α gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, thesgRNA comprises a spacer sequence shown in FIGs.42B, 42K, and 42R (SEQ ID NOs: 107- 132; 319-339; 472-495).

[0299] In some embodiments, the disclosure provides an LNP comprising SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1α gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42B, 42K, and 42R (SEQ ID NOs: 107- 132; 319-339; 472-495).

[0300] In some embodiments, the disclosure provides an LNP comprising LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1α gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence of one of the OCB sequences shown in FIG.42K (SEQ ID NOs: 319-339). In some embodiments, the sgRNA comprises a spacer sequence of OCB02 (SEQ ID NO:341). In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42B, 42K, and 42R (SEQ ID NOs: 107-132; 319-339; 472-495).

[0301] In some embodiments, the disclosure provides an LNP comprising ALC0315, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1α gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42B, 42K, and 42R (SEQ ID NOs: 107- 132; 319-339; 472-495).

[0302] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a Cas9 protein having at least 95% sequence identity to HiFi-Cas9-FP (SEQ ID NO:611 as shown in Figure 32B) and a guide RNA targeting an IL1α gene. In some embodiments, the Cas9 protein has at least 99% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the Cas9 protein is HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42B, 42K, and 42R (SEQ ID NOs: 107-132; 319-339; 472-495).

[0303] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a human IL1α gene comprising a spacer sequence shown in FIG.42B. In some embodiments, the sgRNA comprises a spacer sequence of OHB04 (SEQ ID NO: 136), OHB05 (SEQ ID NO: 137), OHB08 (SEQ ID NO: 140), OHB12 (SEQ ID NO: 144), or OHB13 (SEQ ID NO: 145). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR- associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 proteinhas 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0304] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a canine IL1α gene comprising a spacer sequence shown in FIG.42K. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In someembodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0305] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an equine IL1α gene comprising a spacer sequence shown in FIG.42K. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity toHiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0306] IL1β - In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1β gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 proteinhas at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42C, 42L, and 42S (SEQ ID NOs: 133- 150; 340-347; 496-521).

[0307] In some embodiments, the disclosure provides an LNP comprising SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1β gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In someembodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42C, 42L, and 42S (SEQ ID NOs: 133- 150; 340-347; 496-521).In some embodiments, the sgRNA comprises a spacer sequence of OCB02 (SEQ ID NO:341). In some embodiments, the sgRNA comprises a spacer sequence of OHB04 (SEQ ID NO:136), OHB05 (SEQ ID NO:137), OHB08 (SEQ ID NO:140), OHB12 (SEQ ID NO:144), or OHB13 (SEQ ID NO:145).

[0308] In some embodiments, the disclosure provides an LNP comprising LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1β gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, thespCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence of one of the OCB sequences shown in FIGs.42C, 42L, and 42S (SEQ ID NOs: 133-150; 340-347; 496-521).

[0309] In some embodiments, the disclosure provides an LNP comprising ALC0315, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1β gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ IDNOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42C, 42L, and 42S (SEQ ID NOs: 133- 150; 340-347; 496-521).

[0310] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a Cas9 protein having at least 95% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B) and a guide RNA targeting an IL1β gene. In some embodiments, the Cas9 protein has at least 99% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the Cas9 protein is HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42C, 42L, and 42S (SEQ ID NOs: 133-150; 340-347; 496-521).

[0311] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a human IL1β gene comprising a spacer sequence shown in FIG.42C. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising,from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0312] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a canine IL1β gene comprising a spacer sequence shown in FIG.42L. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, asecond NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0313] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an equine IL1β gene comprising a spacer sequence shown in FIG.42S. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identityto Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0314] IL1R1 - In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1R1 gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments,the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42E, 42M, and 42T (SEQ ID NOs: 177-199; 348-381; 522-550). In some embodiments, the sgRNA comprises a spacer sequence of OCR10 (SEQ ID NO:359).

[0315] In some embodiments, the disclosure provides an LNP comprising SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1R1 gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, theSpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42E, 42M, and 42T (SEQ ID NOs: 177- 199; 348-381; 522-550). In some embodiments, the sgRNA comprises a spacer sequence of OCR10 (SEQ ID NO:359).

[0316] In some embodiments, the disclosure provides an LNP comprising LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1R1 gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In someembodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42E, 42M, and 42T (SEQ ID NOs: 177- 199; 348-381; 522-550). In some embodiments, the sgRNA comprises a spacer sequence of OCR10 (SEQ ID NO:359).

[0317] In some embodiments, the disclosure provides an LNP comprising ALC0315, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1R1 gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ IDNO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42E, 42M, and 42T (SEQ ID NOs: 177- 199; 348-381; 522-550). In some embodiments, the sgRNA comprises a spacer sequence of OCR10 (SEQ ID NO:359).

[0318] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a Cas9 protein having at least 95% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B) and a guide RNA targeting an IL1R1 gene. In some embodiments, the Cas9 protein has at least 99% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the Cas9 protein is HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42E, 42M, and 42T (SEQ ID NOs: 177-199; 348-381; 522-550). In some embodiments, the sgRNA comprises a spacer sequence of OCR10 (SEQ ID NO:359).

[0319] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a human IL1R1 gene comprising a spacer sequence shown in FIG.42E. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0320] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a canine IL1R1 genecomprising a spacer sequence shown in FIG.42M. In some embodiments, the sgRNA comprises a spacer sequence of OCR10 (SEQ ID NO:359). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0321] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an equine IL1R1 gene comprising a spacer sequence shown in FIG.42T. In some embodiments, the nanoparticle is alipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0322] IL1RAP - In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1RAP gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid systemcomponent, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42D, 42N, and 42U (SEQ ID NOs: 151-176; 382-402; 551-580).

[0323] In some embodiments, the disclosure provides an LNP comprising SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1RAP gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In someembodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42D, 42N, and 42U (SEQ ID NOs: 151- 176; 382-402; 551-580).

[0324] In some embodiments, the disclosure provides an LNP comprising LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1RAP gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), aCRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence of one of the OCB sequences shown in FIGs.42D, 42N, and 42U (SEQ ID NOs: 151-176; 382-402; 551-580).

[0325] In some embodiments, the disclosure provides an LNP comprising ALC0315, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL1RAP gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42D, 42N, and 42U (SEQ ID NOs: 151- 176; 382-402; 551-580).

[0326] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a Cas9 protein having at least 95% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B) and a guide RNA targeting an IL1RAP gene. In some embodiments, the Cas9 protein has at least 99% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 811 as shown in Figure 32B). In some embodiments, the Cas9 protein is HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical toone of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42D, 42N, and 42U (SEQ ID NOs: 151-176; 382-402; 551-580).

[0327] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a human IL1RAP gene comprising a spacer sequence shown in FIG.42D. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53,and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0328] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a canine IL1RAP gene comprising a spacer sequence shown in FIG.42N. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0329] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an equine IL1RAP gene comprising a spacer sequence shown in FIG.42U. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0330] IL6ST - In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL6ST gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42F, 42O, and 42V (SEQ ID NOs: 200-233; 403-413; 581-610).

[0331] In some embodiments, the disclosure provides an LNP comprising SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL6ST gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42F, 42O, and 42V (SEQ ID NOs: 200- 233; 403-413; 581-610).

[0332] In some embodiments, the disclosure provides an LNP comprising LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component ata molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL6ST gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence of one of the OCB sequences shown in FIGs.42F, 42O, and 42V (SEQ ID NOs: 200-233; 403-413; 581-610).

[0333] In some embodiments, the disclosure provides an LNP comprising ALC0315, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an IL6ST gene.In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42F, 42O, and 42V (SEQ ID NOs: 200- 233; 403-413; 581-610).

[0334] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a Cas9 protein having at least 95% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B) and a guide RNA targeting an IL6ST gene. In some embodiments, the Cas9 protein has at least 99% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the Cas9 protein is HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). Insome embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42F, 42O, and 42V (SEQ ID NOs: 200-233; 403-413; 581-610).

[0335] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a human IL6ST gene comprising a spacer sequence shown in FIG.42F. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 proteinhas at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0336] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a canine IL6ST gene comprising a spacer sequence shown in FIG.42O. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In someembodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0337] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an equine IL6ST gene comprising a spacer sequence shown in FIG.42V. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises asequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0338] TNFα - In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an TNFΑ gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99%identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46FIGs.42J, and 42Q (SEQ ID NOs: 293-318; 442-471).

[0339] In some embodiments, the disclosure provides an LNP comprising SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an TNFΑ gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42J, and 42Q (SEQ ID NOs: 293-318; 442-471).

[0340] In some embodiments, the disclosure provides an LNP comprising LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an TNFΑ gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence of one of the OHB or OCB sequences shown in FIGs.42J, and 42Q (SEQ ID NOs: 293-318; 442-471).

[0341] In some embodiments, the disclosure provides an LNP comprising ALC0315, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component ata molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an TNFΑ gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42J, and 42Q (SEQ ID NOs: 293-318; 442-471).

[0342] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a Cas9 protein having at least 95% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B) and a guide RNA targeting an TNFΑ gene. In some embodiments, the Cas9 protein has at least 99% sequence identity to HiFi-Cas9-FP (SEQ ID NO: 611 as shownin Figure 32B). In some embodiments, the Cas9 protein is HiFi-Cas9-FP (SEQ ID NO: 611 as shown in Figure 32B). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42J, and 42Q (SEQ ID NOs: 293-318; 442-471).

[0343] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a human TNFΑ gene comprising a spacer sequence shown in FIG.42J. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity toHiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0344] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting a canine TNFΑ gene comprising a spacer sequence shown in FIG.42Q. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 proteinhas at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0345] In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an equine TNFα gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one ofSEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55.

[0346] TGFβ - In some embodiments, the disclosure provides a nanoparticle carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an TGFΒ gene. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid, a neutral / helper lipid, a steroid system component, and a pegylated lipid component. In some embodiments, the ionizable lipid is LP01, SM-102, or ALC0315. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44,47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, the sgRNA comprises a spacer sequence shown in FIGs.42G, and 42P (SEQ ID NOs: 234-262; 414-441).

[0347] In some embodiments, the disclosure provides an LNP comprising SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an TGFΒ gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein is a fusion protein comprising, from N- to C-terminus, a first nuclear localization signal (NLS), a CRISPR-associated endonuclease, a first linker polypeptide having a first length of from 15 to 17 amino acids, a second NLS, a second linker polypeptide having a second length of from 15 to 17 amino acids, and a third NLS. In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9_AA (SEQ ID NO:21). In some embodiments, the SpCas9 protein has at least 95% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has at least 99% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the spCas9 protein has 100% sequence identity to HiFi_Cas9_AA (SEQ ID NO:22). In some embodiments, the SpCas9 protein has at least 95% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has at least 99% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the spCas9 protein has 100% sequence identity to Cas9-FP_AA (SEQ ID NO:23). In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, and 56. In some embodiments, the mRNA comprises a sequence that is at least 99% identical to one of SEQ ID NOs: 28, 31, 34, 37, 40, 43, 46, 49, 52, and 55. In some embodiments, thesgRNA comprises a spacer sequence shown in FIGs.42G, and 42P (SEQ ID NOs: 234-262; 414-441).

[0348] In some embodiments, the disclosure provides an LNP comprising LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5), respectively, the LNP carrying an mRNA encoding a CRISPR-associated endonuclease and a guide RNA targeting an TGFΒ gene. In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is an SpCas9 protein. In some embodiments, the SpCas9 protein...

Claims

CLAIMSWHAT IS CLAIMED:

1. A fusion protein comprising, from N- to C-terminal, a) a first nuclear localization signal (NLS); b) a CRISPR-associated endonuclease c) a first linker polypeptide having a first length of from 15 to 17 amino acids; d) a second NLS; e) a second linker polypeptide having a second length of from 15 to 17 amino acids; and f) a third NLS.

2. The fusion protein of claim 1, wherein the first nuclear localization signal (NLS) is a first portion of a bipartite NLS.

3. The fusion protein of claim 2, wherein the second nuclear localization signal (NLS) is a second portion of the bipartite NLS.

4. The fusion protein according to any one of claims 1 to 3, wherein the third nuclear localization signal (NLS) is a monopartite NLS.

5. The fusion protein according to any one of claims 1 to 4, wherein the first nuclear localization signal (NLS) can bind to an importin-alpha polypeptide.

6. The fusion protein according to any one of claim 1 to 5, wherein the first nuclear localization signal (NLS) comprises bpSV40 T3 NLS.

7. The fusion protein according to any one of claims 1 to 6, wherein the first nuclear localization signal (NLS) comprises a N-terminal amino acid sequence of NLS1_AA (SEQ ID NO: 1).

8. The fusion protein according to any one of claims 1 to 7, wherein the second nuclear localization signal (NLS) can bind to an importin-alpha polypeptide.

9. The fusion protein according to any one of claim 1 to 8, wherein the second nuclear localization signal (NLS) comprises the bipartite SV40 T3 NLS.

10. The fusion protein according to any one of claims 1 to 9, wherein the second nuclear localization signal (NLS) comprises a C-terminal amino acid sequence of NLS2_AA (SEQ ID NO: 2).

11. The fusion protein according to any one of claims 1 to 10, wherein the third nuclear localization signal (NLS) can bind to an importin-alpha polypeptide.

12. The fusion protein according to any one of claims 1 to 11, wherein the third nuclear localization signal (NLS) comprises the c-myc NLS.

13. The fusion protein according to any one of claims 1 to 12, wherein the third nuclear localization signal (NLS) comprises NLS an amino acid sequence of NLS3_AA (SEQ ID NO: 3).

14. The fusion protein according to any one of claims 1 to 13, wherein the CRISPR- associated endonuclease comprises Cas3, Cas9, Cas12a, Cas13a, or Cas13b.

15. The fusion protein according to any one of claims 1 to 14, wherein the CRISPR- associated endonuclease comprises a Cas protein devoid of nucleolytic activity.

16. The fusion protein according to any one of claims 1 to15, wherein the CRISPR- associated endonuclease is a Cas9 endonuclease.

17. The fusion protein according to any one of claims 1 to 16, wherein the CRISPR- associated endonuclease comprises an amino acid sequence having at least 95% identity to Cas9_AA (SEQ ID NO: 21) or HiFi_Cas9_AA (SEQ ID NO:22).

18. The fusion protein according to any one of claims 1 to 17, wherein the CRISPR- associated endonuclease comprises K848A, K1003A, and R1060A amino acid substitutions relative to SEQ ID NO: 21 or 22.

19. The fusion protein according to any one of claims 1 to 18, wherein the CRISPR- associated endonuclease comprises a R691A amino acid substitution relative to SEQ ID NO: 21 or 22.

20. The fusion protein according to any one of claims 1 to 19, wherein the CRISPR- associated endonuclease comprises N497A, R661A, Q695A, and Q926A amino acid substitutions relative to SEQ ID NO: 21 or 22.

21. The fusion protein according to any one of claims 1 to 20, wherein the CRISPR- associated endonuclease comprises N692A, M694A, Q695A, and H698A amino acid substitutions relative to SEQ ID NO: 21 or 22.

22. The fusion protein according to any one of claims 1 to 21, wherein the CRISPR- associated endonuclease comprises R63A and Q768A amino acid substitutions relative to SEQ ID NO: 21 or 22.

23. The fusion protein according to any one of claims 1 to 22, wherein the CRISPR- associated endonuclease comprises M495V, Y515N, K526E and R661Q amino acid substitutions relative to SEQ ID NO: 21 or 22.

24. The fusion protein according to any one of claims 1 to 23, wherein the CRISPR- associated endonuclease comprises F539S, M763I, and K890N amino acid substitutions relative to SEQ ID NO: 21 or 22.

25. The fusion protein according to any one of claims 1 to 24, wherein the CRISPR- associated endonuclease comprises N690C, T769I, G915M and N980K amino acid substitutions relative to SEQ ID NO: 4 or an E1007L amino acid substitution relative to SEQ ID NO: 21 or 22.

26. The fusion protein according to any one of claims 1 to 25, wherein the CRISPR- associated endonuclease comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9_AA (SEQ ID NO: 21) or HiFi_Cas9_AA (SEQ ID NO:22).

27. The fusion protein according to any one of claims 1 to 26, wherein the CRISPR- associated endonuclease comprises an amino acid sequence has 100% identity to Cas9_AA (SEQ ID NO: 21) or HiFi_Cas9_AA (SEQ ID NO:22).

28. The fusion protein of claim 27, comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to Cas9-FP-AA (SEQ ID NO: 23) or HiFi-Cas9-FP (SEQ ID NO: 611).

29. The fusion protein of claim 28, comprising an amino acid sequence having 100% identity to Cas9-FP-AA (SEQ ID NO: 23).

30. The fusion protein according to any one of claims 1 to 29, wherein the first linker polypeptide has a first length of 16 amino acids.

31. The fusion protein according to any one of claims 1 to 30, wherein the first linker polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to Full_Linker_1 (SEQ ID NO: 4).

32. The fusion protein according to any one of claims 1 to 31, wherein the second linker polypeptide has a second length of 16 amino acids.

33. The fusion protein according to any one of claims 1 to 32, wherein the second linker polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to Full_Linker_2 (SEQ ID NO: 5).

34. The fusion protein according to any one of claims 1 to 33, wherein the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least 10, at least 11, at least 12, at least 13, or at least 14 of the amino acids in the amino acid sequence are selected from glycine, alanine, and serine.

35. The fusion protein according to any one of claims 1 to 34, wherein the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least one amino acid is phenylalanine.

36. The fusion protein according to any one of claims 1 to 35, wherein the first linker polypeptide and second linker polypeptide each comprises an amino acid sequence wherein at least one amino acid is glutamate.

37. A polynucleotide encoding a Cas9 endonuclease comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any of the DNA sequences provided in SEQ ID NOs: 60, 63, 66, 69, 72, 75, 78, 81, and 84.

38. A polynucleotide encoding a Cas9 endonuclease comprising a nucleotide sequence having 100% identity to any of the DNA sequences provided in SEQ ID NOs: 60, 63, 66, 69, 72, 75, 78, 81, and 84.

39. The polynucleotide according to any one of claims 37 to 38, further comprising the coding sequence for an initiation site for T7 RNA polymerase-mediated transcription.

40. The polynucleotide according to any one of claims 37 to 39, further comprising a nucleotide sequence encoding a recognition site for a translation initiation complex.

41. The polynucleotide according to any one of claims 37 to 40, further comprising a nucleotide sequence encoding a synthetic NeoUTR3.

42. The polynucleotide according to any one of claims 38 to 41, further comprising a nucleotide sequence encoding a synthetic eIF4G-recruiting aptamer.

43. The polynucleotide according to any one of claims 37 to 42, further comprising a nucleotide sequence encoding an endogenous 5’ UTR of the human hemoglobin α-subunit 1 / 2 (HBA1 / 2).

44. The polynucleotide according to any one of claims 37 to 43, further comprising a nucleotide sequence encoding a minimalistic 5’ UTR.

45. The polynucleotide according to any one of claims 37 to 44, further comprising a nucleotide sequence encoding an endogenous 5’ UTR of hemoglobin β-subunit (HBB).

46. The polynucleotide according to any one of claims 37 to 45, further comprising a nucleotide sequence encoding an a Kozak motif.

47. The polynucleotide according to any one of claims 37 to 46, further comprising a nucleotide sequence encoding a polyadenylation signal.

48. The polynucleotide of claim 37 to 47, further comprising the coding region for a polyadenylation tail.

49. The polynucleotide of claim 48, wherein the polyadenylation tail comprises a linker sequence, wherein the linker sequence comprises a nucleotide sequence encoding at least one type IIS restriction enzyme cleavage site.

50. The polynucleotide of claim 49, wherein the type IIS restriction enzyme cleavage site is a Sap1 restriction enzyme cleavage site.

51. The polynucleotide according to any one of claims 37 to 50, further comprising a single copy of the endogenous 3’ UTR of human hemoglobin α-subunit 1 (HBA1).

52. The polynucleotide according to any one of claims 37 to 51, wherein the polynucleotide comprises a ribonucleic acid (RNA).

53. The polynucleotide according to any one of claims 37 to 52, wherein the polynucleotide comprises a ribonucleic acid having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any of the RNA sequences provided in SEQ ID NOs: 10, 22- 50, 72-74, 96, 99, or 102.

54. The polynucleotide according to any one of claims 37 to 52, wherein the polynucleotide comprises a ribonucleic acid having 100% identity to any of the RNA sequences provided in SEQ ID NOs: 10, 22-50, 72-74, 96, 99, or 102.

55. The RNA of claim 52, wherein the RNA is an mRNA comprising a 5’ terminal cap.

56. The polynucleotide of claim 53, wherein the 5’ terminal cap comprises m7G(5’)ppp(5’)(2’OmeA)pG, m7(3'OMeG)(5’)ppp(5’)(2’OmeA)pG, or m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG.

57. The polynucleotide according to any one of claims 1 to 56, wherein the polynucleotide is a circularRNA.

58. The polynucleotide of claim 57, wherein the polynucleotide comprises a synthetic internal ribosome entry site containing a eukaryotic initiation factor 4G-recruiting aptamer.

59. The polynucleotide of claim 57 or 58, wherein the polynucleotide comprises 5% N6- methyladenosine.

60. The polynucleotide according to any one of claims 58 to 59, wherein the polynucleotide comprises the 3’ UTR of HBA1.

61. The polynucleotide according to any one of claims 37 to 60, wherein the polynucleotide comprises a deoxyribonucleic acid (DNA).

62. A plasmid construct comprising the polynucleotide of claim 61.

63. A composition comprising: (i) a fusion protein according to any one of claims 1 to 36, a polynucleotide according to any one of claims 37 to 61, or a plasmid of claim 62; and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene.

64. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting an IL1α gene.

65. The composition of claim 64, wherein the IL1α gene is a human IL1α gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:107-132 (OHA01-OHA26).

66. The composition of claim 64, wherein the IL1α gene is a canine IL1α gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:319-339 (OCA01-OCA25).

67. The composition of claim 64, wherein the IL1α gene is an equine IL1α gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:471- 495 (OCE01-OCE24).

68. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting an IL1β gene.

69. The composition of claim 68, wherein the IL1β gene is a human IL1β gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:133-150 (OHB01-OHB18).

70. The composition of claim 68, wherein the IL1β gene is a canine IL1β gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:340-347 (OCB01-OCB08).

71. The composition of claim 68, wherein the IL1β gene is an equine IL1β gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:496- 521 (OEB01-OEB26).

72. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting an IL1RAP gene.

73. The composition of claim 72, wherein the IL1RAP gene is a human IL1RAP gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:151- 176 (OHP01-OHP16).

74. The composition of claim 72, wherein the IL1RAP gene is a canine IL1RAP gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:382- 402 (OCP01-OCP12).

75. The composition of claim 72, wherein the IL1RAP gene is an equine IL1RAP gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:551-580 (OEP01-OEP12).

76. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting an IL1R gene.

77. The composition of claim 76, wherein the IL1R gene is a human IL1R gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:177- 199 (OHR01-OHR16).

78. The composition of claim 76, wherein the IL1R gene is an equine IL1R gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:522- 550 (OER01-OER29).

79. The composition of claim 76, wherein the IL1R gene is a canine IL1R gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:348-381 (OCR01-OCR26).

80. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting an IL6ST gene.

81. The composition of claim 80, wherein the IL6ST gene is a human IL6ST gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:200- 233 (OHS01-OHS34).

82. The composition of claim 80, wherein the IL6ST gene is a canine IL6ST gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:403- 413 (OCS01-OCS11).

83. The composition of claim 80, wherein the IL6ST gene is an equine IL6ST gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:581- 610 (OES01-OES30).

84. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting a TGFβ gene.

85. The composition of claim 84, wherein the TGFβ gene is a human TGFβ gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:234- 262 (OHTG01-OHTG29).

86. The composition of claim 84, wherein the TGFβ gene is a canine TGFβ gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:414- 441 (OCTG01-OCTG28).

87. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting a TGFβRI gene.

88. The composition of claim 87, wherein the TGFβRI gene is a human TGFβRI gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:263- 278 (OHTIR-OHTIR16).

89. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting a TGFβRII gene.

90. The composition of claim 89, wherein the TGFβRII gene is a human TGFβRII gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:279-292 (OHTIIR-OHTIIR14).

91. The composition of claim 63, wherein the at least one guide RNA comprises a spacer sequence targeting a TNFα gene.

92. The composition of claim 91, wherein the TNFα gene is a human TNFα gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:293- 318 (OHT01-OHT26).

93. The composition of claim 91, wherein the TNFα gene is a canine TNFα gene and the spacer sequence comprises a sequence selected from the group consisting of SEQ ID NO:442- 471 (OCT01-OCT30).

94. The composition of any one of claims 63-93, wherein the composition is formulated for a non-therapeutic use.

95. The composition of claim 94, wherein the composition is formulated for the diagnosis of a disease or disorder in a subject.

96. The composition of claim 63-93, wherein the composition is a pharmaceutical composition for the prevention or treatment of a disease or disorder in a subject.

97. The pharmaceutical composition of claim 96, wherein the pharmaceutical composition is formulated for local, targeted, or regional administration in the subject.

98. The pharmaceutical composition of claim 96, wherein the pharmaceutical composition is formulated for systemic administration in the subject.

99. The pharmaceutical composition according to any one of claims 96 to 98, wherein the disease or disorder is a genetic disorder.

100. The pharmaceutical composition according to any one of claims 96 to 98, wherein the disease or disorder is a cancer.

101. The pharmaceutical composition according to any one of claims 96 to 98, wherein the disease or disorder is an inflammatory disorder.

102. The pharmaceutical composition according to any one of claims 96 to 98, wherein the disease or disorder is a joint disorder.

103. The composition of claim 102, wherein the joint disorder is arthritis.

104. The composition of claim 102, wherein the joint disorder is osteoarthritis.

105. The composition of claim 102, wherein the joint disorder is rheumatoid arthritis.

106. The composition of claim 102, wherein the joint disorder is post-traumatic arthritis.

107. The composition of claim 102, wherein the joint disorder is gout.

108. The composition of claim 102, wherein the joint disorder is pseudogout.

109. The composition of claim 102, wherein the joint disorder is tendinopathy in any mammalian species.

110. The composition of claim 102, wherein the joint disorder occurs in dogs, cats, or horses.

111. The composition according to any one of claims 63 to 109, wherein the at least one guide RNA targets the coding region for:(i) a transmembrane receptor,(ii) a cytokine, or(iii) a gene associated with the production, blocking, or removal of reactive oxygen species (ROS).

112. The composition of claim 111 wherein the transmembrane receptor is a cytokine receptor.

113. The composition of claim 111 or 112, wherein the cytokine receptor is an interleukin- 1 receptor, an interleukin-6 receptor, or a co-receptor necessary for signal transduction.

114. The composition of claim 113, wherein the at least one guide RNA targets a gene selected from the group consisting of IL1R1, IL1RAP, IL1R5, IL1R6, IL1R7, and IL1R9.

115. The composition of claim 111, wherein the at least one guide RNA comprises a spacer sequence of AAAGCAAUAUCGUAUUACCC.

116. The composition of claim 113, wherein the at least one guide RNA targets an ILR6 gene.

117. The composition according to any one of claims 63 to 116, wherein the cytokine is IL1A, IL1B, or IL-6.

118. The composition according to any one of claims 63 to 117, wherein the at least one guide RNA targets the IL1A or IL1B gene.

119. The composition of claim 118, wherein the at least one guide RNA comprises a spacer sequence of ACUCUUGUUACAGAGCUGGU.

120. The composition according to any one of claims 63 to 118, wherein the at least one guide RNA targets the IL6 gene.

121. The composition according to any one of claims 63 to 119, wherein the gene associated with the production, blocking, or removal of reactive oxygen species (ROS) is an NF-kappa B1 or NF-kappa B2 gene.

122. The composition according to any one of claims 63 to 121, wherein the at least one guide RNA is a single guide RNA (sgRNA).

123. The composition according to any one of claims 63 to 122, wherein the at least one guide RNA targets a human gene.

124. The composition according to any one of claims 63 to 122, wherein the at least one guide RNA targets a canine gene.

125. The composition according to any one of claims 63 to 122, wherein the at least one guide RNA targets an equine gene.

126. The composition according to any one of claims 63 to 122, wherein the at least one guide RNA targets a feline gene.

127. The composition according to any one of claims 63 to 122, wherein the at least one guide RNA targets a mammalian gene.

128. The composition according to any one of claims 63 to 127, wherein the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a viral vector.

129. The composition according to any one of claims 63 to 127, wherein the fusion protein, polynucleotide encoding the fusion protein, or plasmid encoding the fusion protein and the at least one guide RNA or nucleic acid encoding the at least one guide RNA are encapsulated in one or more lipid nanoparticle (LNP).

130. The composition of claim 129, wherein the one or more LNP comprise: a first plurality of LNP encapsulating the fusion protein, polynucleotide encoding the fusion protein, or plasmid encoding the fusion protein; and a second plurality of LNP encapsulating the at least one guide RNA or a nucleic acid encoding at least one guide RNA.

131. The composition of claim 130, wherein the one or more LNP comprise a plurality of LNP encapsulating both the (i) fusion protein, polynucleotide encoding the fusion protein, or plasmid encoding the fusion protein, and (ii) at least one guide RNA or a nucleic acid encoding at least one guide RNA targeting a gene encoding the transmembrane receptor.

132. The composition of any one of claims 130-131, wherein the one or more LNP comprise a component selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1- piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4- piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]- dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8- [(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- -octadeca-9,12-dien-1- yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- ,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z- ,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)), a lipid including a cyclic amine group, and a mixture thereof.

133. The composition of any one of claims 130-132, wherein the one or more LNP comprise a component selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin (SM), and a mixture thereof.

134. The composition of any one of claims 130-133, wherein the one or more LNP comprise a component selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG- DPPC, PEG-DMA, a PEG-DSPE lipid, and a mixture thereof.

135. The composition of any one of claims 130-134, wherein the one or more LNP comprise a component selected from the group consisting of a cholesterol, fecosterol, stigmasterol, stigmastanol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9, 11-dehydroergosterol, tomatidine, tomatine, α- tocopherol, and a mixture thereof.

136. The composition of any one of claims 130-135, wherein the one or more LNP comprises an LP01 ionizable lipid, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component.

137. The composition of any one of claims 130-135, wherein the one or more LNP comprises an SM-102 ionizable lipid, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component.

138. The composition of any one of claims 130-135, wherein the one or more LNP comprises an ALC0315 ionizable lipid, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component.

139. The composition of any one of claims 136-138, wherein the neutral / helper lipid component comprises DSPC.

140. The composition of claim 136-139, wherein the steroid system component comprises cholesterol.

141. The composition of claim 136-140, wherein the steroid system component comprises a glucocorticoid steroid.

142. The composition of claim 141, wherein the glucocorticoid steroid is dexamethasone.

143. The composition of claim 136-142, wherein the pegylated lipid component comprises DMG-PEG.

144. The composition of claim 143, wherein the DMG-PEG is DMG-PEG2000.

145. The composition of any one of claims 136-144, wherein the one or more LNP comprises LP01, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (43-47) : (7.5-10.5) : (42-46) : (1.5-2.5).

146. The composition of any one of claims 136-144, wherein the one or more LNP comprises SM-102, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2).

147. The composition of any one of claims 136-144, wherein the one or more LNP comprises ALC0315, a neutral / helper lipid component, a steroid system component, and a pegylated lipid component at a molar ratio of (48-52) : (8.5-11.5) : (36.5-40.5) : (1-2).

148. The composition according to any one of claims 63 to 127, wherein the fusion protein and the at least one guide RNA or a nucleic acid are encapsulated in a liposome.

149. The composition according to any one of claims 63 to 127, wherein the fusion protein and at least one guide RNA or a nucleic acid are encapsulated in a virus-like particle.

150. A method for altering the genome of a cell comprising contacting the cell with a composition according to any one of claims 63 to 149.

151. The method of claim 150, wherein the cell is synovial cell.

152. The method of claim 151, wherein the cell is a synovial fibroblast, a synoviocyte, a chondrocyte, or a synovial macrophage.

153. The method according to any one of claims 150 to 152, further comprising administering a therapeutically effective amount of the composition to an animal in need thereof.

154. The method according to any one of claims 150 to 153, further comprising administering a therapeutically effective amount of the composition by injection into a joint.

155. The method according to any one of claims 150 to 153, wherein the composition is administered locally to the site of a treatment by a method selected from the group consisting of a surgery, an application of topical ointment, and a combination thereof.

156. A polynucleotide encoding a Cas9 endonuclease comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to amino acids of SEQ ID NO: 21-23, 85-87, 96, or 99.

157. A polynucleotide encoding a Cas9 endonuclease comprising an amino acid sequence having 100% identity to amino acids of SEQ ID NO:4-6, 69-71, 98, or 101.

158. A polynucleotide encoding a Cas9 endonuclease comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to amino acids 44-4381 of SEQ ID NO: 21-23, 85-87, 96, or 99.

159. A polynucleotide encoding a Cas9 endonuclease comprising an amino acid sequence having 100% identity to amino acids 44-4381 of SEQ ID NO: 21-23, 85-87, 96, or 99.

160. A polynucleotide encoding a Cas9 endonuclease comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to amino acids 44-4381 of SEQ ID NO:23.

161. A polynucleotide encoding a Cas9 endonuclease comprising an amino acid sequence having 100% identity to amino acids 44-4381 of SEQ ID NO 23.

162. The polynucleotide according to any one of claims 156-161, further comprising the coding sequence for an initiation site for T7 RNA polymerase-mediated transcription.

163. The polynucleotide according to any one of claims 156-162, further comprising a nucleotide sequence encoding a recognition site for a translation initiation complex.

164. The polynucleotide according to any one of claims 156-163, further comprising a nucleotide sequence encoding a synthetic NeoUTR3.

165. The polynucleotide according to any one of claims 156-164, further comprising a nucleotide sequence encoding a synthetic eIF4G-recruiting aptamer.

166. The polynucleotide according to any one of claims 156-165, further comprising a nucleotide sequence encoding an endogenous 5’ UTR of the human hemoglobin a-subunit 1 / 2 (HBA1 / 2).

167. The polynucleotide according to any one of claims 156-166, further comprising a nucleotide sequence encoding a minimalistic 5’ UTR.

168. The polynucleotide according to any one of claims 156-167, further comprising a nucleotide sequence encoding an endogenous 5’ UTR of hemoglobin P-subunit (HBB).

169. The polynucleotide according to any one of claims 156-168, further comprising a nucleotide sequence encoding an a Kozak motif.

170. The polynucleotide according to any one of claims 156-169, further comprising a nucleotide sequence encoding a polyadenylation signal.

171. The polynucleotide of claim 156-170, further comprising the coding region for a polyadenylation tail.

172. The polynucleotide of claim 171, wherein the polyadenylation tail comprises a linker sequence, wherein the linker sequence comprises a nucleotide sequence encoding at least one type IIS restriction enzyme cleavage site.

173. The polynucleotide of claim 172, wherein the type IIS restriction enzyme cleavage site is a Sap1 restriction enzyme cleavage site.

174. The polynucleotide according to any one of claims 156-173, further comprising a single copy of the endogenous 3’ UTR of human hemoglobin α-subunit 1 (HBA1).

175. The polynucleotide according to any one of claims 156-174, wherein the polynucleotide comprises a ribonucleic acid (RNA).

176. The RNA of claim 175, wherein the RNA is an mRNA comprising a 5’ terminal cap.

177. The polynucleotide of claim 176, wherein the 5’ terminal cap comprises m7G(5’)ppp(5’)(2’OmeA)pG, m7(3'OMeG)(5’)ppp(5’)(2’OmeA)pG, or m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG.

178. The polynucleotide according to any one of claims 156-177, wherein the polynucleotide is a circularRNA.

179. The polynucleotide of claim 178, wherein the polynucleotide comprises a synthetic internal ribosome entry site containing a eukaryotic initiation factor 4G-recruiting aptamer.

180. The polynucleotide of claim 178 or 179, wherein the polynucleotide comprises 5% N6- methyladenosine.

181. The polynucleotide according to any one of claims 178 to 180, wherein the polynucleotide comprises the 3’ UTR of HBA1.

182. The polynucleotide according to any one of claims 156 to 181, wherein the polynucleotide comprises a deoxyribonucleic acid (DNA).

183. A therapeutic LNP comprising a 50 : 10 : 38.5 : 1.5 molar ratio of the ionizable lipid SM- 102 : helper lipid DSPC : cholesterol : DMG-PEG2000, a codon-optimized mRNA encoding an engineered high-fidelity SpCas9 protein fused to bipartite and monopartite nuclear localization signals (NLS) with rationally designed linkers having the sequence of SEQ ID NO:22, and an sgRNA having a spacer sequence of ACUCUUGUUACAGAGCUGGU.

184. A therapeutic LNP comprising a 50 : 10 : 38.5 : 1.5 molar ratio of the ionizable lipid SM- 102 : helper lipid DSPC : cholesterol : DMG-PEG2000, a codon-optimized mRNA encoding an engineered high-fidelity SpCas9 protein fused to bipartite and monopartite nuclear localization signals (NLS) with rationally designed linkers having the sequence of SEQ ID NO:22, and an sgRNA having a spacer sequence of AAAGCAAUAUCGUAUUACCC.

185. A therapeutic LNP comprising a 50 : 10 : 38.5 : 1.5 molar ratio of the ionizable lipid SM- 102 : helper lipid DSPC : cholesterol : DMG-PEG2000, a codon-optimized mRNA encoding an engineered high-fidelity SpCas9 protein fused to bipartite and monopartite nuclear localization signals (NLS) with rationally designed linkers having the sequence of SEQ ID NO:23, and an sgRNA having a spacer sequence of ACUCUUGUUACAGAGCUGGU.

186. A therapeutic LNP comprising a 50 : 10 : 38.5 : 1.5 molar ratio of the ionizable lipid SM- 102 : helper lipid DSPC : cholesterol : DMG-PEG2000, a codon-optimized mRNA encoding an engineered high-fidelity SpCas9 protein fused to bipartite and monopartite nuclear localization signals (NLS) with rationally designed linkers having the sequence of SEQ ID NO:23, and an sgRNA having a spacer sequence of AAAGCAAUAUCGUAUUACCC.

Images