Compositions and methods for reducing complement activation

Abstract

Compositions and methods for reducing complement activation by introducing one or more alterations into a complement component 3 (C3) polynucleotide in a cell. In particular embodiments, the present disclosure features a base editor system (e.g., a fusion protein or complex comprising a programmable DNA binding protein, a nucleobase editor, and a gRNA) for modifying a C3 polynucleotide wherein the modification is associated with a reduction in expression and / or activity of a C3 polypeptide encoded by the polynucleotide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 352,547, filed on June 15, 2022, the entire contents of which are hereby incorporated by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on June 14, 2023, is named 180802-055102PCT_SL.xml and is 1,364,888 bytes in size. Background Art

[0005] The complement system is an important part of the innate immune system and is involved in the clearance of microorganisms and cellular debris, as well as inflammation and the activation of multiple immune pathways. Overactivation of the complement system or inappropriate targeting of autologous cells can lead to disease; however, inhibition of complement system activity has been successfully and safely demonstrated to provide therapeutic benefits to patients suffering from an overactive complement system. Therefore, methods for reducing complement system activation in such patients are needed. Summary of the Invention

[0006] As described below, the present disclosure features compositions and methods for reducing complement activation by introducing one or more changes into complement component 3 (C3) polynucleotides in cells. In specific embodiments, the present disclosure features a base editor system (e.g., a fusion protein or complex comprising a programmable DNA binding protein, a nucleobase editor, and a gRNA) for modifying C3 polynucleotides, wherein the modification is associated with reduced expression and / or reduced activity of a C3 polypeptide encoded by the polynucleotide. Non-limiting examples of changes include base editing.

[0007] In one aspect, the present disclosure features a method for changing the core base of a complement component 3 (C3) polynucleotide. The method includes contacting the C3 polynucleotide with a base editor system, wherein the base editor system contains one or more guide RNAs or one or more polynucleotides encoding the one or more guide RNAs, and a base editor containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain or one or more polynucleotides encoding the base editor, thereby changing the core base of the C3 polynucleotide. The method includes (a), (b), (c), (d) and / or (e). In (a), one or more guide RNAs target base editors to achieve changes in the core bases of the C3 polynucleotides that destroy the splice sites in the C3 polynucleotides. In (b), the deaminase domain is a TadA variant (TadA*) comprising a combination of alterations listed in Tables 5A, 5B, 5C, 5D, 5E, 6A, 6B, 6C, 6D, 6E, 6F, or 7, wherein TadA* is not TadA*7.9 or TadA*7.10, and / or wherein the TadA* variant is a TadA*8.8, TadA*8.17, or TadA*8.20 variant comprising one or more of the amino acid changes V82T, Y147T, and Q154S. In (c), the one or more guide RNAs comprise a nucleic acid sequence comprising at least 10-23 contiguous nucleotides of a spacer nucleic acid sequence listed in any one of Tables 1A, 1B, 1C, 1D, 1E, 1F, and 2. In (d), one or more guide RNAs target the base editor to effect a nucleobase change in the codon encoding an amino acid residue selected from one or more of A741, S742, H743, A747, L746, R748, S749, I1125, H1126, Q1127, and E1128 relative to the following reference sequence: C3 amino acid sequence On the other hand, the present disclosure features a method for changing the core base of a complement component 3 (C3) polynucleotide. The method includes contacting the C3 polynucleotide with one or more guide RNAs or one or more polynucleotides encoding the one or more guide RNAs, and a base editor containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain or one or more polynucleotides encoding the base editor, thereby changing the core base of the complement component 3 (C3) polynucleotide. The method also includes (a) and / or (b). In (a), the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, TadA*8.20_V82T_Y147T_Q154S, rAPOBEC1, and ppAPOBEC. In (b), the one or more guide RNAs contain a spacer corresponding to a guide polynucleotide selected from one or more of gRNA661, gRNA662, gRNA676, gRNA695, gRNA696, gRNA701, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA1793, gRNA1798, gRNA3342, gRNA3343, and gRNA3345. In another aspect, the present disclosure features a method for treating a disease or condition associated with inappropriate activation of the complement system in a subject in need thereof. The method comprises altering the nucleobases of a complement component 3 (C3) polynucleotide in a subject by administering to the subject one or more guide RNAs or one or more polynucleotides encoding the one or more RNAs, and a base editor containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, thereby treating the disease or condition. The method comprises (a), (b), (c), (d) and / or (e). In (a), one or more guide RNAs target the base editor to effectuate an alteration of the nucleobases of the C3 polynucleotide that disrupts a splice site in the C3 polynucleotide. In (b), the deaminase domain is a TadA variant (TadA*) containing a combination of alterations listed in Tables 5A, 5B, 5C, 5D, 5E, 6A, 6B, 6C, 6D, 6E, 6F, or 7, wherein TadA* is not TadA*7.9 or TadA*7.10. In (c), the one or more guide RNAs contain a nucleic acid sequence comprising at least 10-23 consecutive nucleotides of a spacer nucleic acid sequence listed in any one of Tables 1A, 1B, 1C, 1D, 1E, 1F, and 2. In (d), the one or more guide RNAs target a base editor to effect a change in a nucleobase in a codon encoding an amino acid residue selected from one or more of A741, S742, H743, A747, L746, R748, S749, I1125, H1126, Q1127, and E1128 relative to the following reference sequence: C3 amino acid sequence In another aspect, the disclosure features a method for treating a disease or condition associated with inappropriate activation of the complement system in a subject in need thereof. The method comprises administering to the subject one or more guide RNAs or one or more polynucleotides encoding the one or more guide RNAs, and a base editor containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain or one or more polynucleotides encoding the base editor to change the nucleobases of a complement component 3 (C3) polynucleotide in the subject, thereby treating the disease or condition. The method further comprises (a) and (b). In (a), the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, TadA*8.20_V82T_Y147T_Q154S, rAPOBEC1, and ppAPOBEC. In (b), the one or more guide RNAs contain a spacer corresponding to a guide polynucleotide selected from one or more of gRNA661, gRNA662, gRNA676, gRNA695, gRNA696, gRNA701, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA1793, gRNA1798, gRNA3342, gRNA3343, and gRNA3345. In another aspect, the disclosure features a cell produced by the method of any aspect of the disclosure or embodiments thereof. In another aspect, the disclosure features a base editor system containing a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleobases of a spacer listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 2. In another aspect, the disclosure features a polynucleotide or a set of polynucleotides encoding a base editor, a base editor system, or a component thereof of any aspect of the disclosure or embodiments thereof. In another aspect, the disclosure features a kit containing a base editor system or base editor of any aspect of the disclosure, and / or one or more polynucleotides encoding the same or components thereof or embodiments of any aspect of the disclosure. In another aspect, the disclosure features a lipid nanoparticle containing a base editor system or base editor of any aspect of the disclosure, and / or one or more polynucleotides encoding the same or components thereof or embodiments of any aspect of the disclosure. In another aspect, the disclosure features a kit containing one or more polynucleotides comprising or encoding a base editor, wherein the base editor contains a nucleic acid programmable DNA binding protein (napDNAbp) domain, a deaminase domain, and one or more guide polynucleotides or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides contains at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleobases of a spacer listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 2. In another aspect, the disclosure features a pharmaceutical composition containing an effective amount of the base editor system of any aspect of the disclosure or embodiments thereof. In another aspect, the disclosure features a pharmaceutical composition containing one or more polynucleotides comprising or encoding a base editor, wherein the base editor contains a nucleic acid programmable DNA binding protein (napDNAbp) domain, a deaminase domain, and one or more guide polynucleotides or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleobases of a spacer listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 2. In another aspect, the disclosure features a guide polynucleotide comprising a spacer sequence listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 2. In another aspect, the disclosure features a base editor system containing a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, wherein a) the guide polynucleotide contains a spacer corresponding to gRNA661, the napDNAbp domain is SpCas9 or SpCas9 with amino acid changes A1283D and E1250K, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.8 with amino acid change V82T, TadA*8.8 with amino acid changes V82T, Y147T and Q154S, TadA*8.20 with amino acid change V82T, and TadA*8.20 with amino acid changes V82T, Y147T and Q154S; b) the guide polynucleotide contains a spacer corresponding to gRNA676, the napDNAbp domain is SpCas9, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.8 with amino acid changes V82T, Y147T, and Q154S, TadA*8.20 with amino acid changes V82T, and TadA*8.20 with amino acid changes V82T, Y147T, and Q154S; c) the guide polynucleotide contains a spacer corresponding to gRNA696, the napDNAbp domain is SpCas9 or SpCas9 with amino acid changes A1283D and E1250K, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.20 with amino acid change V82T, and TadA*8.20 with amino acid changes V82T, Y147T, and Q154S; d) the guide polynucleotide contains a spacer corresponding to gRNA715, the napDNAbp domain is SpCas9 or SpCas9-VRQR, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, and TadA*8.20 with amino acid change V82T; e) the guide polynucleotide contains a spacer corresponding to gRNA821, the napDNAbp is SpCas9, the deaminase domain is a cytidine deaminase domain, and the base editor further contains two uracil glycosylase inhibitors; f) the guide polynucleotide contains a spacer corresponding to gRNA827, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8; g) the guide polynucleotide contains a spacer corresponding to gRNA828, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8; h) the guide polynucleotide contains a spacer corresponding to gRNA829, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8; i) the guide polynucleotide contains a spacer corresponding to gRNA837, the napDNAbp is SpCas9-VRQR, and the deaminase domain is TadA*8.8; j) the guide polynucleotide contains a spacer corresponding to gRNA838, the napDNAbp is SpCas9-VRQR, and the deaminase domain is TadA*8.8; k) the guide polynucleotide contains a spacer corresponding to gRNA3342, the napDNAbp is SpCas9, and the deaminase is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.8 with amino acid changes V82T, Y147T, and Q154S, TadA*8.17 with amino acid changes V82T, Y147T, and Q154S, TadA*8.17 with amino acid changes V82T, Y147T, and Q154S, TadA*8.20 with amino acid changes V82T, and TadA*8.20 with amino acid changes V82T, Y147T, and Q154S; l) the guide polynucleotide contains a spacer corresponding to gRNA3343, and the napDNAbp is selected from one or more of the following: i) SpCas9-MQKFRAER; ii) SpCas9-MQKFRAER with a combination of amino acid changes I322V, S409I, E427G, R654L, R753G, R1114G, and R1337K; iii) SpCas9-MQKFRAER with a combination of amino acid changes I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, A1283D, and E1250K; and iv) SpCas9-MQKFRAER having a combination of amino acid changes I322V, S409I, E427G, R654L, R753G, and R1114G; and The deaminase domain is TadA*8.20 or TadA*7.10; or m) The guide polynucleotide contains a spacer corresponding to gRNA3345, the napDNAbp domain is SaCas9-KHH, the deaminase domain is a cytidine deaminase domain, and the base editor further contains two uracil glycosylase inhibitors. In another aspect, the disclosure features a method for changing a nucleobase of a complement component 3 (C3) polynucleotide. The method includes contacting the C3 polynucleotide with a base editor system of any aspect of the disclosure or embodiments thereof. In another aspect, the disclosure features a method of treating a disease or condition associated with inappropriate activation of the complement system in a subject in need thereof, comprising altering a C3 polynucleotide in the subject by administering to the subject a base editor system of any aspect of the disclosure or an embodiment thereof. In any aspect of the disclosure or embodiments thereof, the splice site corresponds to any one of the protospacers listed in Table 1A, IB or 1C. In any aspect of the present disclosure or embodiments thereof, the base editor is selected from one or more of ABE8.8, ABE8.13, ABE8.17, ABE8.20, ABE8.8_V82T, ABE8.8_V82T_Y147T_Q154S, ABE8.17_V82T, ABE8.17_V82T_Y147T_Q154S, ABE8.20_V82T, ABE8.20_V82T_Y147T_Q154S, BE4, and those listed in Tables 1A, 1B, 1C, 1D, 1E, 1F, 2, and 28 to 15. In any aspect of the present disclosure or embodiments thereof, one or more guide RNAs or guide polynucleotides target a base editor to effect a change in a nucleobase in a codon encoding an amino acid residue selected from one or more of: A741, S742, H743, A747, L746, R748, S749, I1125, H1126, Q1127, and E1128 relative to the following reference sequence: C3 amino acid sequence In any aspect of the present disclosure or embodiments thereof, the editing rate of the base editor system is greater than 35%. In any aspect of the disclosure or embodiments thereof, the guide RNA or guide polynucleotide contains a spacer containing or containing only about 19 to about 23 nucleotides. In any aspect of the disclosure or embodiments thereof, the spacer contains or contains only 21 nucleotides. In any aspect of the disclosure or embodiments thereof, the nucleobase alteration effects a change in the encoded amino acid residue, wherein the change disrupts the opsonizing effect of C3. In any aspect of the disclosure or embodiments thereof, the nucleobase alteration disrupts splicing of the C3 transcript. In any aspect of the disclosure or embodiments thereof, the nucleobase alteration effects a change in the encoded amino acid residue, wherein the change disrupts cleavage of the C3 polypeptide by the C3 convertase. In any aspect of the disclosure or embodiments thereof, the C3 polynucleotide is in a cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is a primate cell. In embodiments, the primate is a human. In embodiments, the cell is a retinal cell or other cell of the eye, a cell of the CNS, or a liver cell. In any aspect of the present disclosure or embodiments thereof, one or more guide RNAs or guide polynucleotides target a base editor to effect a change in a nucleobase of a C3 polynucleotide that disrupts a splice site in the C3 polynucleotide, wherein the splice site is selected from one or more of those splice sites corresponding to any one of the protospacers listed in Tables 1A, 1B, 1C, 1D, 1E, 1F or 1C. In any aspect of the present disclosure or embodiments thereof, the editing rate is greater than 50%. In any aspect of the present disclosure or embodiments thereof, C3 activity and / or expression is reduced by at least about 50% compared to a control subject without the alteration. In any aspect of the present disclosure or embodiments thereof, C3 activity and / or expression is reduced by at least about 60% compared to a control cell or subject without the alteration. In any aspect of the disclosure or embodiments thereof, inappropriate activation of the complement system is associated with elevated levels of one or more of inflammation, the presence of autoantibodies, neurodegeneration, and microthrombosis. In any aspect of the disclosure or embodiments thereof, inappropriate activation of the complement system is associated with damage to the central nervous system (CNS), eyes, gastrointestinal system, pulmonary system, musculoskeletal system, circulatory system, integumentary system, blood cells, thyroid gland, kidneys, joints, gastrointestinal system, or transplanted organs. In any aspect of the present disclosure or embodiments thereof, the disease or disorder is selected from one or more of the following: acute antibody-mediated rejection, age-related macular degeneration, allergic bronchopulmonary aspergillosis, allergic neuritis, allergic rhinitis, amyotrophic lateral sclerosis, anaphylaxis and scleritis, atopic dermatitis, atypical hemolytic syndrome (aHUS), autoimmune hemolytic anemia, Bechet's disease, bronchiolitis, C3 glomerulopathy, central nervous system (CNS) inflammatory disorders, choroidal neovascularization (CNV), choroiditis, chronic allograft vasculopathy, chronic hepatitis, chronic muscle inflammation, chronic pain, chronic pancreatitis, chronic urticaria, Churg-Strauss syndrome (CNS), inflammatory bowel disease, ... syndrome), conjunctivitis, cyclitis, demyelinating diseases, dermatitis, dermatomyositis, diabetic retinopathy, encephalitis, eosinophilic pneumonia, geographic atrophy, giant cell arteritis, glaucoma, glomerulonephritis, graft or allograft rejection or failure, HELLP syndrome, Henoch-Schonlein purpura, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), IgA nephropathy (IgAN), inflammatory bowel disease, inflammatory arthritis, inflammatory skin diseases, infusion reactions, interstitial pneumonia, iridocyclitis, iritis, ischemia / reperfusion injury, Kawasaki disease disease), keratitis, lupus nephritis, membranoproliferative glomerulonephritis (MPGN), meningitis, microscopic polyangiitis, myasthenia gravis, myocarditis, nasal polyposis, neuromyelitis optica, neuropathic pain, ocular inflammation, osteoarthritis, pancreatitis, panniculitis, paroxysmal nocturnal hemoglobinuria (PNH), pars planitis, pemphigoid, pemphigus, polyarteritis nodosa, polymyositis, primary membranous nephropathy, proliferative vitreoretinopathy, proteinuria, psoriasis, pulmonary fibrosis, nephropathy, respiratory distress syndrome, retinal neovascularization (RNV), retinopathy of prematurity, rheumatoid arthritis (RA), sinusitis, sarcoid, sarcoidosis, scleritis, scleroderma, sclerodermatomyositis, sclerosis, Sjögren's syndrome Systemic lupus erythematosus, systemic scleroderma, Takayasu's arteritis, thyroiditis, thyroidoisis, ulcerative colitis, uveitis, vasculitis, and Wegener's granulomatosis. In any aspect of the disclosure or embodiments thereof, the administration is topical administration. In embodiments, the topical administration is to the eye, cerebrospinal fluid, or liver. In any aspect of the disclosure or embodiments thereof, the C3 polynucleotide is contacted with two or more guide RNAs or guide polynucleotides, and wherein each guide RNA or guide polynucleotide binds to a different position within the C3 polynucleotide. In any aspect of the present disclosure or embodiments thereof, the deaminase is an adenosine deaminase or a cytidine deaminase. In embodiments, the adenosine deaminase converts the target A·T in the C3 polynucleotide to G·C. In embodiments, the cytidine deaminase converts the target C·G in the C3 polynucleotide to T·A. In any aspect of the disclosure or embodiments thereof, the nucleobase change results in a premature stop codon. In any aspect or embodiment of the present disclosure, the napDNAbp domain contains Cas9, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i or Cas12j / CasΦ polynucleotides or functional portions thereof. In any aspect or embodiment of the present disclosure, the napDNAbp domain contains a Cas9 polynucleotide or functional portion thereof having endonuclease activity on both chains of a double-stranded DNA molecule. In any aspect or embodiment of the present disclosure, the napDNAbp domain contains an inactivated (dead) Cas9 (dCas9) or a Cas9 nickase (nCas9). In any aspect of the present disclosure or its embodiment, the napDNAbp domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 (Streptococcus thermophilus 1) Cas9 (St1Cas9), a modified Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In any aspect of the present disclosure or its embodiment, the napDNAbp domain contains a variant of SpCas9 with an altered protospacer adjacent motif (PAM) specificity. In any aspect of the disclosure or embodiments thereof, the cytidine deaminase domain is an APOBEC deaminase domain or a derivative thereof. In any aspect of the disclosure or embodiments thereof, the adenosine deaminase domain is a TadA deaminase domain.

[0008] In any aspect of the disclosure or embodiments thereof, the guide RNA or guide polynucleotide contains a nucleic acid analog. In any aspect of the disclosure or embodiments thereof, the guide RNA or guide polynucleotide contains one or more of 2'-OMe and phosphorothioate.

[0009] In any aspect of the present disclosure or its embodiment, the base editor further contains one or more uracil glycosylase inhibitors (UGI).In any aspect of the present disclosure or its embodiment, the base editor further contains one or more nuclear localization sequences (NLS).

[0010] In any aspect of the disclosure or embodiments thereof, the napDNAbp is a nuclease-inactive variant or a nickase variant.

[0011] In any aspect of the present disclosure or its embodiments, the deaminase domain is capable of deaminating cytidine or adenine in DNA. In any aspect of the present disclosure or its embodiments, the deaminase domain is a cytidine deaminase domain. In embodiments, the cytidine deaminase is an APOBEC deaminase or a derivative thereof. In any aspect of the present disclosure or its embodiments, the deaminase domain is an adenosine deaminase domain. In embodiments, the adenosine deaminase is a TadA*8 variant. In embodiments, the adenosine deaminase is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.1 2. TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23 or TadA*8.24. In any aspect of the present disclosure or embodiments thereof, the deaminase is an adenosine deaminase selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, and TadA*8.20_V82T_Y147T_Q154S. In any aspect of the present disclosure or embodiments thereof, the deaminase domain is a monomer or a heterodimer. In any aspect of the present disclosure or embodiments thereof, the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, TadA*8.20_V82T_Y147T_Q154S, rAPOBEC1, and ppAPOBEC. In any aspect of the disclosure or embodiments thereof, one or more guide RNAs or guide polynucleotides contain a spacer corresponding to a guide polynucleotide selected from one or more of gRNA676, gRNA661, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA3342, gRNA3343, and gRNA3345. In any aspect of the disclosure or embodiments thereof, the napDNAbp domain contains a Cas9 variant. In any aspect of the disclosure or embodiments thereof, the Cas9 variant contains one or more of the amino acid changes A1283D and E1250K relative to the SpCas9 reference amino acid sequence. In any aspect of the disclosure or embodiments thereof, the Cas9 variant contains one or more of the following combinations of amino acid changes relative to the spCas9 reference amino acid sequence: I322V, S409I, E427G, R654L, R753G, and R1114G; I322V, S409I, E427G, R654L, R753G, and R1114G; G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, and A1283D; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R220A, and R221A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D 1136Y, R1337K, A1283D, R765A, and Q768A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A, Q768A, K772A, and K775A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, R1337 In any aspect of the present disclosure or embodiments thereof, the Cas9 variant is SaCas9-KHH, SpCas9-MQKFRAER, or SpCas9-VRQR. In any aspect or embodiment thereof of the present disclosure, one or more guide RNAs or guide polynucleotides contain a nucleic acid analog. In any aspect or embodiment thereof of the present disclosure, one or more guide RNAs or guide polynucleotides contain one or more of 2'-OMe and phosphorothioate. In any aspect or embodiment thereof of the present disclosure, one or more guide RNAs or guide polynucleotides contain one of the following nucleotide sequences: End-mod SpCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUsmUsmUsmU (SEQ ID NO: 943); End-mod SaCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU (SEQ ID NO: 944); H M01: mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ IDNO: 855); H M07: mNsmNsmNsmNmNmNmNmNmNmNmNNNNNNNNNNNmGUUUUAGmAmGmCmUmAmGmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmG mGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 856); NLS (bpsv 40): mNsmNsmNsNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsmU (SEQ ID NO:857); LONG EST: mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUA AGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGUGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 859); NLS + LONG ST: mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAA GGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 860); or LONGEST + G OLD: mNsmNsmNsNNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 861). The term "mN" represents a 2'-OMe modification of a nucleotide "N", and "Ns" represents a nucleotide "N" that is linked to the following nucleotide via phosphorothioate (PS). In any aspect of the present disclosure, or embodiments thereof, one or more guide RNAs or guide polynucleotides are covalently linked to a peptide having the following amino acid sequence at the 5' end: CKRTADGSEFESPKKKRKV (SEQ ID NO: 858). In any aspect of the present disclosure or embodiments thereof, the base editor contains a linker peptide between the deaminase domain and the napDNAbp domain, wherein the linker peptide contains the amino acid sequence KGPKPKKEESEK (SEQ ID NO: 940). In any aspect of the present disclosure or embodiments thereof, one or more guide RNAs or guide polynucleotides target the base editor to effect a change in a nucleobase in the start codon of the C3 polynucleotide. In any aspect of the present disclosure or embodiments thereof, the method comprises administering to the subject a lipid nanoparticle containing one or more guide RNAs or guide polynucleotides and an mRNA molecule encoding a base editor. In any aspect of the present disclosure or embodiments thereof, the method further comprises administering to the subject a second time one or more guide RNAs or guide polynucleotides or one or more polynucleotides encoding the one or more guide RNAs or guide polynucleotides, and base editors or one or more polynucleotides encoding the base editors. The second administration is about or at least about 1 month, 6 months, or one year after the first administration. In any aspect of the present disclosure or embodiments thereof, the napDNAbp domain is a nickase. In any aspect of the present disclosure or embodiments thereof, the polynucleotide encoding the base editor is codon optimized. In any aspect of the disclosure or embodiments thereof, the kit further contains written instructions for using the kit to treat a disease or condition associated with inappropriate activation of the complement system in a subject. In any aspect or embodiments thereof provided herein, the method is not a process for modifying the germline genetic identity of a human. definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. The following references provide general definitions of many terms used in the present disclosure for technical personnel: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have their meanings as described below. By "complement component 3 (C3) polypeptide" is meant a C3 protein having at least about 85% amino acid sequence identity to Ensembl Accession No. ENSP00000245907 provided below, or a fragment thereof that is capable of activating the complement system. >ENSG00000125730:ENST00000245907.11 Peptide:ENSP00000245907 Peptide:protein_coding "Complement component 3 (C3) polynucleotide" means a nucleic acid molecule or fragment thereof that encodes a C3 polypeptide, as well as introns, exons, 3' untranslated region, 5' untranslated region, and regulatory sequences associated with its expression. In embodiments, a C3 polynucleotide is a genomic sequence, cDNA, mRNA, or gene associated with and / or required for C3 expression. An exemplary C3 nucleotide sequence from Homo sapiens is provided below (see Ensembl Accession Nos. ENST00000245907.11 and ENSG00000125730):

[0012] >ENSG00000125730:ENST00000245907.11cds:protein_coding

[0013] >19 dna: Chromosome: GRCh38:19:6677704: (SEQ 6730562: sequence shown in bold)

[1252] "Adenine" or "9H-purin-6-amine" means a purine nucleobase having the molecular formula C5H5N5, which has the structure And corresponds to CAS No. 73-24-5.

[1253] "Adenosine" or "4-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2(1H)-one" means an adenine molecule linked to a ribose sugar via a glycosidic bond, which has the structure And corresponds to CAS number 65-46-3. Its molecular formula is C 10 H 13 N5O4.

[1254] "Adenosine deaminase" or "adenine deaminase" means a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or catalyzes the hydrolytic deamination of deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). Adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism (e.g., eukaryotes, prokaryotes), including but not limited to algae, bacteria, fungi, plants, invertebrates (e.g., insects) and vertebrates (e.g., amphibians, mammals). In some embodiments, the adenosine deaminase is an adenosine deaminase variant with one or more changes, and is capable of deaminating adenine and cytosine in a target polynucleotide (e.g., DNA, RNA), and can be referred to as a "dual deaminase." Non-limiting examples of dual deaminases include those described in PCT / US22 / 22050. In some embodiments, the target polynucleotide is single-stranded or double-stranded. In some embodiments, adenosine deaminase variants can deaminate adenine and cytosine in DNA. In some embodiments, adenosine deaminase variants can deaminate adenine and cytosine in single-stranded DNA. In some embodiments, adenosine deaminase variants can deaminate adenine and cytosine in RNA. In embodiments, adenosine deaminase variants are selected from those described in PCT / US2020 / 018192, PCT / US2020 / 049975, PCT / US2017 / 045381 and PCT / US2020 / 028568, the complete contents of which are incorporated herein by reference in their entirety for all purposes. "Adenosine deaminase activity" means catalyzing the deamination of adenine or adenosine in a polynucleotide to guanine. In some embodiments, adenosine deaminase variants as provided herein retain adenosine deaminase activity (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19)). "Adenosine base editor (ABE)" means a base editor comprising an adenosine deaminase. "Adenosine base editor (ABE) polynucleotide" means a polynucleotide encoding an ABE. "Adenosine base editor 8 (ABE8) polypeptide" or "ABE8" means a base editor comprising an adenosine deaminase or an adenosine deaminase variant as defined herein, comprising one or more of the alterations listed in Table 5B, one of the combinations of alterations listed in Table 5B, or alterations at one or more of the amino acid positions listed in Table 5B, wherein such alterations are relative to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or the corresponding positions in another adenosine deaminase. In embodiments, ABE8 comprises alterations at amino acids 82 and / or 166 of SEQ ID NO: 1. In some embodiments, ABE8 comprises further alterations relative to the reference sequence, as described herein. "Adenosine Base Editor 8 (ABE8) polynucleotide" means a polynucleotide encoding an ABE8 polypeptide. "Administering" herein refers to providing one or more of the compositions described herein to a patient or subject. "Administering" herein refers to providing one or more compositions described herein to a patient or subject. For example, but not limited to, administration (e.g., injection) of the composition can be performed by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, and intrasternal infusion or injection. Alternatively or concurrently, administration can be by oral route. "Agent" means any small molecule compound, antibody, nucleic acid molecule or polypeptide, or a fragment thereof. "Alteration" means a change in the level, structure or activity of an analyte, gene or polypeptide, as detected by standard methods known in the art (such as those described herein). As used herein, alteration includes a change in expression level (e.g., an increase or decrease). In embodiments, expression level increases or decreases by 10%, 25%, 40%, 50% or more. In some embodiments, alteration includes insertion, deletion or substitution of a nucleobase or amino acid (e.g., by genetic engineering). "Ameliorate" means to reduce, inhibit, attenuate, eliminate, arrest or stabilize the development or progression of a disease. "Analog" refers to a molecule that is different but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while having certain biochemical modifications relative to the naturally occurring polypeptide that enhance the analog's function. Such biochemical modifications may increase the analog's protease resistance, membrane permeability, or half-life without altering, for example, ligand binding. Analogs may include unnatural amino acids. "Base editor (BE)" or "nucleobase editor polypeptide (NBE)" means an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase modification polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cpf1) that binds to a guide polynucleotide (e.g., a guide RNA (gRNA)). Representative nucleic acid and protein sequences of base editors include those having about or at least about 85% sequence identity to any base editor sequence provided in the sequence listing (such as those corresponding to SEQ ID NOs: 2-11). "Base editing activity" means an action to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting A·T to G·C. The term "base editor system" refers to an intermolecular complex for editing the core base of a target nucleotide sequence. In various embodiments, the base editor (BE) system includes (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating the core base in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) bound to the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system includes a core base editor domain selected from adenosine deaminase or cytidine deaminase, and a domain with nucleic acid sequence-specific binding activity. In some embodiments, the base editor system includes (1) a base editor (BE), which includes a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more core bases in the target nucleotide sequence; and (2) one or more guide RNAs bound to the nucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine or cytosine base editor (CBE). In some embodiments, the base editor system (e.g., a base editor system comprising a cytidine deaminase) comprises a uracil glycosylase inhibitor or other agents or peptides that inhibit the inosine base excision repair system (e.g., uracil stabilizing proteins (such as provided in WO2022015969, the disclosure of which is incorporated herein by reference in its entirety for all purposes)). "BE3" means a base editor comprising a cytidine deaminase domain, a nucleic acid programmable DNA binding protein domain (napDNAbp), and a single uracil glycosylase inhibitor domain. In some embodiments, npDNAbp is the SpCas9 (D10A) nickase domain. "BE4" means a base editor comprising a cytidine deaminase domain, a nucleic acid programmable DNA binding protein domain (napDNAbp), and two uracil glycosylase inhibitor domains. In some embodiments, npDNAbp is the SpCas9 (D10A) nickase domain. The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease that comprises a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9 and / or a gRNA binding domain of Cas9). Cas9 nucleases are sometimes also referred to as casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. The term "conservative amino acid substitution" or "conservative mutation" refers to an amino acid that is replaced by another amino acid with a common property. A functional approach to defining the common property between individual amino acids is to analyze the normalized frequency of amino acid changes between the corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to this type of analysis, amino acid groups can be defined, in which the amino acids within the group are preferentially exchanged with each other and are therefore most similar to each other in terms of their impact on overall protein structure (Schulz, GE and Schirmer, RH, the same). Non-limiting examples of conservative mutations include amino acid substitutions, such as lysine replacing arginine, and conversely, making it possible to maintain a positive charge; glutamic acid replacing aspartic acid, and conversely, making it possible to maintain a negative charge; serine replacing threonine, making it possible to maintain free-OH; and glutamine replacing asparagine, making it possible to maintain free-NH2.

[1255] The terms "coding sequence" or "protein coding sequence", used interchangeably herein, refer to a segment of a polynucleotide that encodes a protein. A coding sequence may also be referred to as an open reading frame. The region or sequence is bounded by a start codon closer to the 5' end and a stop codon closer to the 3' end. Useful stop codons for the base editors described herein include the following: TAG, TAA, and TGA.

[1256] "Complex" means a combination of two or more molecules, the interaction of which depends on intermolecular forces. Non-limiting examples of intermolecular forces include covalent and non-covalent interactions. Non-limiting examples of non-covalent interactions include hydrogen bonds, ionic bonds, halogen bonds, hydrophobic bonds, van der Waals interactions (e.g., dipole-dipole interactions, dipole-induced dipole interactions, and London dispersion forces) and π-effects. In one embodiment, the complex comprises a polypeptide, a polynucleotide, or a combination of one or more polypeptides and one or more polynucleotides. In one embodiment, the complex comprises one or more polypeptides (e.g., a base editor comprising a nucleic acid programmable DNA binding protein such as Cas9 and a deaminase) and a polynucleotide (e.g., a guide RNA) that associate to form a base editor. In one embodiment, the complex is held together by hydrogen bonds. It should be understood that one or more components of a base editor (e.g., a deaminase or a nucleic acid programmable DNA binding protein) may be covalently or non-covalently associated. As an example, a base editor may include a deaminase covalently linked (e.g., by a peptide bond) to a nucleic acid programmable DNA binding protein. Alternatively, the base editor may comprise a non-covalently associated deaminase and a nucleic acid programmable DNA binding protein (e.g., wherein one or more components of the base editor are provided in trans and associated directly or via another molecule such as a protein or nucleic acid). In one embodiment, one or more components of the complex are held together by hydrogen bonds.

[1257] "Cytosine" or "4-aminopyrimidin-2(1H)-one" means a purine nucleobase having the molecular formula C4H5N3O, which has the structure And corresponds to CAS No. 71-30-7.

[1258] "Cytidine" means a cytosine molecule linked to a ribose sugar via a glycosidic bond, which has the structure And corresponds to CAS number 65-46-3. Its molecular formula is C9H 13 N3O5.

[1259] "Cytidine base editor (CBE)" means a base editor comprising a cytidine deaminase.

[1260] "Cytidine base editor (CBE) polynucleotide" means a polynucleotide encoding a CBE.

[1261] "Cytidine deaminase" or "cytosine deaminase" means a polypeptide or fragment thereof that is capable of deaminating cytidine or cytosine. In an embodiment, cytidine or cytosine is present in a polynucleotide. In one embodiment, the cytidine deaminase converts cytosine to uracil or converts 5-methylcytosine to thymine. The terms "cytidine deaminase" and "cytosine deaminase" are used interchangeably throughout the application. Sea lamprey (Petromyzon marinus) cytosine deaminase 1 (PmCDA1) (SEQ ID NOs: 13-14), activation-induced cytidine deaminase (AICDA) (SEQ ID NOs: 15-21), and APOBEC (SEQ ID NOs: 12-61) are exemplary cytidine deaminases. Other exemplary cytidine deaminase (CDA) sequences are provided in the sequence listing as SEQ ID NOs: 62-189. Non-limiting examples of cytidine deaminases include those described in PCT / US20 / 16288, PCT / US2018 / 021878, 180802-021804 / PCT, PCT / US2018 / 048969, and PCT / US2016 / 058344. "Cytosine deaminase activity" means catalyzing the deamination of cytosine or cytidine. In one embodiment, the polypeptide with cytosine deaminase activity converts an amino group into a carbonyl group. In one embodiment, cytosine deaminase converts cytosine into uracil (i.e., C to U) or converts 5-methylcytosine into thymine (i.e., 5mC to T). In some embodiments, a cytosine deaminase as provided herein has increased cytosine deaminase activity relative to a reference cytosine deaminase (e.g., at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more).

[1262] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or fragment thereof that catalyzes a deamination reaction.

[1263] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, sequence changes in a polynucleotide or polypeptide are detected. In another embodiment, the presence of an indel is detected. "Detectable label" means a composition that, when attached to a molecule of interest, renders the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens. "Disease" means any illness or condition that damages or interferes with the normal function of a cell, tissue, or organ. Exemplary diseases include diseases that can be treated by introducing changes to a complement component 3 polynucleotide in a cell, thereby resulting in a decrease in the activity and / or expression of a complement 3 polypeptide in the cell. In some cases, the disease is a disease associated with inappropriate activation of the complement system in a subject. Non-limiting examples of diseases associated with inappropriate activation of the complement system include blood disorders, transplantation or graft rejection, inflammatory diseases or conditions, eye diseases or conditions, kidney diseases or conditions, cardiac disorders, respiratory diseases or conditions, autoimmune disorders, inflammatory bowel disease or conditions, arthritis, neurodegenerative diseases or conditions, musculoskeletal diseases or conditions associated with inflammation, disorders affecting the epidermal system, diseases or conditions affecting the central nervous system, diseases or conditions affecting the circulatory system, diseases or conditions affecting the gastrointestinal system, diseases or conditions affecting the thyroid gland, chronic pain, allergies, and lung disease. Other non-limiting examples of diseases associated with inappropriate activation of the complement system include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic syndrome (aHUS), HELLP syndrome, autoimmune hemolytic anemia, transplant rejection, ischemia / reperfusion injury, transplant injury, hyperacute rejection, transplant rejection or failure, acute antibody-mediated rejection, chronic inflammation, chronic allograft vasculopathy, chronic rejection of a transplant or graft, age-related macular degeneration (e.g., wet or dry age-related macular degeneration), diabetic retinopathy, glaucoma, uveitis, autoimmune diseases, myasthenia gravis, neuromyelitis optica (NMO), kidney disease, membranoproliferative glomerulonephritis (MPGN) (e.g., MPGN IgA nephropathy (IgAN), primary membranous nephropathy, C3 glomerulopathy, proteinuria, neurodegenerative diseases, neuropathic pain, sinusitis, nasal polyposis, cancer, sepsis, respiratory distress syndrome, allergic reaction, infusion reaction, respiratory disease or disorder (e.g., asthma or chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis or asthma), Th2-related disorders (e.g., disorders associated with high levels or high activation of CD4+ helper T cells of the Th2 subtype), disorders associated with high levels or inappropriate activity of CD4+ helper T cells of the Th17 subtype, inflammatory bowel disease (e.g.,Crohn's disease or ulcerative colitis), inflammatory skin diseases, chronic inflammatory diseases, psoriasis, atopic dermatitis, systemic scleroderma, sclerosis, Behçet's disease, dermatomyositis, polymyositis, multiple sclerosis (MS), dermatitis, meningitis, encephalitis, uveitis, osteoarthritis, lupus nephritis, rheumatoid arthritis (RA), Sjögren's syndrome, vasculitis, inflammatory disorders of the central nervous system (CNS), chronic hepatitis, chronic pancreatitis, glomerulonephritis, sarcoidosis, thyroiditis, pathological immune response to tissue / organ transplantation, bronchiolitis, Hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), periodontitis, gingivitis, disorders associated with excessive or inappropriate activity of IgE-producing cells, neuromyelitis optica, pemphigoid, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic pneumonitis, interstitial pneumonia, sarcoidosis, Wegener's granulomatosis, bronchiolitis obliterans, allergic rhinitis, inflammatory joint disorders (e.g., arthritis such as rheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis, spondyloarthropathies, Reither's syndrome syndrome or gout), dermatomyositis, polymyositis, chronic muscle inflammation, pemphigus, systemic lupus erythematosus, dermatomyositis, scleroderma, sclerodermatomyositis, Sjögren's syndrome, chronic urticaria, demyelinating diseases, amyotrophic lateral sclerosis, chronic pain, stroke, allergic neuritis, Huntington's disease, Alzheimer's disease, Parkinson's disease, circulatory system diseases, polyarteritis nodosa, Wegener's granulomatosis, giant cell arteritis, Chargar-Strauss syndrome, microscopic polyangiitis, Henoch-Schonlein purpura, Takayasu arteritis, Kawasaki disease, Behçet's disease, ulcerative colitis, thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease) disease), postpartum thyroiditis), myocarditis, hepatitis (e.g., hepatitis C), pancreatitis, glomerulonephritis (e.g., membranoproliferative glomerulonephritis or membranous glomerulonephritis), panniculitis, eye diseases, choroidal neovascularization (CNV), retinal neovascularization (RNV), eye inflammation, retinopathy of prematurity, proliferative vitreoretinopathy, uveitis, keratitis, conjunctivitis and scleritis, geographic atrophy, conjunctivitis, keratitis, scleritis, iritis, iridocyclitis, cyclitis, pars planitis, choroiditis, persistent asthma, and allergic asthma. "Dual editing activity" or "dual deaminase activity" means having adenosine deaminase activity and cytidine deaminase activity. In one embodiment, the base editor with dual editing activity has both A→G and C→T activity, wherein the two activities are approximately equal or within about 10% or 20% of each other. In another embodiment, the A→G activity of the dual editor is no more than about 10% or 20% higher than the C→T activity. In another embodiment, the A→G activity of the dual editor is no more than about 10% or 20% lower than the C→T activity. In some embodiments, the adenosine deaminase variant mainly has cytosine deaminase activity and has almost no adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity and has no significant or detectable adenosine deaminase activity. "Effective amount" means the amount of an agent or active compound (e.g., a base editor as described herein) required to improve the symptoms of a disease relative to an untreated patient or an individual not suffering from the disease (i.e., a healthy individual), or the amount of an agent or active compound sufficient to induce a desired biological response. The effective amount of the active compound used to practice the present disclosure for treating a disease varies according to the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective" amount. In one embodiment, the effective amount is the amount of the base editor of the present disclosure that is sufficient to introduce a change in a gene of interest in a cell (e.g., in vitro or in vivo). In one embodiment, the effective amount is the amount of the base editor required to achieve a therapeutic effect. Such a therapeutic effect does not need to be sufficient to change the pathogenic genes in all cells of the subject, tissue, or organ, but only needs to change the pathogenic genes in about 1%, 5%, 10%, 25%, 50%, 75% or more cells present in the subject, tissue, or organ. In one embodiment, the effective amount is sufficient to improve one or more symptoms of the disease. "Fragment" means a portion of a polypeptide or nucleic acid molecule. The portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the reference nucleic acid molecule or polypeptide full length. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides or amino acids. In some embodiments, a fragment is a functional fragment. "Guiding polynucleotide" means a polynucleotide or polynucleotide complex that is specific to a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guiding polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. "Hybridization" means hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds. "Inappropriate activation" in the context of C3 means any increase in complement activation associated with a disease or condition. In one embodiment, inappropriate activation is activation that is increased or elevated locally (e.g., in an organ or tissue, such as in the central nervous system or eye) or systemically relative to a healthy reference (e.g., a healthy subject). In some cases, "inappropriate activation" is activation associated with chronic (e.g., lasting more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks) inflammation in a subject. In some cases, inappropriate activation is activation that is directed to a tissue, cell, or organ of a subject and / or causes undesirable damage to a tissue, cell, or organ of a subject. In embodiments, a disease or condition associated with inappropriate activation of the complement system can be treated by any of the methods or compositions provided herein to reduce or eliminate the expression and / or activity of a C3 polypeptide. In one embodiment, complement activation is detected by measuring the levels of C3 polypeptide and / or cleaved C3 polypeptide (e.g., C3a fragment or C3b fragment), where inappropriate activation can be determined as high levels of C3 polypeptide and / or cleaved C3 polypeptide relative to healthy reference subjects. By "increase" is meant a positive change of at least 10%, 25%, 50%, 75%, or 100%, or about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or about 100-fold. The term "inhibitor of base repair" or "base repair inhibitor" or "IBR" or their grammatical equivalents refers to a protein that is capable of inhibiting the activity of a nucleic acid repair enzyme (eg, a base excision repair enzyme). An "intein" is a fragment of a protein that is capable of self-cleavage and joining the remaining fragment (the extein) with peptide bonds in a process known as protein splicing. The terms "isolated," "purified," or "biologically pure" refer to a substance that is free to varying degrees from components that normally accompany it as found in its native state. "Isolate" indicates a degree of separation from the original source or surrounding environment. "Purified" indicates a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, the nucleic acids and peptides of the present disclosure are purified if they are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces essentially a single band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications may produce different isolated proteins that can be purified separately. "Isolated polynucleotide" means a nucleic acid molecule that is free of the genes that flank the genes in the naturally occurring genome of the organism from which the nucleic acid molecule of the present disclosure is derived. Thus, this term includes, for example, recombinant DNA that is incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). In addition, this term includes RNA molecules transcribed from recombinant DNA molecules as well as those that are part of a hybrid gene encoding another polypeptide sequence. "Isolated polypeptide" means a polypeptide of the present disclosure that has been separated from its naturally associated components. Typically, a polypeptide is isolated when it is at least 60% free by weight of proteins and naturally occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, or at least 99% by weight of a polypeptide of the present disclosure. Isolated polypeptides of the present disclosure can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding such polypeptides, or by chemically synthesized proteins. Purity can be measured by any appropriate method (e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis). As used herein, the term "linker" refers to a molecule that connects two moieties. In one embodiment, the term "linker" refers to a covalent linker (eg, a covalent bond) or a non-covalent linker. "Marker" means any protein or polynucleotide that has an alteration in expression, level, structure, or activity associated with a disease or condition. In embodiments, the disease or condition is associated with inappropriate activation of the complement system. In some cases, the marker is a C3 polynucleotide or polypeptide. As used herein, the term "mutation" refers to a residue in a sequence (e.g., nucleic acid or amino acid sequence) that is replaced by another residue, or a deletion or insertion of one or more residues in the sequence. Mutation is generally described herein by identifying the original residue, then identifying the position of the residue in the sequence, and then identifying the identity of the newly substituted residue. The various methods for carrying out amino acid substitutions (mutations) provided herein are well known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds comprising core bases and acidic moieties (e.g., polymers of nucleosides, nucleotides or nucleotides). Typically, polymeric nucleic acids (e.g., nucleic acid molecules comprising three or more nucleotides) are linear molecules in which adjacent nucleotides are interconnected via phosphodiester bonds. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more single nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably and refer to polymers of nucleotides (e.g., strings of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. Nucleic acid can be naturally occurring, for example, in the case of genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, for example recombinant DNA or RNA, artificial chromosome, engineered genome or its fragment, or synthetic DNA, RNA, DNA / RNA hybrid, or comprise non-naturally occurring nucleotides or nucleosides.In addition, term " nucleic acid ", " DNA ", " RNA " and / or similar term comprise nucleic acid analogs, for example have the analog of the component except phosphodiester backbone.Nucleic acid can be purified from natural origin, uses recombinant expression system to produce and optionally purifying, chemosynthesis etc.In appropriate cases, for example, in the case of chemosynthetic molecule, nucleic acid comprises nucleoside analogs, such as the analog with chemically modified base or sugar and backbone modification.Unless otherwise indicated, otherwise nucleotide sequence presents with 5 ' to 3 ' direction. In some embodiments, the nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages). The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to an amino acid sequence that facilitates the import of proteins into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application No. PCT / EP2000 / 011690, filed November 23, 2000, by Plank et al. and published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, such as described by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 190), KRPAATKKAGQAKKKK (SEQ ID NO: 191), KKTELQTTNAENKTKKL (SEQ ID NO: 192), KRGINDRNFWRGENGRKTR (SEQ ID NO: 193), RKSGKIAAIVVKRPRK (SEQ ID NO: 194), PKKKRKV (SEQ ID NO: 195), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 196), PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328), or RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 329). The terms "nucleobase," "nitrogenous base," or "base," used interchangeably herein, refer to nitrogenous biological compounds that form nucleosides, which in turn are components of nucleotides. The ability of nucleobases to form base pairs and stack on each other directly leads to long helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as primary or canonical. Adenine and guanine are derived from purines, and cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other modified (non-basic) bases. Non-limiting examples of modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxymethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagens, and both are produced by deamination (replacing an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by deamination of cytosine. "Nucleoside" consists of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). "Nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Non-limiting examples of modified nucleobases and / or chemical modifications that a modified nucleobase may comprise are as follows: pseudouridine, 5-methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, 2'-O-methylthioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), constrained ethyl (S-cEt), 2'-O-methyl ('M'), 2'-O-methyl-3'-phosphorothioate ('MS'), 2'-O-methyl-3'-phosphorothioate ('MSP'), 5-methoxyuridine, phosphorothioate, and N1-methylpseudouridine. The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or a guide polynucleotide (e.g., gRNA) that directs the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can associate with a guide RNA that directs the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ (Cas12j / Casphi).Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas12j / CasΦ, Cpf1, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm 6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they may not be specifically listed in the present disclosure. See, e.g., Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type VCRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are hereby incorporated by reference. Exemplary nucleic acid programmable DNA binding proteins and nucleic acid sequences encoding nucleic acid programmable DNA binding proteins are provided in the sequence listing as SEQ ID NOs: 197-245, 254-260, and 378. As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and deamination of adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase). As used herein, "obtaining" in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent. "Subject" or "patient" means a mammal, including but not limited to a human or non-human mammal. In embodiments, the mammal is a bovine, equine, canine, ovine, rabbit, rodent, non-human primate, or feline. In one embodiment, a "patient" refers to a mammalian subject who has a higher than average likelihood of developing a disease or condition. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that can benefit from the treatments disclosed herein. Exemplary human patients can be male and / or female. A "patient in need thereof" or "subject in need thereof" as used herein refers to a patient diagnosed with, at risk of or having, predetermined to have, or suspected of having a disease or condition. The terms "pathogenic mutation," "pathogenic variant," "disease causing mutation," "disease-causing variant," "deleterious mutation," or "predisposing mutation" refer to genetic changes or mutations that are associated with a disease or condition or increase an individual's susceptibility or vulnerability to a disease or condition. In some embodiments, the pathogenic mutation comprises a substitution of at least one wild-type amino acid with at least one pathogenic amino acid in the protein encoded by the gene. In some embodiments, the pathogenic mutation is in a termination region (e.g., a stop codon). In some embodiments, the pathogenic mutation is in a non-coding region (e.g., an intron, a promoter, etc.). By "Borneo orangutan (Pongo pygmaeus) (Orangutan) APOBEC (ppAPOBEC) polypeptide" is meant a cytidine deaminase polypeptide having at least about 85% amino acid sequence identity to the exemplary ppAPOBEC polypeptide sequences provided below, or a fragment thereof having cytidine deaminase activity. Exemplary ppAPOBEC polypeptide sequences: MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR (SEQ ID NO: 941). By "Borneo orangutan (Pongo pygmaeus) (Orangutan) APOBEC (ppAPOBEC) polynucleotide" is meant a nucleic acid molecule or fragment thereof that encodes a ppAPOBEC polypeptide, as well as introns, exons, 3' untranslated region, 5' untranslated region, and regulatory sequences associated with its expression. In embodiments, the ppAPOBEC polynucleotide is a genomic sequence, cDNA, mRNA, or gene associated with and / or required for ppAPOBEC expression. Exemplary ppAPOBEC nucleotide sequences are provided below. Exemplary ppAPOBEC polynucleotide sequences: ATGACCTCTGAGAAGGGCCCTAGCACAGGCGACCCCACCCTGCGGCGGAGAATCGAGAGCTGGGAGTTCGACGTGTTCTACGACCCTAGAGAACTGAGAAAGGAAACCTGCCTGCTGTACGAGATCAAGTGGGGCATGAGCAGAAAGATCTGGCGGAGCTCTGGCAAGAACACCACC AACCACGTGGAAGTGAATTTCATCAAGAAGTTCACCAGCGAGAGAAGGTTCCACAGCAGCATCAGCTGCAGCATCACCTGGTTCCTGAGCTGGTCCCCTTGCTGGGAATGCAGCCAGGCCATCAGAGAGTTCCTGAGCCAACACCCCGGAGTGACACTGGTGATCTACGTGGCCAGA CTGTTCTGGCACATGGACCAGAGAAACAGACAGGGCCTGAGAGATCTGGTCAACAGCGGCGTGACTATCCAGATCATGCGGGCCAGCGAGTACTACCACTGTTGGCGGAACTTCGTGAACTACCCCCCCGGCGATGAGGCCCACTGGCCTCAGTACCCTCCTCTGTGGATGATGCTG TACGCCCTGGAACTGCACTGCATCATCCTGTCTCTGCCTCCATGTCTGAAGATCTCTAGAAGATGGCAGAACCACCTGGCCTTCTTCAGACTGCACCTGCAGAATTGCCACTACCAGACCATCCCCCCCCACATCCTGCTGGCTACAGGCCTGATCCACCCTTCTGTGACCTGGAGA (SEQ ID NO: 942). The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature but is a product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence. By "reduction" is meant a negative change of at least 10%, 25%, 50%, 75% or 100%. So-called " reference " means standard or control conditions. In one embodiment, the reference is wild type or healthy cells. In other embodiments and without limitation, the reference is untreated cells that have not been subjected to test condition treatment, or subjected to placebo or normal saline, culture medium, buffer and / or control vector treatment without the polynucleotide of interest. In embodiments, the reference is a healthy subject or cell without inappropriate activation of the complement system. In some cases, the reference is unedited or untreated cells (e.g., hepatocytes), tissues (e.g., components of the central nervous system or organs such as liver, eyes,) and / or subjects. A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will typically be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will typically be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer therebetween. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein. The terms "RNA programmable nuclease" and "RNA-guided nuclease" refer to nucleases that form a complex (e.g., bind or associate) with one or more RNAs that are not cleavage targets. In some embodiments, when in a complex with RNA, the RNA programmable nuclease may be referred to as a nuclease-RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Csnl) from Streptococcus pyogenes (e.g., SEQ ID NO: 197), Cas9 (NmeCas9; SEQ ID NO: 208) from Neisseria meningitidis, Nme2Cas9 (SEQ ID NO: 209), Streptococcus constellatus (Streptococcus constellatus) (ScoCas9) or a derivative thereof (e.g., a sequence having at least about 85% sequence identity to Cas9, such as Nme2Cas9 or spCas9). Amino acids can generally be divided into groups based on the following common side chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, He; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. In some embodiments, conservative substitutions may include exchanging a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions may include exchanging a member of one of these classes for another class. The term "single nucleotide polymorphism (SNP)" is a variation of a single nucleotide occurring at a specific position in the genome, wherein each variation exists to a certain extent in the population (e.g., >1%). SNPs can be located in the coding region of a gene, the non-coding region of a gene, or the intergenic region (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs within the coding sequence do not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs in the coding region: synonymous and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not in the protein coding region can still affect gene splicing, transcription factor binding, messenger RNA degradation, or non-coding RNA sequence. Gene expression affected by such SNPs is referred to as eSNP (expression SNP) and can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations of a single nucleotide, do not have any frequency restrictions, and can occur in somatic cells. Somatic single nucleotide variations can also be referred to as single nucleotide changes. "Specifically binds" means a nucleic acid molecule, polypeptide, polypeptide / polynucleotide complex, compound or molecule that recognizes and binds a polypeptide and / or nucleic acid molecule of the present disclosure but does not substantially recognize and bind other molecules in a sample (e.g., a biological sample). "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence. In one embodiment, the reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, the reference sequence is any of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence is at least about 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or even 99.99% identical to the sequence being compared at the amino acid level or nucleic acid level. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software assigns degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Nucleic acid molecules that can be used in the disclosed methods include any nucleic acid molecules encoding polypeptides of the present disclosure or their functional fragments. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will generally exhibit substantial identity. Polynucleotides having "substantial identity" to the endogenous sequence are generally capable of hybridizing with at least one chain of a double-stranded nucleic acid molecule. Nucleic acid molecules that can be used in the disclosed methods include any nucleic acid molecules encoding polypeptides of the present disclosure or their functional fragments. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will generally exhibit substantial identity. Polynucleotides having "substantial identity" to the endogenous sequence are generally capable of hybridizing with at least one chain of a double-stranded nucleic acid molecule. "Hybridization" means pairing under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). "Split" means to separate into two or more fragments. A "split polypeptide" or "split protein" refers to a protein provided as an N-terminal fragment and a C-terminal fragment that are translated into two separate polypeptides from a nucleotide sequence. The polypeptides corresponding to the N-terminal and C-terminal portions of the split protein can be spliced ​​in some embodiments to form a "reconstructed" protein. In an embodiment, the split polypeptide is a nucleic acid programmable DNA binding protein (e.g., Cas9) or a base editor. The term "target site" refers to a sequence within a modified nucleic acid molecule. In embodiments, the modification is deamination of a base. The deaminase can be a cytidine or adenine deaminase. A fusion protein or base editing complex comprising a deaminase can include a dCas9-adenosine deaminase fusion protein, a Cas12b-adenosine deaminase fusion, or a base editor disclosed herein. As used herein, the term "treat / treating / treatment, etc." refers to reducing or ameliorating a condition and / or symptoms associated therewith or obtaining a desired pharmacological and / or physiological effect. It will be understood that treating a condition or illness does not require the complete elimination of the condition, illness, or symptoms associated therewith, although this is not excluded. In some embodiments, the effect is therapeutic, i.e., but not limited to, the effect partially or completely reduces, weakens, eliminates, alleviates, relieves, reduces the intensity of the disease and / or adverse symptoms attributable to the disease, or cures the disease and / or adverse symptoms attributable to the disease. In some embodiments, the effect is preventive, i.e., the effect protects or prevents the occurrence or recurrence of a disease or illness. To this end, the methods disclosed herein comprise administering a therapeutically effective amount of a composition as described herein. So-called "uracil glycosylase inhibitors" or "UGIs" refer to agents that inhibit the uracil excision repair system. A base editor comprising a cytidine deaminase converts cytosine to uracil, which is then converted to thymine by DNA replication or repair. In various embodiments, uracil DNA glycosylase (UGI) prevents base excision repair that changes U back to C. In some cases, cells and / or polynucleotides are contacted with UGI and a base editor to prevent base excision repair that changes U back to C. An exemplary UGI comprises the following amino acid sequence: >splP14739IUNGI_BPPB2 uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQID NO: 231). In some embodiments, the agent that inhibits the uracil excision repair system is uracil stabilizing protein (USP). See, for example, WO 2022015969 A1, which is incorporated herein by reference. 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. As used herein, the term "vector" refers to a means for introducing a nucleic acid molecule into a cell to thereby obtain a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, lipid nanoparticles, and episomes. Recitation of a list of chemical groups in any definition of a variable herein includes defining the variable as any single group or combination of listed groups. Reference herein to an embodiment of a variable or aspect includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. All terms should be understood as they are understood by those skilled in the art. Unless defined otherwise, 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 disclosure belongs. In this application, the use of the singular includes the plural, unless otherwise specifically stated. It must be noted that, as used in this specification, the singular forms "a (kind)" and "the (said)" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms such as "include", "includes" and "included" is not limited. As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended. This wording indicates the presence of specified elements, features, components, and / or method steps, but does not exclude the presence of other elements, features, components, and / or method steps. In some embodiments, any embodiment designated as "comprising" a particular component or element is also contemplated as "consisting of" or "consisting essentially of the particular component or element. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure. The term "about" or "approximately" means that the particular value is within an acceptable error range as determined by one of ordinary skill in the art, which will depend in part on the manner in which the value is measured or determined, ie, the limitations of the measurement system. Reference in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least some of the embodiments, but not necessarily all embodiments. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A bar graph is provided showing the A>G base editing rate of complement component 3 (C3) when the indicated guides were screened with the indicated ABE8.8 base editors at the corresponding target site knockout. The dotted line indicates that the editing rate is 40%. Figure 2 A bar graph is provided showing the C>T base editing rate at the relevant target site for the indicated guides of complement component 3 (C3) screened with the indicated BE4 base editors. The dotted line indicates that the editing rate is 50%. Figure 3 A bar graph is provided showing the concentration of human C3 protein measured by ELISA in PXB cell hepatocyte supernatants 2 days prior to transfection. Each condition was run in triplicate, as indicated by each point in the graph. The bars show the mean C3 protein concentration, and the error bars represent the standard deviation. Figure 4A bar graph is provided showing the concentration of human C3 protein measured by ELISA in PXB cell hepatocyte supernatants 7 days after transfection. Each condition was run in triplicate, as indicated by each point in the graph. The bars show the mean C3 protein concentration, and the error bars represent the standard deviation.

[1264] Figure 5 Provided are overlapping bar graphs and graphs showing the editing rate at the target site in PXB cell hepatocytes assessed by next-generation DNA sequencing (NGS) 16 days after transfection (squares, right axis) and the human C3 protein concentration assessed by ELISA 13 days after transfection (columns, left axis). Each condition was run in triplicate as indicated by each point in the figure. The dotted line represents the average human C3 protein concentration of cells contacted with sgRNA_088_ABE8.8_SpCas9.

[1265] Figure 6 Provided are overlaid bar graphs and graphs showing the editing rate at the targeted site in PXB cell hepatocytes assessed by NGS 16 days after transfection (squares, right axis) and the human C3 protein concentration assessed by ELISA 16 days after transfection (bars, left axis). Each condition was run in triplicate as indicated by each point in the figure. The dotted line represents the average human C3 protein concentration of cells contacted with sgRNA_088_ABE8.8_SpCas9.

[1266] Figure 7 A bar graph is provided showing the A>G base editing rates at the relevant target sites for the indicated complement component 3 (C3) knockout (KO) guides screened with five ABE8 deaminase variants. The deaminase variants were tested in three independent experiments, which are separated by two gaps in the bar graph. Each editing experiment included a positive control sgRNA_088_ABE8.8_SpCas9 condition. Figure 7 In the figure, each group of five bars corresponds to ABE8.8, ABE8.17, ABE8.13, ABE8.20 and ABE8.20_V82T from left to right.

[1267] Figure 8A bar graph is provided showing the A>G base editing rates at the relevant target sites for the specified C3KO guides with different spacer lengths. The standard spacer length for all guides was 20 bp. The guides were tested in two independent experiments separated from each other by a gap in the bar graph. Each editing experiment included a positive control sgRNA_088_ABE8.8_SpCas9 condition. The gRNA676 and gRNA696 spacer length series were ordered from Synthego, and the remaining spacer length series and positive controls were ordered from IDT. sgRNA_088 has a standard spacer length of 20 bp and was ordered from IDT. Figure 8 In the figure, each group of five bars corresponds to 20 bp, 21 bp, 22 bp, 23 bp and 19 bp from left to right.

[1268] Figure 9 A bar graph is provided showing the concentration of human complement component 3 (C3) protein measured by ELISA in PXB cell hepatocyte supernatants immediately before transfection. Each condition was run in triplicate. The condition transfected with 800 ng of total RNA is represented by dots, and the condition transfected with 1200 ng of total RNA is represented by squares. The bars represent the mean C3 protein concentration, and the error bars represent the standard deviation. Figure 10 Overlaid bar graphs and graphs are provided showing the editing rate at the target site in PXB cell hepatocytes assessed by NGS 13 days after transfection (diamonds, right axis) and the human C3 protein concentration assessed by ELISA 13 days after transfection (columns, left axis). Each condition was run in triplicate as indicated at each point in the figure. The dotted line represents the average human C3 protein concentration of cells exposed to sgRNA_088_ABE8.8_SpCas9 at a transfection dose of 1200 ng. Figure 10 In the case of , a single column corresponds to 800 ng. Figure 10 For the pair of bars shown in , the left bar corresponds to 800 ng and the right bar corresponds to 1200 ng. Figure 11 A bar graph is provided showing human C3 protein concentrations measured by ELISA in PHH co-culture supernatants prior to transfection. Each condition was run in triplicate. The conditions involving transfection with 800 ng of total RNA are represented by dots, and the conditions involving transfection with 1200 ng of total RNA are represented by squares. The bars represent the mean C3 protein concentration, and the error bars represent the standard deviation. Figure 12Overlaid bar graphs and graphs are provided showing the editing rate at the targeted site in primary human hepatocyte (PHH) co-cultures assessed by NGS 13 days after transfection (diamonds, right axis) and the human C3 protein concentration assessed by ELISA 13 days after transfection (bars, left axis). Each condition was run in duplicate or triplicate as indicated for each point in the figure. The dotted line represents the average human C3 concentration of cells exposed to sgRNA_088_ABE8.8_SpCas9 at a transfection dose of 1200 ng. Figure 12 In the case of , a single column corresponds to 800 ng. Figure 12 For the pair of bars shown in , the left bar corresponds to 800 ng and the right bar corresponds to 1200 ng. Figure 13 A bar graph is provided showing the target A>G base editing rate at the targeted site in primary cynomolgus monkey hepatocyte (PCH) monolayers assessed by next-generation DNA sequencing (NGS) 3 days after transfection. Each condition was run in triplicate as indicated by each point in the figure. Error bars represent standard deviations. Figure 14 A bar graph is provided showing the concentration of cynomolgus monkey C3 protein measured by ELISA in primary cynomolgus monkey hepatocyte co-culture supernatants before transfection. Each condition was run in triplicate, as indicated by each point in the graph. The bars show the mean C3 protein concentration, and the error bars represent the standard deviation. Figure 15 Provided are overlaid bar graphs and graphs showing the editing rate at the target site in primary cynomolgus monkey hepatocyte co-cultures assessed by NGS 13 days after transfection (squares, right axis) and cynomolgus monkey C3 protein concentration assessed by ELISA 7 days after transfection (bars, left axis). Each condition was run in triplicate as indicated by each point in the figure. The dotted line represents the average cynomolgus monkey C3 concentration of cells contacted with sgRNA_088_ABE8.8_SpCas9. Figure 16 Provided are overlapping bar graphs and graphs showing the editing rate at the target site in primary cynomolgus monkey hepatocyte co-cultures assessed by NGS 13 days after transfection (squares, right axis) and the cynomolgus monkey C3 protein concentration assessed by ELISA 13 days after transfection (bars, left axis). Each condition was run in triplicate as indicated by each point in the figure. The dotted line represents the average cynomolgus monkey C3 concentration of cells contacted with sgRNA_088_ABE8.8_SpCas9. Figure 17A bar graph is provided showing the base editing rates of C3 guides designed to disrupt opsonization. On-target base editing rates are in black and represent the editing efficiency at the site of interest indicated below the sample name. Bystander editing rates are in gray and represent the maximum editing rate detected within the protospacer at sites other than the site indicated below the sample name. Figure 18 A bar graph is provided showing the base editing rates of C3 guides designed to disrupt C3 convertase cleavage of C3. Mutation #1 (black) indicates the editing efficiency at the first mutated residue of this guide, see the "Mutated Residue" column in Table 2. Mutation #2 (grey) indicates the editing efficiency at the second mutated residue of this guide, see the "Mutated Residue" column in Table 2. Figure 19 A depiction of the chemical structure of pegacetacoplan ("APL2") is provided, assuming that n is between about 800 and 1100 and that the polyethylene glycol (PEG) is about 40 kD. Figure 19 The two amino acid sequences depicted in the chemical structure of each correspond to SEQ ID NO: 854. Figure 20 Bar graphs are provided showing target A>G % base editing and human C3 protein concentrations (normalized to day zero (0) concentrations) in FRG liver humanized mice administered the indicated base editor systems at the indicated total combined doses of guide polynucleotide RNA and mRNA encoding the base editors on day 14 post-transfection. Figure 20 Each shaded column in the corresponds to an hC3 protein concentration measurement, and each set of data points to the left of each column (whose mean is represented by a horizontal line) corresponds to an on-target base editing measurement. Figure 21 A bar graph is provided showing C3 RNA levels normalized to GAPDH RNA levels in FRG liver humanized mice administered the indicated base editor systems at the indicated total combined doses of guide polynucleotide RNA and mRNA encoding the base editor. Figure 21 Each set of three bars in the , from left to right, corresponds to the levels of mRNA detected using probes spanning exons 4-5 (first set of 3 bars), exons 19-20 (second set of 3 bars), exons 27-38 (third set of 3 bars), and exons 38-39 (fourth set of 3 bars). Figure 22 Provided are bar graphs showing target A>G % base editing and human C3 protein concentrations (normalized to day zero (0) concentrations) in FRG liver humanized mice administered the indicated base editor systems at day 14 post-transfection. Figure 22Each shaded column in the corresponds to an hC3 protein concentration measurement, and each set of data points to the left of each column (whose mean is represented by a horizontal line) corresponds to an on-target base editing measurement. Figure 23 Graphs are provided showing on-target A>G base editing in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) at the indicated doses of the indicated base editor systems, where "base editing dose" represents the combined mass of the administered guide polynucleotide and mRNA encoding the base editor. Figure 24 Provided are graphs showing the maximum A>G base editing in HEK293T cells administered the indicated base editor systems using the indicated doses of guide polynucleotides (gRNA). Figure 25 Provided are bar graphs showing target A>G base editing (%) in primary human hepatocyte (PHH) monolayers administered the indicated base editor systems at a dose of 2.5 ng or 10 ng (i.e., the total combined mass of the guide polynucleotide and the mRNA encoding the base editor). The left group of bars corresponds to the 2.5 ng dose, and the right group of bars corresponds to the 10 ng dose. Figure 26 Provided are bar graphs showing on-target A>G base editing (%) in primary human hepatocyte (PHH) monolayers administered the indicated base editor systems at a dose of 5 ng (i.e., the total combined mass of the guide polynucleotide and the mRNA encoding the base editor). Figure 27 Provided are bar graphs showing on-target A>G base editing (%) in primary human hepatocyte (PHH) monolayers administered the indicated base editor systems at a dose of 20 ng (i.e., the total combined mass of the guide polynucleotide and the mRNA encoding the base editor). Figure 28 Bar graphs are provided showing target A>G % base editing and human C3 protein concentrations (normalized to day zero (0) concentrations) in FRG liver humanized mice administered the indicated base editor systems at the indicated total combined doses of guide polynucleotide RNA and mRNA encoding the base editors on day 14 post-transfection. Figure 28 Each shaded column in the corresponds to an hC3 protein concentration measurement, and each set of data points to the left of each column (whose mean is represented by a horizontal line) corresponds to an on-target base editing measurement. Figure 29Bar graphs are provided showing target A>G % base editing and human C3 protein concentrations (normalized to day zero (0) concentrations) in FRG liver humanized mice administered the indicated base editor systems at the indicated total combined doses of guide polynucleotide RNA and mRNA encoding the base editors on day 14 post-transfection. Figure 29 Each shaded column in the corresponds to an hC3 protein concentration measurement, and each set of data points to the left of each column (whose mean is represented by a horizontal line) corresponds to an on-target base editing measurement. Figure 30 A bar graph is provided showing target A>G % base editing and human C3 protein concentration (normalized to day zero (0) concentration) in FRG liver humanized mice administered the indicated base editor systems at the indicated total combined doses of guide polynucleotide RNA and mRNA encoding the base editors at day 14 post-transfection. Figure 30 In the present study, RNA levels were measured using a probe for C3 spanning exons 4 and 5. Figure 31 A bar graph is provided showing C3 RNA levels normalized to GAPDH RNA levels in FRG liver humanized mice administered the indicated base editor systems at the indicated doses (total combined mass of guide polynucleotide RNA and mRNA encoding the base editor). Figure 31 In the present study, RNA levels were measured using a probe for C3 spanning exons 19 and 20. Figure 32 A bar graph is provided showing C3 RNA levels normalized to GAPDH RNA levels in FRG liver humanized mice administered the indicated base editor systems at the indicated doses (total combined mass of guide polynucleotide RNA and mRNA encoding the base editor). Figure 32 In the present study, RNA levels were measured using a probe for C3 spanning exons 27 and 28. Figure 33 A bar graph is provided showing C3 RNA levels normalized to GAPDH RNA levels in FRG liver humanized mice administered the indicated base editor systems at the indicated doses (total combined mass of guide polynucleotide RNA and mRNA encoding the base editor). Figure 33 In the present study, RNA levels were measured using a probe for C3 spanning exons 38 and 39. Figure 34 Provided are bar graphs showing on-target A>G base editing (%) by the indicated base editors in Hek293T cells. Figure 35A bar graph is provided showing target A>G base editing (%) of the indicated base editors in Hek293T cells. The numbers listed below each group of bars indicate, from left to right, the corresponding position of the target nucleotide upstream of the C3 start codon corresponding to each bar. Figure 36 Provided are bar graphs showing the concentration of human C3 protein in PHH cells administered with the indicated base editors. Figure 37 Provided are bar graphs showing the editing rate (%) and human C3 protein concentration of primary human hepatocyte (PHH) co-cultures administered the indicated base editor systems targeting the C3 start codon or the TATA box for base editing. Figure 37 In the figure, the light grey columns correspond to the human C3 (hC3) protein concentration, and the dark grey columns correspond to the editing rate. Figure 38 Provided are bar graphs showing on-target A>G base editing (%) of PHH monolayers administered with the indicated base editor systems. Figure 39 Provided are bar graphs showing negligible variability between PHH co-cultures administered the indicated base editors prior to transfection with the base editors. Figure 40 Provided are bar graphs showing editing rates (%) and hC3 concentrations of PHH co-cultures administered the indicated base editor systems, where light grey bars represent protein concentrations and dark grey bars represent editing rates. Figure 41 Provided are graphs showing target A>G base editing (%) in HepG2 cells administered the indicated base editors at the indicated base editing doses (i.e., the combined mass of the guide polynucleotide and the mRNA encoding the base editor). DETAILED DESCRIPTION Provided herein are base editors, endonucleases, and guide RNAs (gRNAs) for editing, modifying, or changing target polynucleotides. In a specific embodiment, the base editors or endonucleases of the present disclosure modify complement component 3 (C3) polynucleotides. In a specific embodiment, the base editors of the present disclosure modify termination codons, missense mutations, or indels (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide insertions or deletions (indels)) by changing the splice sites introduced into C3 polynucleotides or destroying C3 polynucleotides. These changes are associated with a decrease in the activity or level of C3 polypeptides and / or polynucleotides in cells. The invention of the present disclosure is based at least in part on the discovery that the complement system can be activated through a variety of different pathways, and each of these pathways requires protein C3 to achieve amplification and function of the complement pathway. The present invention is also based at least in part on the discovery that base editing (e.g., destroying a splice acceptor or splice donor, or introducing a stop codon, missense mutation, or insertion / deletion change) can be used to reduce the expression of C3 polypeptides in cells associated with a dysregulated complement system (e.g., inappropriate activation). Specifically, reducing the activity and / or expression of C3 polypeptides in subjects diagnosed with a disease or condition associated with excessive activation of the complement system can be an effective therapeutic strategy. Any base editing system and / or endonuclease and method provided herein can be used to achieve this reduction in activity and / or expression. Therefore, the present disclosure features compositions and methods for editing C3 polynucleotides. Editing of C3 polynucleotides is associated with a reduction in the expression and / or activity of C3 polypeptides in cells, tissues, and / or body fluids of the subject, as well as symptoms associated with excessive activation of the complement system or other pathogenic activation in the subject. Thus, as described in the examples provided herein, a base editor system was successfully developed to disrupt complement system activity by functionally disrupting C3 at the genetic level. C3 disruption was performed using two separate approaches: 1) silencing the C3 gene and 2) generating mutations that disrupt specific C3 functions (e.g., opsonization or cleavage by the C3 convertase). In an embodiment, the method of the present disclosure includes disrupting the splicing of C3 polynucleotide transcripts. For example, a base editor or base editor system provided herein can be used to edit a nucleobase in a splicing acceptor located at the 5' of a C3 polynucleotide exon. In some embodiments, the target sequence is a splicing acceptor in an intron of an intron sequence adjacent to an exon of a C3 polynucleotide, and is associated with changes in the splicing acceptor compared to a wild-type splicing acceptor. In some embodiments, deamination of A or C nucleobases in a splicing acceptor results in interrupted splicing of an mRNA transcript during or after transcription. In some embodiments, the subject has or is likely to suffer from an imbalanced and / or overactivated complement system and any disease or condition associated therewith. In some cases, the methods of the present disclosure comprise modifying a C3 polynucleotide to introduce an amino acid change into the C3 polypeptide encoded thereby. In embodiments, the amino acid change disrupts cleavage of the C3 polypeptide by the C3 convertase to produce the C3b fragment. In embodiments, the C3 convertase cleaves the C3 polypeptide between residues R748 and S749. In some cases, the amino acid change disrupts opsonization. In embodiments, the amino acid change disrupts the opsonization of the C3b fragment. In embodiments, the altered amino acid is at position C1010, Q1013, E1128, and / or H1126. In some cases, the altered amino acid is R748. In some cases, the methods of the present disclosure include modifying C3 polynucleotides to introduce stop codons, indels, or missense mutations that are associated with reduced levels or activity of C3 polynucleotides and / or polypeptides. The alterations can be achieved by base editor systems or endonucleases (e.g., Cas9), such as those described herein. In some embodiments, the present disclosure provides base editors or nucleases, which effectively generate expected mutations, such as point mutations or insertions and deletions, in nucleic acid molecules (e.g., nucleic acids in the subject's genome), without generating a large number of unexpected mutations, such as unexpected point mutations. In some embodiments, the expected mutation is a mutation generated by a specific base editor (e.g., an adenosine base editor or a cytidine base editor) or an endonuclease (e.g., Cas9) combined with a guidance polynucleotide (e.g., gRNA), and the guidance polynucleotide is specifically designed to generate an expected mutation. In some embodiments, the expected mutation is an adenine (A) to guanine (G) point mutation in the non-coding region of a gene. In some embodiments, the expected mutation is a cytosine (C) to thymine (T) point mutation in the non-coding region of a gene. In some embodiments, the expected mutation is a mutation in the splicing acceptor in the non-coding region 5' of a gene exon associated with a disease or disorder. In some cases, the expected mutation is an insertion and deletion mutation. In some embodiments, the expected mutation is an adenine (A) to guanine (G) point mutation in a splice acceptor in the 5' non-coding region of a gene exon associated with a disease or condition. In some embodiments, the expected mutation is a missense mutation. The expected mutation may include introducing a stop codon into a polynucleotide sequence. In some embodiments, the expected mutation is a mutation that disrupts the normal splicing of a complete transcript of a gene, for example, an A to G change in a splice acceptor located in the 5' non-coding region of a pathogenic gene or disease-associated gene exon. In some embodiments, the expected mutation is a mutation in a splice acceptor that disrupts the splicing of a gene transcript and results in an alternative transcript encoding a truncated and / or non-functional protein product. In some embodiments, any of the base editors or endonucleases provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations: unintended point mutations) greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations: unintended point mutations) of at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or greater. In some embodiments, multiple base pairs in one or more genes are edited using the methods provided herein to form at least one expected mutation. In some embodiments, the formation of at least one expected mutation is in the splicing acceptor 5' of the disease-associated gene exon and results in the destruction of the splicing of the mRNA transcript of the disease-associated gene. It should be understood that any method or combination of methods provided herein can be used to complete multiple editing. The present disclosure provides methods for treating subjects diagnosed with a dysregulated and / or overactivated complement system or any disease or condition associated therewith. For example, in some embodiments, a method is provided comprising administering an effective amount of a nucleobase editor (e.g., an adenosine deaminase base editor or a cytidine deaminase base editor) or an endonuclease to a subject having or prone to a dysregulated and / or overactivated complement system to effect a change in a C3 polynucleotide sequence. The complement system and C3 Complement is a system composed of a large number of plasma- and cell-bound proteins that play a vital role in innate and adaptive immunity. The proteins of the complement system act in a series of enzymatic cascades through multiple protein interactions and cleavage events. The complement system plays an important role in protecting the body from infectious agents. The complement system contains more than 30 serum and cell proteins that participate in three major pathways: the classical pathway, the alternative pathway, and the lectin pathway. The classical pathway is usually triggered by binding a complex of antigen and IgM or IgG antibodies to C1 (although some other activators can also initiate the pathway). Activated C1 cuts C4 and C2 to produce C4a and C4b in addition to C2a and C2b. C4b and C2a combine to form the C3 convertase, which cuts C3 at a defined cleavage site to form C3a and C3b. C3b is bound to the C3 convertase to produce the C5 convertase, which cuts C5 into C5a and C5b. C3a, C4a, and C5a are anaphylatoxins and can mediate a variety of reactions in acute inflammatory reactions. C3a and C5a are also chemotactic factors that attract immune system cells such as neutrophils. More detailed information about C3 is provided in Ricklin et al. "Complement component C3 - The 'SwissArmyKnife' of innate immunity and host defense." Immunol Rev. 2016 Nov;274(1):33-58; and Janssen et al., "Structures of complement component C3 provide insights into the function and evolution of immunity." Nature. 2005 Sep 22;437(7058):505-11, the disclosures of which are incorporated herein by reference in their entirety for all purposes. The alternative pathway is usually initiated and amplified on the microbial surface and various complex polysaccharides. In this approach, C3 is hydrolyzed to C3 (H2O) (spontaneously occurring at low levels) resulting in the binding of factor B, which is cut by factor D to generate the fluid-phase C3 convertase, which activates complement by cutting C3 into C3a and C3b. C3b binds to a target (such as a cell surface) and forms a complex with factor B, which is then cut by factor D to obtain the C3 convertase. Surface-bound C3 convertase cuts and activates other C3 molecules, resulting in rapid C3b deposition near the activation site, and results in the formation of other C3 convertases, which then generate other C3b. This process results in a cycle of C3 cutting and C3 convertase formation, which significantly amplifies the reaction. C3 cutting and another molecule of C3b bind to the C3 convertase to produce the C5 convertase. The C3 and C5 convertases of this approach are regulated by cellular molecules CR1, DAF, MCP, CD59, and fH. The modes of action of these proteins include decay-accelerating activity (ie, the ability to sequester convertases), the ability to serve as cofactors in the degradation of C3b or C4b by Factor I, or both. Normally, the presence of complement regulatory proteins on the cell surface prevents significant complement activation from occurring there. The C5 convertase produced in both pathways cleaves C5 to produce C5a and C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzes C9 polymerization to form the C5b-9 membrane attack complex (MAC), also known as the terminal complement complex (TCC). The MAC itself inserts into the target cell membrane and causes cell lysis. A small amount of MAC on the cell membrane can have multiple consequences in addition to cell death. If the TCC is not inserted into the membrane, it can circulate in the blood as soluble sC5b-9 (sC5b-9). The level of sC5b-9 in the blood can serve as an indicator of complement activation. The lectin complement pathway can be initiated by the binding of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) to carbohydrates. The MB1-1 gene (called LMAN-1 in humans) encodes a type I integral membrane protein located in the intermediate region between the endoplasmic reticulum and the Golgi apparatus. The MBL-2 gene encodes a soluble mannose-binding protein found in serum. In the human lectin pathway, MASP-1 and MASP-2 participate in the proteolysis of C4 and C2, generating the aforementioned C3 convertase. Diseases and / or conditions associated with undesirable increased activation of the complement system Inappropriate activation of the complement system can lead to various diseases and / or conditions in a subject. For example, inappropriate activation of the complement system in a subject damages cells, leading to increased inflammation, the presence of autoantibodies, neurodegeneration, and microthrombosis, among other things. Inappropriate activation of the complement system is associated with damage to the central nervous system (CNS), eyes, blood cells (e.g., red blood cells and white blood cells and platelets), and transplanted organs, as well as damage to other organs or tissues, which may be associated with the presence of microemboli. Therefore, effective treatment of such diseases and / or conditions may include altering the C3 nucleotide sequence to reduce and / or eliminate the expression and / or activity of the C3 polypeptide in the subject, thereby reducing activation of the complement system in organs, cells, and / or tissues. In an embodiment, the organ or tissue is an eye, kidney, a component of the central nervous system, a heart, or a thyroid gland. Non-limiting examples of diseases associated with inappropriate activation of the complement system include blood disorders, transplant or graft rejection, inflammatory diseases or conditions, eye diseases or conditions, kidney diseases or conditions, cardiac disorders, respiratory / pulmonary diseases or conditions, autoimmune disorders, inflammatory bowel diseases or conditions, arthritis, neurodegenerative diseases or conditions, musculoskeletal diseases or conditions associated with inflammation, disorders affecting the integumentary system, diseases or conditions affecting the central nervous system, diseases or conditions affecting the circulatory system, diseases or conditions affecting the gastrointestinal system, diseases or conditions affecting the thyroid gland, chronic pain, allergies, and lung diseases. Other non-limiting examples of diseases associated with inappropriate activation of the complement system include acute antibody-mediated rejection, age-related macular degeneration (e.g., wet or dry age-related macular degeneration), allergic asthma, allergic bronchopulmonary aspergillosis, allergic neuritis, allergic rhinitis, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, atopic dermatitis, atypical hemolytic syndrome (aHUS), autoimmune diseases, Autoimmune hemolytic anemia, Behçet's disease, Behçet's disease, bronchiolitis, bronchiolitis obliterans, C3 glomerulopathy, cancer, central nervous system (CNS) inflammatory disorders, choroidal neovascularization (CNV), choroiditis, chronic allograft vasculopathy, chronic hepatitis, chronic inflammation, chronic inflammatory diseases, chronic muscle inflammation, chronic pain, chronic pancreatitis, chronic rejection of transplants or grafts, chronic urticaria, Tschag-Strauss syndrome, conjunctivitis, cyclitis, demyelinating diseases, dermatitis, dermatomyositis, diabetic retinopathy, circulatory system diseases, diseases associated with excessive or inappropriate activity of IgE-producing cells, disorders associated with high levels or inappropriate activity of CD4+ helper T cells of the Th17 subtype, encephalitis, eosinophilic pneumonia, eye disorders, geographic atrophy, macrocytopenia arteritis, gingivitis, glaucoma, glomerulonephritis, glomerulonephritis (e.g., membranoproliferative glomerulonephritis or membranous glomerulonephritis), transplant rejection or failure, HELLP syndrome, Henoch-Schonlein purpura, hepatitis (e.g., hepatitis C), Huntington's disease, hyperacute rejection, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), IgA nephropathy (IgAN), inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), inflammatory joint disorders (e.g., arthritis such as rheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis, spondyloarthropathies, Reiter's syndrome, or gout), inflammatory skin diseases, infusion reactions, interstitial pneumonitis, iridocyclitis, iritis, ischemia / reperfusion injury, Kawasaki disease, keratitis, lupus nephritis, membranoproliferative glomerulonephritis (MPGN) (e.g.,MPGN type I, II, or III), meningitis, microscopic polyangiitis, multiple sclerosis (MS), myasthenia gravis, myocarditis, nasal polyposis, neurodegenerative diseases, neuromyelitis optica (NMO), neuropathic pain, ocular inflammation, osteoarthritis, pancreatitis, panniculitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pars planitis, pathological immune responses to tissue / organ transplantation, pemphigoid, pemphigus, and periodontitis, persistent asthma, polyarteritis nodosa, polymyositis, primary Membranous nephropathy, proliferative vitreoretinopathy, proteinuria, psoriasis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury, kidney disease, respiratory disease or disorder (e.g., asthma or chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis or asthma), respiratory distress syndrome, retinal neovascularization (RNV), retinopathy of prematurity, rheumatoid arthritis (RA), sinusitis, sarcoidosis, sarcoidosis, scleritis, scleroderma, sclerodermatomyositis, sclerosis, sepsis, Sjögren's syndrome, Sjoren's syndrome, stroke, systemic lupus erythematosus, systemic scleroderma, Takayasu's arteritis, Th2-related disorders (e.g., disorders associated with high levels or high activation of CD4+ helper T cells of the Th2 subtype), thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease, postpartum thyroiditis), thyroidoisis, transplant injury, transplant rejection, ulcerative colitis, uveitis, vasculitis, and Wegener's granulomatosis. In embodiments, the methods of the present disclosure comprise reducing complement-mediated hemolysis in a subject. Other non-limiting examples of diseases include Creutzfeldt-Jakob disease, Pick's disease, mild cognitive impairment, fibromyalgia, frontotemporal dementia, dementia with Lewy bodies, multiple system atrophy, chronic inflammation, demyelinating polyneuropathy, Guillain-Barré syndrome, multifocal motor neuropathy, non-alcoholic fatty liver disease (NAFLD) such as non-alcoholic steatohepatitis (NASH), and Stargardt macular dystrophy.

[1269] The methods and compositions of the present disclosure are suitable for use in treating any of the above-mentioned disease or condition embodiments associated with inappropriate activation of the complement system. In various instances, the methods comprise introducing modifications into a C3 polynucleotide resulting in reduced expression and / or activity of a C3 polypeptide in a cell.

[1270] Target gene editing

[1271] Tables 1A to 1F and 2 below list exemplary spacer sequences for guide RNAs suitable for use in producing polynucleotide editors described herein (e.g., missense mutations, introduction of stop codons, splice site disruption mutations, etc.). In order to produce polynucleotide editors, cells (e.g., cells in a subject or cells from a subject) are contacted with one or more of the spacer sequences listed in Tables 1A to 1F or 2 below or one or more guide RNAs of their fragments, and contacted with a nucleic acid programmable DNA binding protein (napDNAbp) and one or more deaminases with cytidine deaminase and / or adenosine deaminase activity (e.g., " double deaminases " with cytidine and adenosine deaminase activity) of a core base editor polypeptide or complex. In embodiments, a base editor and / or endonuclease is introduced into a cell using a polynucleotide sequence (e.g., mRNA) encoding a base editor and / or endonuclease. Tables 1A to 1F and 2 below list representative guide RNA spacer sequences that can be used in combination with specified base editors. The guide RNA containing the spacer sequences listed in Tables 1A to 1F and 2 can be used to target the target sequences listed in Tables 1A to 1F and 2 to achieve the editing listed in Tables 1A to 1F and 2. In some embodiments, the gRNA comprises nucleotide analogs. In some cases, the gRNA is added directly to the cell. These nucleotide analogs can inhibit the degradation of the gRNA in the cell process. Tables 1A to 1F and 2 provide target sequences to be used for gRNA. Further exemplary spacer sequences suitable for the gRNA sequences used in the methods provided herein include fragments of any spacer provided in Tables 1A to 1F and 2 and modifications provided in Tables 1A to 1F and 2 to include any spacer extended or truncated at the 3' end and / or 5' end. In an embodiment, the spacer sequences of Tables 1A to 1F and 2 can be modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides extended or truncated at the 3' end and / or 5' end. In various cases, it is advantageous for the spacer sequence to include 5' and / or 3' "G" nucleotides. In some cases, for example, any of the spacer sequences or guide polynucleotides provided herein include or further include a 5' "G," wherein, in some embodiments, the 5' "G" is complementary or non-complementary to the target sequence. In some embodiments, a 5' "G" is added to a spacer sequence that does not already contain a 5' "G." For example, when the guide RNA is expressed under the control of a U6 promoter, etc., it may be advantageous for the guide RNA to include a 5' terminal "G" because the U6 promoter prefers a "G" at the transcription start site (see Cong, L. et al. "Multiplex genome engineering using CRISPR / Cas systems. Science 339:819-823 (2013) doi:10.1126 / science.1231143). In some cases, a 5' terminal "G" is added to a guide polynucleotide that is to be expressed under the control of a promoter, but if or when the guide polynucleotide is not to be expressed under the control of a promoter, a 5' terminal "G" is optionally not added to the guide polynucleotide. In some embodiments, the guidance polynucleotides of the present disclosure contain a scaffold having one of the following chemical modification schemes, wherein "N" represents any nucleotide, "mN" represents a 2'-OMe modification of nucleotide "N", and "Ns" represents that nucleotide "N" is linked to the following nucleotides via phosphorothioate (PS): End-mod SpCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUsmUsmUsmU (SEQ ID NO: 943) End-mod SaCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU(SEQ ID NO: 944) HM01: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ IDNO: 855) HM07: mNsmNsmNsmNmNmNmNmNmNmNNNNNNNNNNNmGUUUUAGmAmGmCmUmAmGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 856) NLS (bpsv40): mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsmU-NHC6-CrossL-ac-CKRTADGSEFESPKKKRKV (SEQ ID NO: 857 and 858) LONGEST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGUGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsm UsmU(SEQ ID NO:859)

[1272] NLS+LONGEST:

[1273] mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUC AmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU-NHC5-CrossL-CKRTADGSEFESPKKK RKV (SEQ ID NO:860 and 858)

[1274] LONGEST+GOLD:

[1275] mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGC UAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU(SEQ ID NO:861)

[1276] Exemplary guide RNA sequences are provided in Tables 1A to 1F and 2 below.

[1277] Table 1A. List of C3 knockout (KO) guides screened in Hek293T cells, the associated protospacers, the editors associated with each, and the mechanism of KO generation.

[1278]

[1279]

[1280]

[1281]

[1282]

[1283]

[1284]

[1285]

[1286]

[1287]

[1288]

[1289]

[1290]

[1291] Table 1B. List of cynomolgus monkey alternative C3 KO guides, the protospacer associated with each guide, the related human C3 KO guides, the editor associated with each, and the mechanism of KO generation.

[1292]

[1293]

[1294] Table 1C. List of C3 KO length series guides including protospacer sequences, spacer sequences, corresponding 20 bp guide names, editors used for each, and spacer lengths.

[1295]

[1296]

[1297]

[1298] Table 1D. List of C3 knockout (KO) re-modified guides screened in FRG (Fah- / -Rag2- / -Il2rg- / -) liver humanized mice including protospacer sequences, spacer sequences, associated terminal modification guide names, editors associated with each, and chemical modification protocols.

[1299]

[1300]

[1301]

[1302] ABE9.48 represents a base editor containing the TadA*9.48 deaminase domain corresponding to TadA*8.8 with changes V82T, Y147T, and Q154S; ABE9.52 represents a base editor containing the TadA*9.52 deaminase domain corresponding to TadA*8.20 with changes V82T, Y147T, and Q154S.

[1303] Table 1E. List of heavily modified cynomolgus monkey replacement C3 knockout (KO) guides including the protospacer associated with each guide, the related human C3 KO guides, the editors associated with each, and the chemical modification scheme.

[1304]

[1305] Table 1F. List of start codon-targeted and TATA box (e.g., -80 to -98 bp upstream of the start codon of C3)-targeted C3 knockout (KO) guides screened in Hek293T cells including the relevant protospacer, the editors associated with each, and the mechanism of KO generation.

[1306]

[1307]

[1308] Table 2. List of C3 mutant guides, protospacers, spacers, editors associated with each, target residues, and strategies for C3 functional disruption screened in Hek293T cells. Ops. - Opsonization.

[1309]

[1310]

[1311] Nucleobase editors

[1312] The nuclear base editors that can be used for the methods and compositions described herein edit, modify or change the target nucleotide sequence of a polynucleotide. The nuclear base editors described herein generally contain a polynucleotide programmable nucleotide binding domain and a nuclear base editing domain (e.g., adenosine deaminase, cytidine deaminase or dual deaminase). When combined with a bound guidance polynucleotide (e.g., gRNA), the polynucleotide programmable nucleotide binding domain can specifically bind to the target polynucleotide sequence, thereby positioning the base editor to the target nucleic acid sequence that needs to be edited. Polynucleotide programmable nucleotide binding domain The polynucleotide programmable nucleotide binding domain binds to a polynucleotide (e.g., RNA, DNA). The polynucleotide programmable nucleotide binding domain of the base editor itself may comprise one or more domains (e.g., one or more nuclease domains). In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain comprises an endonuclease or an exonuclease. Disclosed herein are base editors comprising a polynucleotide programmable nucleotide binding domain containing all or part (e.g., a functional portion) of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein (e.g., a Cas protein) as a domain, also referred to as a "CRISPR protein-derived domain" of a base editor). The CRISPR protein-derived domain incorporated into the base editor may be modified compared to the wild-type or native version of the CRISPR protein. The CRISPR protein-derived domain may comprise one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to the wild-type or native version of the CRISPR protein. Cas proteins useful herein include Class 1 and Class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1 (e.g., SEQ ID NO: 232), Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ, CARF, DinG, homologs thereof, or modified versions thereof. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. Vectors encoding CRISPR enzymes that are mutated relative to the corresponding wild-type enzyme can be used, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. A Cas protein (e.g., Cas9, Cas12) or a Cas domain (e.g., Cas9, Cas12) can refer to a polypeptide or domain having at least or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology with a wild-type exemplary Cas polypeptide or Cas domain. Cas (e.g., Cas9, Cas12) can refer to a wild-type or modified form of a Cas protein, which can comprise amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof. In some embodiments, the CRISPR protein-derived domain of the base editor may include all or part (e.g., a functional part) of Cas9 from: Corynebacterium ulcerans (NCBI Ref: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Ref: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus. Some aspects of the present disclosure provide high-fidelity Cas9 domains. High-fidelity Cas9 domains are known in the art and are described in, for example, Kleinstiver, BP et al. "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects." Nature 529, 490-495 (2016); and Slaymaker, IM et al. "Rationally engineered Cas9 nucleases with improved specificity." Science 351, 84-88 (2015); the entire contents of each of the above documents are incorporated herein by reference. An exemplary high-fidelity Cas9 domain is provided in the sequence listing as SEQ ID NO: 233. In some embodiments, any of the Cas9 fusion proteins or complexes provided herein comprise one or more of D10A, N497X, R661X, Q695X, and / or Q926X mutations, or corresponding mutations in any of the amino acid sequences provided herein, wherein X is any amino acid. Typically, Cas9 proteins, such as Cas9 from Streptococcus pyogenes (spCas9), require a "protospacer adjacent motif (PAM)" or PAM-like motif, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The presence of the NGG PAM sequence is required for binding to specific nucleic acid regions, where the "N" in "NGG" is adenosine (A), thymidine (T), or cytosine (C), and G is guanosine. In some embodiments, any fusion protein or complex provided herein may contain a Cas9 domain capable of binding to a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and are apparent to those skilled in the art. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, BP et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, BP et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference. In some embodiments, the napDNAbp is a circular permutation mutant (eg, SEQ ID NO: 238). In some embodiments, the polynucleotide programmable nucleotide binding domain includes a nickase domain. The term "nickase" herein refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that can cut only one of the two chains in a double-stranded nucleic acid molecule (e.g., DNA). For example, when the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 may include a D10A mutation and histidine at position 840. In another example, the nickase domain derived from Cas9 includes an H840A mutation, and the amino acid residue at position 10 is still D. In some embodiments, Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, That is, Cas9 is a nickase, referred to as "nCas9" protein (for "nickase" Cas9; SEQ ID NO: 201). Cas9 nickase can be a Cas9 protein that can only cut one chain of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any one of the Cas9 nickases provided herein. Based on the present disclosure and knowledge in the art, additional suitable Cas9 nickases will be apparent to those skilled in the art, and are within the scope of the present disclosure. Also provided herein is a base editor comprising a polynucleotide programmable nucleotide binding domain that is catalytically inactive (i.e., cannot cut a target polynucleotide sequence). For example, in the case of a base editor comprising a Cas9 domain, Cas9 may include a D10A mutation and an H840A mutation. In other embodiments, the catalytically inactive polynucleotide programmable nucleotide binding domain includes a point mutation (e.g., D10A or H840A) and a deletion of all or part (e.g., a functional portion) of a nuclease domain. dCas9 domains are known in the art and are described in, for example, Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference. The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to a DNA sequence of 2-6 base pairs immediately adjacent to a DNA sequence targeted by a nucleic acid programmable DNA binding protein. In some embodiments, the PAM can be a 5' PAM (i.e., upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., downstream of the 5' end of the protospacer). The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAC. Y is a pyrimidine; N is any nucleotide base; and W is A or T.

[1313] The base editors provided herein may comprise a CRISPR protein-derived domain capable of binding to a nucleotide sequence containing a canonical or non-canonical protospacer-adjacent motif (PAM) sequence.

[1314] In some embodiments, the PAM is an "NRN" PAM, wherein the "N" in "NRN" is adenine (A), thymine (T), guanine (G), or cytosine (C), and R is adenine (A) or guanine (G); or the PAM is a "NYN" PAM, wherein the "N" in NYN is adenine (A), thymine (T), guanine (G), or cytosine (C), and Y is cytidine (C) or thymine (T), e.g., as described in RT Walton et al., 2020, Science, 10.1126 / science.aba8853 (2020), the entire contents of which are incorporated herein by reference.

[1315] Several PAM variants are described in Table 3 below.

[1316] Table 3. Cas9 proteins and corresponding PAM sequences. N is A, C, T, or G; and V is A, C, or G.

[1317]

[1318]

[1319] In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM Cas9 variant comprises one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively referred to as "MQKFRAER") or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) D1135V, G1218R, R1335Q, and T1337R (collectively referred to as VRQR) or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) D1135V, G1218R, R1335E, and T1337R (collectively referred to as VRER) or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from saCas9 (SEQ ID NO: 218) E782K, N968K, and R1015H (collectively referred to as KHH) . In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R (collectively referred to as "MQKSER") or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R (collectively referred to as "MQKSER") of spCas9 (SEQ ID No: 197) or corresponding mutations in another Cas9. In some embodiments, the CRISPR protein-derived domain of the base editor comprises all or part (e.g., a functional portion) of a Cas9 protein having a canonical PAM sequence (NGG). In other embodiments, the Cas9-derived domain of the base editor may employ a non-canonical PAM sequence. Such sequences have been described in the art and will be apparent to those skilled in the art. For example, Cas9 domains that bind to non-canonical PAM sequences have been described in Kleinstiver, BP et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, BP et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); RT Walton et al. “Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants” Science 10.1126 / science.aba8853 (2020); Hu et al. “Evolved Cas9 variants with broad PAM compatibility and high DNA specificity,” Nature, 2018 Apr. 5, 556(7699), 57-63; Miller et al., “Continuous evolution of SpCas9 variants compatible with non-G PAMs” Nat. Biotechnol., 2020 Apr;38(4):471-481; the entire contents of each are incorporated herein by reference. Fusion protein or complex comprising NapDNAbp and cytidine deaminase and / or adenosine deaminase Some aspects of the present disclosure provide fusion proteins or complexes comprising a Cas9 domain or other nucleic acid programmable DNA binding protein (e.g., Cas12) and one or more cytidine deaminases, adenosine deaminases or cytidine adenosine deaminase domains. It should be understood that the Cas9 domain can be any Cas9 domain or Cas9 protein (e.g., dCas9 or nCas9) provided herein. In some embodiments, any Cas9 domain or Cas9 protein (e.g., dCas9 or nCas9) provided herein can be fused to any cytidine deaminase and / or adenosine deaminase provided herein. The domains of the base editors disclosed herein can be arranged in any order. In some embodiments, the fusion protein or complex comprising cytidine deaminase or adenosine deaminase and napDNAbp (e.g., Cas9 or Cas12 domain) does not comprise a linker sequence. In some embodiments, a linker is present between cytidine or adenosine deaminase and napDNAbp. In some embodiments, cytidine or adenosine deaminase and napDNAbp are fused via any linker provided herein. For example, in some embodiments, cytidine or adenosine deaminase and napDNAbp are fused via any linker provided herein. Should be appreciated that fusion rotein of the present disclosure or complex can comprise one or more other features.For example, in some embodiments, fusion rotein or complex can comprise inhibitor, cytoplasm localization sequence, export sequence (such as nuclear export sequence) or other localization sequences, and can be used for dissolving, purifying or detecting the sequence tag of fusion rotein or complex.Suitable protein tag provided herein includes but is not limited to biotin carboxylase carrier protein (BCCP) label, myc label, calmodulin label, FLAG label, hemagglutinin (HA) label, polyhistidine tag (also referred to as histidine tag or His label), maltose binding protein (MBP) label, nus label, glutathione-S-transferase (GST) label, green fluorescent protein (GFP) label, thioredoxin label, S label, Softag (for example, Softag 1, Softag 3), streptococcus label, biotin ligase label, F1AsH label, V5 label and SBP label.Other suitable sequence will be apparent to those skilled in the art.In some embodiments, fusion rotein or complex comprise one or more His tags. Exemplary, but non-limiting, fusion proteins are described in International PCT Application Nos. PCT / US2017 / 045381, PCT / US2019 / 044935, and PCT / US2020 / 016288, each of which is incorporated herein by reference in its entirety. Fusion proteins or complexes with internal insertions Provided herein are fusion proteins or complexes comprising heterologous polypeptides fused to nucleic acid programmable nucleic acid binding proteins (e.g., napDNAbp). The heterologous polypeptide can be fused to napDNAbp at the C-terminus of napDNAbp, the N-terminus of napDNAbp, or inserted into an internal position of napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., cytidine or adenosine deaminase) or a functional fragment thereof. For example, the fusion protein may comprise a deaminase flanking the N-terminal fragment and the C-terminal fragment of a Cas9 or Cas12 (e.g., Cas12b / C2c1) polypeptide. The deaminase can be a circular permutation mutant deaminase. In some embodiments, the deaminase is a circular permutation mutant TadA, circular at amino acid residue 116, 136, or 65, as numbered by the TadA reference sequence. The fusion protein or complex may comprise more than one deaminase. The fusion protein or complex may comprise, for example, 1, 2, 3, 4, 5 or more deaminases. The deaminase in the fusion protein or complex may be adenosine deaminase, cytidine deaminase, or a combination thereof. In some embodiments, the napDNAbp in the fusion protein or complex contains a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. The Cas9 polypeptide can be a circularly arranged Cas9 protein. A heterologous polypeptide (e.g., a deaminase) can be inserted into a suitable position of napDNAbp (e.g., Cas9 or Cas12 (e.g., Cas12b / C2c1)), for example, such that napDNAbp retains its ability to bind to a target polynucleotide and a guide nucleic acid. A deaminase (e.g., an adenosine deaminase, a cytidine deaminase, or an adenosine deaminase and a cytidine deaminase (dual deaminase)) can be inserted into napDNAbp without impairing the function of the deaminase (e.g., base editing activity) or the function of napDNAbp (e.g., the ability to bind to a target nucleic acid and a guide nucleic acid). In some embodiments, a deaminase (e.g., an adenosine deaminase, a cytidine deaminase, or both an adenosine deaminase and a cytidine deaminase) is inserted into a region of the Cas9 polypeptide containing an above-average level of Factor B (e.g., a higher level of Factor B than the total protein or protein domain containing the disordered region). Cas9 polypeptide positions containing an above-average level of Factor B can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 as numbered in the Cas9 reference sequence above. Regions of the Cas9 polypeptide containing an above-average level of Factor B can include, for example, residues 792-872, 792-906, and 2-791 as numbered in the Cas9 reference sequence above. In some embodiments, a heterologous polypeptide (e.g., a deaminase) is inserted into a flexible loop of a Cas9 polypeptide. The flexible loop portion can be selected from 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300 numbered by the above-mentioned Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide. The flexible loop portion can be selected from: 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 numbered by the above-mentioned Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide.

[1320] should amino acid residues.

[1321] A heterologous polypeptide (e.g., an adenine deaminase) can be inserted into the region of the Cas9 polypeptide corresponding to amino acid residues 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002-1003, 943-947, 530-537, 568-579, 686-691, 1242-1247, 1298-1300, 1066-1077, 1052-1056, or 1060-1077, as numbered in the above-mentioned Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide.

[1322] A heterologous polypeptide (e.g., adenine deaminase) can be inserted to replace the deleted region of the Cas9 polypeptide. The deleted region may correspond to the N-terminal or C-terminal portion of the Cas9 polypeptide. Exemplary internal fusion base editors are provided in Table 4A below:

[1323] Table 4A: Insertion loci in Cas9 proteins

[1324] BE ID Modification Other IDs IBE001 Cas9 TadA insertion 1015 ISLAY01 IBE002 Cas9 TadA insertion 1022 ISLAY02 IBE003 Cas9 TadA insertion 1029 ISLAY03 IBE004 Cas9 TadA insertion 1040 ISLAY04 IBE005 Cas9 TadA insertion 1068 ISLAY05 IBE006 Cas9 TadA insertion 1247 ISLAY06 IBE007 Cas9 TadA insertion 1054 ISLAY07 IBE008 Cas9 TadA insertion 1026 ISLAY08 IBE009 Cas9 TadA insertion 768 ISLAY09 IBE020 delta HNH TadA 792 ISLAY20 IBE021 N-terminal fusion with a single TadA helix truncated at 165 ISLAY21 IBE029 TadA-circular arrangement mutant 116ins1067 ISLAY29 IBE031 TadA-circular arrangement mutant 136 inserted into 1248 ISLAY31 IBE032 TadA-circular permutation mutant 136 inserted into 1052 ISLAY32 IBE035 delta 792-872 TadA insertion ISLAY35 IBE036 delta 792-906 TadA insertion ISLAY36 IBE043 TadA-circular permutation mutant 65 inserted into 1246 ISLAY43 IBE044 TadA insertion C-terminal truncation 2 791 ISLAY44

[1325] A heterologous polypeptide (e.g., a deaminase) can be inserted into the structure or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., a deaminase) can be inserted between two structure or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., a deaminase) can be inserted in place of the structure or functional domain of a Cas9 polypeptide, for example, after the domain is deleted from the Cas9 polypeptide. The structure or functional domain of a Cas9 polypeptide may include, for example, RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH. The fusion protein may include a linker between the deaminase and the napDNAbp polypeptide. The linker can be a peptide or non-peptide linker. For example, the linker can be XTEN, (GGGS)n (SEQ ID NO: 246), SGGSSGGS (SEQ ID NO: 330), (GGGGS)n (SEQ ID NO: 247), (G)n, (EAAAK)n (SEQ ID NO: 248), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 249). In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein includes a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of napDNAbp are connected to the deaminase with a linker. In some embodiments, the N-terminal and C-terminal fragments are joined to the deaminase domain without a linker. In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase, but does not include a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase, but does not comprise a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the napDNAbp in the fusion protein or complex is a Cas12 polypeptide (e.g., Cas12b / C2c1) or a functional fragment thereof that can associate with a nucleic acid (e.g., gRNA) that guides Cas12 to a specific nucleic acid sequence. The Cas12 polypeptide can be a variant Cas12 polypeptide. In other embodiments, the N-terminal or C-terminal fragment of the Cas12 polypeptide includes a nucleic acid programmable DNA binding domain or a RuvC domain. In other embodiments, the fusion protein contains a joint between the Cas12 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the joint is GGSGGS (SEQ ID NO: 250) or GSSGSETPGTSESATPESSG (SEQ ID NO: 251). In other embodiments, the joint is a rigid joint. In other embodiments of aspects of the present disclosure, the joint is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO: 252) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO: 253).

[1326] In other embodiments, the fusion protein or complex contains a nuclear localization signal (e.g., a bipartite nuclear localization signal). In other embodiments, the amino acid sequence of the nuclear localization signal is MAPKKKRKVGIHGVPAA (SEQ ID NO: 261). In other embodiments of aspects of the present disclosure, the nuclear localization signal is encoded by the following sequence:

[1327] ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGA GTCCCAGCAGCC (SEQ ID NO: 262). In other embodiments, the Cas12b polypeptide contains a mutation that silences the catalytic activity of the RuvC domain. In other embodiments, the Cas12b polypeptide contains D574A, D829A and / or D952A mutations.

[1328] In some embodiments, the fusion protein or complex comprises a napDNAbp domain (e.g., a Cas12-derived domain) with an internally fused nucleobase editing domain (e.g., all or part (e.g., a functional part) of a deaminase domain (e.g., an adenosine deaminase domain). In some embodiments, the napDNAbp is Cas12b. In some embodiments, the base editor comprises a BhCas12b domain with an internally fused TadA*8 domain inserted at the locus provided in Table 4B below.

[1329] Table 4B: Insertion loci in Cas12b proteins

[1330]

[1331]

[1332] In some embodiments, the base editing system described herein is an ABE with TadA inserted into Cas9. The polypeptide sequences of the relevant ABEs with TadA inserted into Cas9 are provided in the accompanying sequence listing as SEQ ID NOs: 263-308.

[1333] Exemplary, but non-limiting, fusion proteins are described in International PCT Application No. PCT / US2020 / 016285 and U.S. Provisional Application Nos. 62 / 852,228 and 62 / 852,224, the contents of which are incorporated herein by reference in their entirety.

[1334] A to GEdit

[1335] In some embodiments, the base editor described herein comprises an adenosine deaminase domain. Such an adenosine deaminase domain of the base editor can facilitate the editing of adenine (A) nucleobase to guanine (G) nucleobase by deaminating A to form inosine (I), which exhibits the base pairing properties of G. In some embodiments, the A to G base editor further comprises an inosine base excision repair inhibitor, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without being bound by any particular theory, a UGI domain or a catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of deaminated adenosine residues (e.g., inosine), which can improve the activity or efficiency of the base editor.

[1336] The base editor comprising adenosine deaminase can act on any polynucleotide, including DNA, RNA and DNA-RNA hybrids. In one embodiment, the adenosine deaminase domain of the base editor includes all or part (e.g., a functional part) of ADAT, which includes one or more mutations that allow ADAT to deaminate the target A in DNA. For example, the base editor may include all or part (e.g., a functional part) of ADAT from Escherichia coli (Escherichia coli) (EcTadA), comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I156F or a corresponding mutation in another adenosine deaminase. Exemplary ADAT homologous polypeptide sequences are provided in the sequence table as SEQ ID NO: 1 and 309-315. Adenosine deaminase can be derived from any suitable organism (e.g., Escherichia coli). In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenine deaminase is a naturally occurring adenosine deaminase comprising one or more mutations corresponding to any mutation provided herein (e.g., mutations in ecTadA). The corresponding residues in any homologous protein can be identified by, for example, sequence alignment and determination of homologous residues. Mutations in any naturally occurring adenosine deaminase (e.g., having homology to ecTadA) corresponding to any mutation described herein (e.g., any mutation identified in ecTadA) can be generated accordingly. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the amino acid sequences shown in any of the adenosine deaminases provided herein. It should be understood that the adenosine deaminases provided herein may include one or more mutations (e.g., any mutation provided herein). The present disclosure provides any deaminase domain having a certain percentage of identity plus any mutation described herein or a combination thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 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, or more mutations compared to a reference sequence or any adenosine deaminase provided herein.

[1337] It will be understood that any mutation provided herein (e.g., based on a TadA reference sequence, such as TadA*7.10 (SEQ ID NO: 1)) can be introduced into other adenosine deaminases, such as Escherichia coli TadA (ecTadA), Staphylococcus aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). In some embodiments, the TadA reference sequence is TadA*7.10 (SEQ ID NO: 1). It will be apparent to the skilled artisan that additional deaminases can be similarly aligned to identify homologous amino acid residues that can be mutated as provided herein. Thus, any mutation identified in the TadA reference sequence can occur in other adenosine deaminases (e.g., ecTadA) having homologous amino acid residues. It will also be understood that any mutation provided herein can occur alone or in any combination in the TadA reference sequence or another adenosine deaminase.

[1338] In some embodiments, the adenosine deaminase comprises an alteration or group of alterations selected from those listed in Tables 5A to 5E below:

[1339] Table 5A. Adenosine deaminase variants. Residue positions in the E. coli TadA variant (TadA*) are indicated.

[1340]

[1341]

[1342] Table 5B. Adenosine deaminase variants. Residue positions in E. coli TadA variants (TadA*) are indicated. Changes were made with reference to TadA*7.10 (first row).

[1343]

[1344]

[1345] Table 5C. Adenosine deaminase variants. Change reference TadA*7.10. Additional details of TadA*9 adenosine deaminase are described in International PCT Application No. PCT / US2020 / 049975, which is incorporated herein by reference in its entirety for all purposes.

[1346]

[1347]

[1348] In some embodiments, the adenosine deaminase comprises M1I, S2A, S2E, V4D, V4E, V4M, F6S, H8E, H8Y, E9Y, M12S, R13H, R13I, R13Y, T17L, T17S, L18A, L18E, A19N, R21N, K20K, K20R, R21A, G22P, W23D, R23H, W23G, W23Q, W23L, W23R, D24E, D24G, E25F, E25M, E25D, E25A, E25G, E25R, E25V, E25S, E25Y, R26D, R26E, R26G, R26N, R26Q, R26C, R26L, R26K, R26W, E27V, E27D, P29V, V3 0G, L34S, L34V, L36H, H36L, H36N, N37N, N37T, N37S, N38G, N38R, W45A, W45L, W45N, N46N, R46W, R46F, R46Q, R46M, R47A, R47Q, R47F, R47K, R47P, R47W, R4 7M, P48T, P48L, P48A, P48I, P48S, I49G, I49H, I49V, I49F, I49H, G50L, R51H, R51L, R51N, L51W, R51Y, H52D, H52Y, D53P, P54C, P54T, A55H, T55A, A56E, A5 6S, E59A, E59G, E59I, E59Q, E59W, M61A, M61I, M61L, M61V, L63S, L63V, Q65V, G66C, G67D, G67L, G67V, L68Q, M70H, M70Q, L84F, M70V, M70L, E70A, M70V, Q7 1M, Q71N, Q71L, Q71R, N72A, N72K, N72S, N72D, N72Y, Y73G, Y73I, Y73K, Y73R, Y73S, R74A, R74Q, R74G, R74K, R74L, R74N, I76D, I76F, I76I, I76N, I76T, I7 6Y, D77G, A78I, T79M, L80M, L80Y, V82A, V82S, V82G, V82T, L84E, L84F, L84Y, E85K, E85G, E85P, E85S, S87C, S87L, S87V, V88A, V88M, C90S, A91A, A91G, A9 1S, A91V, A91T, G92T, A93I, M94A, M94V, M94L, M94I, M94H, I95S, I95G, I95L,I95H、I95V、H96A、H96L、H96R、H96S、S97C、S97G、S97I、S97M、S97R、S97S、R98K、R98I、R98N、R98Q、G100R、G100V、R101V、R101R、V102A、V102F、V102I、V102V、D103A、F104G、D104N、F104V、F104I、F104L、A106T、V106Q、V106F、V106W、V106M、A106A、A106Q、A106F、A106G、A106W、A106M、A106V、A106R、R107C、R107G、R107P、R107K、R107A、R107N、R107W、R107H、R107S、D108N、D108F、D108G、D108V、D108A、D108Y、D108H、D108I、D108K、D108L、D108M、D108Q、N108Q、N108F、N108W、N108M、N108K、D108K、D108F、D108M、D108Q、D108R、D108W、D108S、A109H、A109K、A109R、A109S、A109T、A109V、K110G、K110H、K110I、K110R、K110T、T111A、T111G、T111H、T111R、G112A、A114G、A114H、A114V、G115S、L117M、L117N、L117V、M118D、M118G、M118K、M118N、M118V、D119L、D119N、D119S、D119V、V120H、V120L、H122H、H122N、H122P、H122R、H122S、H122Y、H123C、H123G、H123P、H123V、H123Y、Y123H、P124G、P124I、P124L、P124W、G125H、G125I、G125A、G125M、G125K、M126D、M126H、M126K、M126I、M126N、M126O、M126S、M126Y、N127H、N127S、N127D、N127K、N127R、H128R、R129H、R129Q、R129V、R129I、R129E、R129V、I132I、I132F、T133V、T133E、T133G、T133K、E134A、E134E、E134G、E134I、G135G、G135V、I136G、I136L、I136T、l137A、l137D、l137E、L137M, l137S, A138D, A138E, A138G, S138A, A138N, A138S, A138T, A138V, A138Y, D139E, D139I, D139C, D139L, D139M, E140A, E140C, E140L, E140R, A142N, A142D, A142G, A142A, A142L, A142S, A142T, A142N, A142S, A142V, A143D, A143E, A143G,, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, A143R, C146R, S146A, S146C, S146D, S146F, S146R, S146T, D147D, D147L, D147F, D147G, D147Y, Y147T, Y147R, Y147D, D147R, F148L, F148F, F148R, F148Y, F149C, F149M, F149R, F149Y, M151F, M151P, M151R, M151V, R152C, R152F, R152H, R152P, R152R, R153C, R 153Q, R153R, R153V, Q154E, Q154H, Q154M, Q154R, Q154L, Q154S, Q154V, E155F, E155G, E155I, E155K, E155P, E155V, E155D, I156A, I156F, I 156D, I156K, I156N, I156R, I156Y, E157A, E157F, E157I, E157P, E157T, E157V, N157K, K157N, K157R, A158Q, A158K, A158V, Q159F, Q159K, Q1 59L, Q159N, K160A, K160S, K160E, K160K, K160N, K161I, K161A, K161N, K161Q, K161S, K161T, A162D, A162Q, R162H, R162P, A162S, Q163G, Q163H, Q163N, Q163R, S164I, S164R, S164Y, S165A, S165D, S165I, S165T, S165Y, T166D, T166K, T166I, T166N, T166P, T166R, D167S and / or D167N mutations, and one or more of any alternative mutations at the corresponding positions, or relative to the TadA reference sequence from R26, W23, E27, H36, R47, P48, R51, H52, R74, I76, V82,any substitution of V88, M94, I95, H96, A106, D108, A109, K110, T111, A114, D119, H122, H123, M126, N127, A142, S146, D147, F149, R152, Q154, E155, I156, E157, K161, T166 and / or D167, or substitution of 2-50 amino acids in the TadA reference sequence, the substitutions being selected from W23R, E27D, H36L, R 47K, P48A, R51H, R51L, I76F, I76Y, V82S, A106V, D108G, A109S, K110R, T111H, A114V, D119N, H122R, H122N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, I156F, K157N, K161N, T166I and D167N, or one or more corresponding mutations in another adenosine deaminase. Additional mutations are described in U.S. Patent Application Publication No. 2022 / 0307003 A1 and International Patent Application Publication Nos. WO 2023 / 288304 A2 and WO 2023 / 034959 A2, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In embodiments, variants of TadA*7.10 comprise one or more alterations selected from any of those provided herein. In a specific embodiment, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is selected from Staphylococcus aureus (S. aureus) TadA, Bacillus subtilis (B. subtilis) TadA, Salmonella typhimurium (S. typhimurium) TadA, Shewanella putrefaciens (S. putrefaciens) TadA, Haemophilus influenzae F3031 (H. influenzae) TadA, Caulobacter crescentus (C. crescentus) TadA, Geobacter sulfurreducens (G. sulfurreducens) TadA, or TadA*7.10. In some embodiments, TadA*8 is a variant as shown in Table 5D. Table 5D shows certain amino acid position numbers in the TadA amino acid sequence and the amino acids present in these positions in the TadA-7.10 adenosine deaminase. Table 5D also shows the amino acid changes relative to TadA-7.10 in the TadA variants after phage-assisted discontinuous evolution (PANCE) and phage-assisted continuous evolution (PACE), As described in M. Richter et al., 2020, Nature Biotechnology, doi.org / 10.1038 / s41587-020-0453-z, the entire contents of which are incorporated herein by reference. In some embodiments, TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d or TadA*8e. In some embodiments, TadA*8 is TadA*8e. In one embodiment, the adenosine deaminase is TadA*8, which comprises or consists essentially of SEQ ID NO: 316 or a fragment thereof having adenosine deaminase activity.

[1349] Table 5D. Selected TadA*8 variants

[1350]

[1351] In some embodiments, the TadA variant is a variant as shown in Table 5E. Table 5E shows certain amino acid position numbers in the TadA amino acid sequence and the amino acids present at these positions in the TadA*7.10 adenosine deaminase. In some embodiments, the TadA variant is MSP605, MSP680, MSP823, MSP824, MSP825, MSP827, MSP828, or MSP829. In some embodiments, the TadA variant is MSP828. In some embodiments, the TadA variant is MSP829.

[1352] Table 5E. TadA variants

[1353]

[1354]

[1355] In a specific embodiment, the fusion protein or complex comprises a single (e.g., provided as a monomer) TadA* (e.g., TadA*8 or TadA*9). Throughout this disclosure, adenosine deaminase base editors comprising a single TadA* domain are represented by the terms ABEm or ABE#m, where "#" is an identification number (e.g., ABE8.20m) and where "m" represents "monomer". In some embodiments, TadA* is linked to a Cas9 nickase. In some embodiments, the fusion protein or complex of the present disclosure comprises a heterodimer of wild-type TadA (TadA(wt)) linked to TadA*. Throughout this disclosure, adenosine deaminase base editors comprising a single TadA* domain and a TadA(wt) domain are represented by the terms ABEd or ABE#d, where "#" is an identification number (e.g., ABE8.20d) and where "d" represents "dimer". In other embodiments, the fusion protein or complex of the present disclosure comprises a heterodimer of TadA*7.10 connected to TadA*. In some embodiments, the base editor is ABE8, which comprises a TadA* variant monomer. In some embodiments, the base editor is ABE, which comprises a heterodimer of TadA* and TadA (wt). In some embodiments, the base editor is ABE, which comprises a heterodimer of TadA* and TadA*7.10. In some embodiments, the base editor is ABE, which comprises a heterodimer of TadA*. In some embodiments, TadA* is selected from Tables 5A to 5E.

[1356] In some embodiments, adenosine deaminase is expressed as a monomer. In other embodiments, adenosine deaminase is expressed as a heterodimer. In some embodiments, the deaminase or other polypeptide sequence lacks methionine, for example when included as a component of a fusion protein. This may change the numbering of positions. However, the skilled artisan will understand that such corresponding mutations refer to the same mutation.

[1357] Any mutation provided herein and any additional mutation (e.g., based on the ecTadA amino acid sequence) can be introduced into any other adenosine deaminase. Any mutation provided herein can occur alone or in any combination in a TadA reference sequence or another adenosine deaminase (e.g., ecTadA).

[1358] Details of the A to G nucleobase editing protein are described in International PCT Application No. PCT / US2017 / 045381 (WO2018 / 027078) and Gaudelli, NM et al., "Programmable base editing of A·T to G·Cin genomic DNA without DNA cleavage", Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference. C to T Edit In some embodiments, the base editor disclosed herein comprises a fusion protein or a complex comprising a cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example, when the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be replaced by a thymidine base (e.g., by a cell repair mechanism) to obtain a C:G to T:A conversion. In other embodiments, the deamination of C in a nucleic acid to U by a base editor cannot be accompanied by a substitution of U to T. The target C deamination in the polynucleotide to produce U is a non-limiting example of a base editing type that can be performed by a base editor as described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate the conversion of cytosine (C) bases into guanine (G) bases. For example, the U of the polynucleotide produced by deaminating cytidine by the cytidine deaminase domain of the base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by uracil DNA glycosylase (UDG) domain), thereby producing an abasic site. The nucleobase opposite the abasic site can then be replaced (e.g., by a base repair mechanism) by another base (such as C) by, for example, a trans-damage polymerase. Although the nucleobase opposite the abasic site is typically replaced by C, other substitutions (e.g., A, G, or T) may also occur. Therefore, in some embodiments, the base editor described herein comprises a deamination domain (e.g., a cytidine deaminase domain) capable of deaminating a target C in a polynucleotide to U. In addition, as described below, the base editor may comprise another domain that, in some embodiments, promotes the conversion of U obtained by deamination to T or G. For example, a base editor comprising a cytidine deaminase domain may further comprise a uracil glycosylase inhibitor (UGI) domain to mediate the replacement of U by T, thereby completing a C to T base editing event. In another example, the base editor may comprise a uracil stabilizing protein as described herein. In another example, a base editor may be incorporated into a trans-lesion polymerase to improve the efficiency of C to G base editing, because the trans-lesion polymerase may contribute to the incorporation of C opposite the abasic site (i.e., resulting in the incorporation of G at the abasic site, thereby completing a C to G base editing event). Base editors containing a cytidine deaminase as a domain can deaminize target C in any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In some embodiments, the cytidine deaminase of the base editor comprises all or part (e.g., a functional part) of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is an evolutionarily conserved family of cytidine deaminases. Members of this family are C to U editing enzymes. The N-terminal domain of the APOBEC-like protein is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D ("APOBEC3E" now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. Other exemplary deaminases that can be fused to Cas9 according to aspects of the present disclosure are provided below. In an embodiment, the deaminase is an activation-induced deaminase (AID). It should be understood that in some embodiments, an active domain of the corresponding sequence can be used, for example, a domain without a localization signal (nuclear localization sequence, without a nuclear export signal, a cytoplasmic localization signal). Some aspects of the present disclosure are based on the recognition that modulating the catalytic activity of the deaminase domain of any fusion protein or complex described herein (e.g., by making point mutations in the deaminase domain) affects the persistence of the fusion protein (e.g., base editor) or complex. For example, mutations that reduce, but do not eliminate, the catalytic activity of the deaminase domain within a base editing fusion protein or complex can make the deaminase domain less likely to catalyze the deamination of residues adjacent to the target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent unwanted deamination of residues adjacent to a specific target residue, which can reduce or prevent off-target effects. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1; D316R, D317R, R320A, R320E, R313A, W285A, W285Y, and R326E of hAPOBEC3G; and any alternative mutations at corresponding positions, or one or more corresponding mutations in another APOBEC deaminase. Many modified cytidine deaminases are commercially available, including but not limited to SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177). In some embodiments, the deaminase incorporated into the base editor comprises all or a portion (e.g., a functional portion) of an APOBEC1 deaminase. In some embodiments, the fusion protein or complex of the present disclosure includes one or more cytidine deaminase domains. In some embodiments, the cytidine deaminase provided herein can deaminate cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the cytidine deaminase provided herein can deaminate cytosine in DNA. The cytidine deaminase can be derived from any suitable organism. In some embodiments, the cytidine deaminase is a naturally occurring cytidine deaminase that includes one or more mutations corresponding to any mutation provided herein. Those skilled in the art will be able to identify the corresponding residues in any homologous protein, for example, by sequence alignment and determination of homologous residues. Therefore, those skilled in the art will be able to produce mutations corresponding to any mutation described herein in any naturally occurring cytidine deaminase. In some embodiments, the cytidine deaminase is from a prokaryotic organism. In some embodiments, the cytidine deaminase is from a bacterium. In some embodiments, the cytidine deaminase is from a mammal (e.g., people). In some embodiments, the cytidine deaminase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity to any cytidine deaminase amino acid sequence shown herein. It should be understood that the cytidine deaminase provided herein may include one or more mutations (e.g., any mutation provided herein). Some embodiments provide polynucleotide molecules encoding any of the aforementioned aspects or cytidine deaminase core base editor polypeptides as described herein. In some embodiments, the polynucleotide is codon optimized.

[1359] In embodiments, a fusion protein of the present disclosure comprises two or more nucleic acid editing domains.

[1360] Details of C to T nucleobase editing proteins are described in International PCT Application No. PCT / US2016 / 058344 (WO2017 / 070632) and Komor, AC et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference.

[1361] Cytidine Adenosine Base Editor (CABE)

[1362] In some embodiments, the base editors described herein comprise an adenosine deaminase variant with increased cytidine deaminase activity. Such base editors may be referred to as "cytidine adenosine base editors (CABE)" or "cytosine base editors derived from TadA* (CBE-T)", and their corresponding deaminase domains may be referred to as "TadA* (T acting on DNA cytosine)". ADC)" domain. In some cases, the adenosine deaminase variant has both adenine and cytosine deaminase activity (i.e., is a dual deaminase). In some embodiments, the adenosine deaminase variant deaminates adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant deaminates adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variant deaminates adenine and cytosine in RNA. In some embodiments, the adenosine deaminase variant predominantly deaminates cytosine in DNA and / or RNA (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all deaminases catalyzed by the adenosine deaminase variant, or the amount of cytosine deaminases catalyzed by the variant is equal to the amount of adenine deaminases catalyzed by the variant). In some embodiments, the adenosine deaminase variant has approximately equal cytosine and adenosine deaminase activities (e.g., the two activities are within about 10% or 20% of each other). In some embodiments, the adenosine deaminase variant has primarily cytosine deaminase activity and has little or no adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity and has no significant or detectable adenosine deaminase activity. In some embodiments, the target polynucleotide is present in a cell in vitro or in vivo. In some embodiments, the cell is a bacterial, yeast, fungal, insect, plant, or mammalian cell. In some embodiments, the CABE comprises a bacterial TadA deaminase variant (e.g., ecTadA). In some embodiments, the CABE comprises a truncated TadA deaminase variant. In some embodiments, the CABE comprises a fragment of a TadA deaminase variant. In some embodiments, the CABE comprises a TadA*8.20 variant. In some embodiments, the adenosine deaminase variants of the present disclosure are TadA adenosine deaminase enzymes comprising one or more alterations that increase cytosine deaminase activity (e.g., an increase of at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) while maintaining adenosine deaminase activity (e.g., at least about 30%, 40%, 50% or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19). In some cases, the adenosine deaminase variants comprise one or more alterations that increase cytosine deaminase activity relative to the activity of a reference adenosine deaminase (e.g., an increase of at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) and include undetectable adenosine deaminase activity or an adenosine deaminase activity that is less than 30%, 20%, 10% or 5% of the activity of a reference adenosine deaminase. In some embodiments, the reference adenosine deaminase is TadA*8.20 or TadA*8.19. In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising two or more alterations at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167 of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO: 1, or corresponding alterations in another deaminase. In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising one or more alterations selected from the group consisting of S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, or a corresponding alteration in another deaminase. , I49N, I49Q, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, A114C, G115M, M118L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P. In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising an amino acid change or combination of amino acid changes selected from those listed in any one of Tables 6A to 6F. The residue identities of exemplary adenosine deaminase variants capable of deaminating adenine and / or cytidine in a target polynucleotide (e.g., DNA) are provided in Tables 6A to 6F below. Further examples of adenosine deaminase variants include the following 1.17 variants (see Table 6A): 1.17+E27H; 1.17+E27K; 1.17+E27S; 1.17+E27S+I49K; 1.17+E27G; 1.17+I49N; 1.17+E27G+I49N; and 1.17+E27Q. In some embodiments, any amino acid change provided herein is substituted with a conservative amino acid. Additional mutations known in the art can also be added to any adenosine deaminase variant provided herein. In some embodiments, a base editor system comprising a CABE as provided herein has at least about 30%, 40%, 50%, 60%, 70% or more C to T editing activity in a target polynucleotide (e.g., DNA). In some embodiments, a base editor system comprising a CABE as provided herein has increased C to T base editing activity (e.g., at least about 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more) relative to a reference base editor system comprising a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19).

[1363] Table 6A. Adenosine deaminase variants. Reference TadA*8.20 indicates mutations. "S" indicates "surface" and "NAS" indicates "near active site."

[1364]

[1365] Table 6A (continued). Adenosine deaminase variants. Reference TadA*8.20 indicates mutations. "I" indicates "internal," "S" indicates "surface," and "NAS" indicates "near active site."

[1366]

[1367]

[1368] Table 6B. Adenosine deaminase variants. Reference TadA*8.20 indicates mutations.

[1369]

[1370]

[1371]

[1372]

[1373]

[1374]

[1375] Table 6C. Adenosine deaminase variants. Reference variant 1.2 (Table 6A) represents the mutation.

[1376]

[1377]

[1378] Table 6C. (Continued)

[1379]

[1380]

[1381] Table 6D. Adenosine deaminase variants. Reference TadA*8.20 indicates mutations.

[1382]

[1383]

[1384] Table 6E. Hybrid constructs. Reference TadA*7.10 indicates mutations.

[1385]

[1386]

[1387] Table 6F. Base editor variants. Reference TadA*8.19 / 8.20 represents mutations.

[1388] AA Position: 17 27 48 49 76 82 84 118 142 147 149 166 167 ABE8.19m / 8.20m T E A I Y / I S F M A Y F T D 1.1+8e(B879) H I M Y 1.2+8e(B879) H K I Y 1.12+8e(B879) A H M I L Y 1.17+8e(B879) A G T E Y 1.18+8e(B879) G Y 1.19+8e(B879) G N Y 1.1+8e(B882) H I M D Y I N 1.2+8e(B882) H K I D Y I N 1.12+8e(B882) A H M I L D Y I N 1.17+8e(B882) A G T E D Y I N 1.18+8e(B882) G D Y I N 1.19+8e(B882) G N D Y I N

[1389] Guide polynucleotide

[1390] The polynucleotide programmable nucleotide binding domain can specifically bind to the target polynucleotide sequence when bound to the bound guide polynucleotide (e.g., gRNA) (i.e., via complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby positioning the base editor to the target nucleic acid sequence that needs to be edited. In some embodiments, the target polynucleotide sequence includes single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence includes RNA. In some embodiments, the target polynucleotide sequence includes a DNA-RNA hybrid. In one embodiment, the guide polynucleotide described herein can be RNA or DNA. In one embodiment, the guide polynucleotide is gRNA. In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide comprises two or more separate polynucleotides that can interact with each other via, for example, complementary base pairing (e.g., dual guide polynucleotides, dual gRNAs). For example, the guide polynucleotide can comprise a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA) or can comprise one or more transactivating CRISPR RNAs (tracrRNAs). The guide polynucleotide can comprise natural or non-natural nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some cases, the targeting region of the guide nucleic acid sequence (e.g., spacer) can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNA (gRNA) is a short synthetic RNA that consists of a scaffold sequence necessary for Cas binding and a spacer of about 20 nucleotides defined by the user, which defines the genomic target to be modified. Exemplary gRNA scaffold sequences are provided as SEQ ID NOs: 317-327 and 425 in the sequence table. Therefore, technicians can change the specific genomic target of the Cas protein, depending in part on how specific the gRNA targeting sequence is to the genomic target compared to the rest of the genome. In embodiments, the length of the spacer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides. The length of the spacer of the gRNA can be or can be about 19, 20 or 21 nucleotides. In some embodiments, the composition comprises a plurality of gRNAs that all target the same exon or a plurality of gRNAs that target different exons. The exons and / or introns of a gene can be targeted. GRNA or guidance polynucleotides can target about 20 nucleotides or less than about 20 nucleotides (e.g., at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides) or about 1-100 between any number of nucleotides (e.g., 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100) nucleic acid sequence. The target nucleic acid sequence can be the 20 bases immediately 5' of the first nucleotide adjacent to the PAM or can be about 20 bases immediately 5' of the first nucleotide adjacent to the PAM. The gRNA can target the nucleic acid sequence. The target nucleic acid can be at least or about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides. A guide polynucleotide can comprise standard ribonucleotides, modified ribonucleotides (eg, pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs. In some embodiments, the base editor system may include multiple guide polynucleotides, such as gRNAs. For example, gRNAs may target one or more target loci (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs) contained in the base editor system. Multiple gRNA sequences can be arranged in series and can be separated by directional repeats. Modified polynucleotides To enhance expression, stability, and / or genome / base editing efficiency, and / or to reduce possible toxicity, base editor coding sequences (e.g., mRNA) and / or guide polynucleotides (e.g., gRNA) can be modified to include one or more modified nucleotides and / or chemical modifications, such as pseudouridine, 5-methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, 2'-O-methylthioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), tri-constrained ethyl (S-cEt), 2'-O-methyl ('M'), 2'-O-methyl-3'-thiophosphorothioate ('MS'), 2'-O-methyl-3'-thiophosphoroacetate ('MSP'), 5-methoxyuridine, thiophosphorothioate, and N1-methyl pseudouridine. Chemically protected gRNA can enhance in vivo and ex vivo stability and editing efficiency. Methods of using chemically modified mRNA and guide RNA are known in the art and are described, for example, by Jiang et al., Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). doi.org / 10.1038 / s41467-020-15892-8, Callum et al., N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Nucleic Acids Research, Vol. 48, No. 6, April 6, 2020, p. 35, and Andries et al., Journal of Controlled Release, Vol. 217, Nov. 10, 2015, pp. 337-344, each of which is herein incorporated by reference in its entirety. In some embodiments, the guide polynucleotide comprises one or more modified nucleotides at the 5' end and / or 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleotides at the 5' end and / or 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleotides at the 5' end and / or 3' end of the guide. In some embodiments, the guide comprises at least about 50%-75% modified nucleotides. In some embodiments, the guide comprises at least about 85% or more modified nucleotides. In some embodiments, at least about 1-5 nucleotides at the 5' end of the gRNA are modified and at least about 1-5 nucleotides at the 3' end of the gRNA are modified. In some embodiments, at least about 3-5 consecutive nucleotides at the 5' and 3' ends of the gRNA are modified. In some embodiments, at least about 20% of the nucleotides present in the direct repeat or inverted repeat are modified. In some embodiments, at least about 50% of the nucleotides present in the direct repeat sequence or inverted repeat sequence are modified. In some embodiments, at least about 50-75% of the nucleotides present in the direct repeat sequence or inverted repeat sequence are modified. In some embodiments, at least about 100 nucleotides present in the direct repeat sequence or inverted repeat sequence are modified. In some embodiments, at least about 20% or more of the nucleotides present in the hairpin present in the gRNA scaffold are modified. In some embodiments, at least about 50% or more of the nucleotides present in the hairpin present in the gRNA scaffold are modified. In some embodiments, the guide comprises a variable length spacer. In some embodiments, the guide comprises a 20-40 nucleotide spacer. In some embodiments, the guide comprises a spacer comprising at least about 20-25 nucleotides or at least about 30-35 nucleotides. In some embodiments, the spacer comprises a modified nucleotide. In some embodiments, the guide comprises two or more of the following: ● at least about 1-5 nucleotides at the 5' end of the gRNA are modified and at least about 1-5 nucleotides at the 3' end of the gRNA are modified; ● at least about 20% of the nucleotides present in the direct repeat or inverted repeat are modified; ● at least about 50-75% of the nucleotides present in the direct repeat or inverted repeat are modified; ● at least about 20% or more of the nucleotides present in the hairpin present in the gRNA scaffold are modified; ● variable length spacers; and ● A spacer comprising a modified nucleotide. In several embodiments, the gRNA contains many modified nucleotides and / or chemical modifications ("heavy modifications"). Such heavy modifications can increase base editing by about 2-fold in vivo or in vitro. In an embodiment, the gRNA comprises a 2'-O-methyl or a phosphorothioate modification. In one embodiment, the gRNA comprises a 2'-O-methyl and a phosphorothioate modification. In one embodiment, the modification increases base editing by at least about 2-fold. The guide polynucleotide may comprise one or more modifications to provide new or enhanced characteristics to the nucleic acid. The guide polynucleotide may comprise a nucleic acid affinity tag. The guide polynucleotide may comprise synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives and / or modified nucleotides. The gRNA or guide polynucleotide can also be modified with the following: 5' adenylate, 5' guanosine-triphosphate cap, 5' N7-methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' phosphorothioate, 5' phosphate, 5' phosphorothioate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 2'-O-methylthio PACE (MSP), 2'-O-methyl-PACE (MP) and constrained ethyl (S-cEt), 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bibiotin, PC biotin, psoralen C2, psoralen C6, TINA, 3' DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar modification analog, wobble / universal base, fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof. In some cases, the RNA gRNA enhanced by thiophosphate can inhibit RNase A, RNase T1, calf serum nuclease or any combination thereof. These properties can allow PS-RNA gRNA to be used in applications with a high probability of being exposed to nucleases in vivo or in vitro. For example, a thiophosphate (PS) bond can be introduced between the last 3-5 nucleotides at the 5' or 3' end of the gRNA, which can inhibit exonuclease degradation. In some cases, thiophosphate bonds can be added throughout the gRNA to reduce endonuclease attack. Fusion proteins or complexes containing a nuclear localization sequence (NLS) In some embodiments, the fusion protein or complex provided herein further comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as nuclear localization sequences (NLS). In one embodiment, a dichotomous NLS is used. In some embodiments, the NLS comprises an amino acid sequence that helps to import a protein (comprising an NLS) into the nucleus (e.g., by nuclear transport). In some embodiments, the NLS is fused to the N-terminus or C-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus or N-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of a Cas12 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of a cytidine or adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more joints. In some embodiments, the NLS is fused to the fusion protein in the absence of a joint. In some embodiments, the NLS comprises the amino acid sequence of any NLS sequence provided herein or mentioned. Additional nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in Plank et al., PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS is present in a linker or linker-flanking NLS, such as described herein. A bipartite NLS comprises two clusters of basic amino acids separated by a relatively short spacer sequence (thus bipartite - 2 parts, whereas a monocomponent NLS is not). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 191), is a prototype of the ubiquitous bipartite signal: two clusters of basic amino acids separated by a spacer of approximately 10 amino acids. The sequence of an exemplary bipartite NLS is as follows: PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328). In some embodiments, any fusion protein or complex provided herein comprises an NLS comprising EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO 328). In some embodiments, any adenosine base editor provided herein (e.g., ABE variant A, ABE variant B, ABE variant C, ABE variant D, ABE variant E, ABE variant F, ABE variant G, ABE variant H, ABE variant I, ABE variant J, ABE variant K, or ABE variant D) comprises an NLS comprising the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO: 328). In some embodiments, the NLS is located at the C-terminal portion of the adenosine base editor. In some embodiments, the NLS is located at the C-terminus of the adenosine base editor. Additional domains The base editors described herein may include any domain that helps promote the editing, modification or change of the core bases of the polynucleotides. In some embodiments, the base editor includes a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a core base editing domain (e.g., a deaminase domain) and one or more additional domains. In some embodiments, the additional domain may contribute to the enzyme or catalytic function of the base editor, the binding function of the base editor, or an inhibitor (e.g., enzyme) of the cellular mechanism that may interfere with the desired base editing results. In some embodiments, the base editor includes a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcription activator or a transcription repressor domain. In some embodiments, the base editor comprises a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the cellular DNA repair response to the presence of U:G heteroduplex DNA can result in reduced efficiency of base editing in the cell. In such embodiments, uracil DNA glycosylase (UDG) can catalyze the removal of U from the DNA in the cell, which can initiate base excision repair (BER), mainly causing the U:G pair to revert to a C:G pair. In such embodiments, BER can be suppressed in a base editor comprising one or more domains that bind to a single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base and / or promote the repair of the non-edited chain. Therefore, the present disclosure contemplates a base editor fusion protein or complex comprising a UGI domain and / or a uracil stabilizing protein (USP) domain. Base editor system Provided herein are systems, compositions, and methods for editing nuclear bases using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE), which comprises a polynucleotide programmable nucleotide binding domain and a nuclear base editing domain (e.g., a deaminase domain) for editing nuclear bases; and (2) a guidance polynucleotide (e.g., a guide RNA) bound to the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system is a cytidine base editor (CBE) or an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA or RNA binding domain. In some embodiments, the nuclear base editing domain is a deaminase domain. In some embodiments, the deaminase domain can be a cytidine deaminase or a cytosine deaminase. In some embodiments, the deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, the base editor can deaminate cytidine in DNA. The use of the base editor system provided herein comprises the following steps: (a) contacting the target nucleotide sequence of the subject's polynucleotide (e.g., double-stranded or single-stranded DNA or RNA) with a base editor system comprising a base editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleic acid (e.g., gRNA), wherein the target nucleotide sequence comprises a targeted base pair; (b) inducing the chain separation of the target region; (c) converting the first base of the target base pair in the single strand of the target region to a second base; and (d) cutting no more than one chain of the target region, wherein the third base complementary to the first base is replaced by a fourth base complementary to the second base. It should be understood that in some embodiments, step (b) is omitted. In some embodiments, the targeted base pair is a plurality of base pairs in one or more genes. In some embodiments, the base editor system provided herein is capable of multiple editing of a plurality of base pairs in one or more genes. In some embodiments, a plurality of base pairs are located in the same gene. In some embodiments, the plurality of nucleobase pairs are located in one or more genes, wherein at least one gene is located in a different locus. The components of the base editor system (e.g., deaminase domains, guide RNAs, and / or polynucleotide programmable nucleotide binding domains) may be covalently or non-covalently associated with each other. For example, in some embodiments, the deaminase domain may be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain, optionally wherein the polynucleotide programmable nucleotide binding domain is compounded with a polynucleotide (e.g., a guide RNA). In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or connected to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain may target the deaminase domain to a target nucleotide sequence by non-covalent interaction or association with the deaminase domain. For example, in some embodiments, the core base editing component (e.g., a deaminase component) comprises an additional heterologous portion or domain that is capable of interacting, associating, or forming a complex with a corresponding heterologous portion, antigen, or domain that is a portion of a polynucleotide programmable nucleotide binding domain and / or a guide polynucleotide (e.g., a guide RNA) compounded therewith. In some embodiments, the polynucleotide programmable nucleotide binding domain and / or the guide polynucleotide (e.g., guide RNA) compounded therewith comprises another heterologous portion or domain that can interact, associate or form a complex with a corresponding heterologous portion, antigen or domain that is a part of a core base editing domain (e.g., a deaminase component). In some embodiments, another heterologous portion may be able to bind to, interact with, associate with or form a complex with a polypeptide. In some embodiments, another heterologous portion may be able to bind to, interact with, associate with or form a complex with a polynucleotide. In some embodiments, another heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, another heterologous portion may be able to bind to a polypeptide linker. In some embodiments, another heterologous portion may be able to bind to a polynucleotide linker. Another heterologous portion may be a protein domain.In some embodiments, the additional heterologous portion includes a polypeptide, such as the 22 amino acid RNA binding domain of phage lambda anti-terminator protein N (N22p), a 2G12 IgG homodimer domain, an ABI, an antibody (e.g., an antibody that binds a component of a base editor system or a heterologous portion thereof) or a fragment thereof (e.g., heavy chain domain 2 (CH2) of IgM (MHD2) or heavy chain domain 2 (EHD2) of IgE, an immunoglobulin Fc region, heavy chain domain 3 (CH3) of IgG or IgA, heavy chain domain 4 (CH4) of IgM or IgE, Fab, Fab2, minibody and / or ZIP antibody), a barnase-barstar dimer domain, a Bcl-xL domain, a calcineurin A (CAN) domain, a cardiac phospholamban transmembrane pentamer domain, a collagen domain, a ComRNA binding protein domain (e.g., SfMu Com coat protein domain and SfMu Com binding protein domain), cyclophilin-Fas fusion protein (CyP-Fas) domain, Fab domain, Fc domain, cellulose protein foldon domain, FK506 binding protein (FKBP) domain, FKBP binding domain (FRB) of mTOR, foldon domain, fragment X domain, GAI domain, GID1 domain, glycophorin A transmembrane domain, GyrB domain, Halo tag, HIV Gp41 trimerization domain, HPV45 oncoprotein E7 C-terminal dimerization domain, hydrophobic polypeptide, K homology (KH) domain, Ku protein domain (e.g., Ku heterodimer), leucine zipper, LOV domain, mitochondrial antiviral signaling protein CARD filament domain, MS2 coat protein domain (MCP), non-natural RNA aptamer ligand that binds to the corresponding RNA motif / aptamer, parathyroid hormone dimerization domain, PP7 coat protein (PCP) domain, PSD95-Dlgl-zo-1 (PDZ) domain, PYL domain, SNAP tag, SpyCatcher portion, SpyTag portion, streptavidin domain, streptavidin binding protein domain, streptavidin binding protein (SBP) domain, telomerase Sm7 protein domain (e.g., Sm7 homoheptamer or monomeric Sm-like protein) and / or fragments thereof. In embodiments, the additional heterologous portion comprises a polynucleotide (e.g., an RNA motif) such as an MS2 phage operator stem-loop (e.g., MS2, MS2 C-5 mutant, or MS2 F-5 mutant), a non-natural RNA motif, a PP7 operator stem-loop, a SfMu phate Com stem-loop, a sterile alpha motif, a telomerase Ku binding motif, a telomerase Sm7 binding motif, and / or fragments thereof.Non-limiting examples of additional heterologous moieties include polypeptides having at least about 85% sequence identity to any one or more of SEQ ID NOs: 380, 382, ​​384, 386-388, or fragments thereof. Non-limiting examples of additional heterologous moieties include polynucleotides having at least about 85% sequence identity to any one or more of SEQ ID NOs: 379, 381, 383, 385, or fragments thereof. In some cases, the components of the base editing system are associated with each other through the interaction of leucine zipper domains (e.g., SEQ ID NOs: 387 and 388). In some cases, the components of the base editing system are associated with each other through polypeptide domains (e.g., FokI domains), which associate to form a protein complex containing about, at least about, or no more than about 1, 2 (i.e., dimerization), 3, 4, 5, 6, 7, 8, 9, 10 polypeptide domain units, optionally including changes that reduce or eliminate their activity. In some cases, the components of the base editing system associate with each other through the interaction of multimeric antibodies or fragments thereof (e.g., heavy chain domain 2 (CH2) of IgG, IgD, IgA, IgM, IgE, IgM (MHD2) or IgE (EHD2), immunoglobulin Fc region, heavy chain domain 3 (CH3) of IgG or IgA, heavy chain domain 4 (CH4) of IgM or IgE, Fab and Fab2). In some cases, the antibody is dimeric, trimeric or tetrameric. In embodiments, the dimeric antibody binds to a polypeptide or polynucleotide component of the base editing system. In some cases, the components of the base editing system associate with each other through the interaction of a polynucleotide binding protein domain with a polynucleotide. In some cases, the components of the base editing system associate with each other through the interaction of one or more polynucleotide binding protein domains with self-complementary and / or mutually complementary polynucleotides, such that the complementary binding of the polynucleotides to each other associates the polynucleotide binding protein domains to which they are each bound. In some cases, the components of the base editing system are associated with each other through the interaction of a polypeptide domain with a small molecule (e.g., a chemical inducer of dimerization (CID), also referred to as a "dimerizer"). Non-limiting examples of CIDs include those disclosed in Amara et al., "A versatile synthetic dimerizer for the regulation of protein-protein interactions," PNAS, 94: 10618-10623 (1997); and Voβ et al., "Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells," Current Opinion in Chemical Biology, 28: 194-201 (2015), the disclosures of each of which are incorporated herein by reference in their entirety for all purposes. In some embodiments, the base editor inhibits base excision repair (BER) of the edited chain. In some embodiments, the base editor protects or binds to the unedited chain. In some embodiments, the base editor includes UGI activity or USP activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease.

[1391] The base editor of the present disclosure may include any domain, feature or amino acid sequence that contributes to the editing of the target polynucleotide sequence. For example, in some embodiments, the base editor includes a nuclear localization sequence (NLS). In some embodiments, the NLS of the base editor is located between the deaminase domain and the polynucleotide programmable nucleotide binding domain. In some embodiments, the NLS of the base editor is located at the C-terminus of the polynucleotide programmable nucleotide binding domain.

[1392] The protein domain contained in the fusion protein can be a heterologous functional domain. Non-limiting examples of protein domains that can be contained in the fusion protein include deaminase domains (e.g., cytidine deaminase or adenosine deaminase), uracil glycosylase inhibitor (UGI) domains, epitope tags, and reporter gene sequences.

[1393] In some embodiments, an adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, an ABE is generated by replacing the APOBEC1 component of BE3 with natural or engineered Escherichia coli TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, the ABE comprises an evolved TadA variant. In some embodiments, the base editor is ABE8.1, which comprises the following sequence or a fragment thereof having adenosine deaminase activity or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: SEQ ID NO: 331. Other ABE8 sequences are provided in the attached sequence listing (SEQ ID NOs: 332-354).

[1394] In some embodiments, the base editor comprises an adenosine deaminase variant comprising an amino acid sequence comprising an alteration relative to the ABE7*10 reference sequence as described herein. The term "monomer" as used in Table 7 refers to a monomeric form of TadA*7.10 comprising the alteration. The term "heterodimer" as used in Table 7 refers to a specified wild-type E. coli TadA adenosine deaminase fused to TadA*7.10 comprising the alteration.

[1395] Table 7. Adenosine deaminase base editor variants

[1396]

[1397] In some embodiments, the base editor comprises a domain containing all or a portion (e.g., a functional portion) of a uracil glycosylase inhibitor (UGI) or a uracil stabilizing protein (USP) domain. connector In certain embodiments, a linker can be used to connect any peptide or peptide domain of the present disclosure. The linker can be as simple as a covalent bond, or it can be a polymeric linker of many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.). In some embodiments, any of the fusion proteins provided herein comprise a cytidine or adenosine deaminase and a Cas9 domain fused to each other via a linker. Various linker lengths and flexibilities between the cytidine or adenosine deaminase and the Cas9 domain can be employed (e.g., ranging from very flexible linker formats (GGGS)n (SEQ ID NO: 246), (GGGGS)n (SEQ ID NO: 247), and (G)n to more rigid linker formats (EAAAK)n (SEQ ID NO: 248), (SGGS)n (SEQ ID NO: 355), SGSETPGTSESATPES (SEQ ID NO: 249) (see, e.g., Guilinger JP et al. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; herein incorporated by reference in its entirety), and (XP)n) to obtain the optimal activity length for the cytidine or adenosine deaminase nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, the cytidine deaminase or adenosine deaminase and Cas9 domains of any fusion protein provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which may also be referred to as an XTEN linker. In some embodiments, the domain of the base editor is fused via a linker comprising the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 356), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 357) or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 358). In some embodiments, the domain of the base editor is fused via a linker (also referred to as an XTEN linker) comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 355). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 359). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 360). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 361). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 362). In some embodiments, the linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, or 5-7 amino acids in length, e.g., PAPAP (SEQ ID NO: 363), PAPAPA (SEQ ID NO: 364), PAPAPAP (SEQ ID NO: 365), PAPAPAPA (SEQ ID NO: 366), P(AP)4 (SEQ ID NO: 367), P(AP)7 (SEQ ID NO: 368), P(AP)10 (SEQ ID NO: 369) (see, e.g., Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan 25; 10(1):439; incorporated herein by reference in its entirety). Such proline-rich linkers are also referred to as "rigid" linkers. Nucleic acid-programmable DNA-binding proteins with guide RNA Provided herein are compositions and methods for base editing in cells. Also provided herein are compositions comprising guiding polynucleic acid sequences, such as guiding RNA sequences, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more guide RNAs as provided herein. In some embodiments, the compositions for base editing as provided herein also include polynucleotides encoding base editors (e.g., C-base editors or A-base editors). For example, the composition for base editing may include an mRNA sequence encoding a combination of BE, BE4, ABE and one or more guide RNAs provided. The composition for base editing may include a base editor polypeptide and one or more combinations of any guide RNAs provided herein. This composition can be used to achieve base editing in cells by different delivery methods, such as by electroporation, nuclear transfection, viral transduction or transfection. In some embodiments, a composition for base editing comprises a combination of an mRNA sequence encoding a base editor and one or more guide RNA sequences for electroporation provided herein. Some aspects of the present disclosure provide systems comprising any fusion protein or complex provided herein and a guide RNA bound to a nucleic acid programmable DNA binding protein (napDNAbp) domain (e.g., Cas9 (e.g., dCas9, nuclease-active Cas9 or Cas9 cutter) or Cas12) of the fusion protein or complex. These complexes are also referred to as ribonucleoproteins (RNPs). In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15-100 nucleotides long and comprises a sequence of at least 10 consecutive nucleotides complementary to the target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is an RNA sequence. In some embodiments, the target sequence is a sequence in the genome of bacteria, yeast, fungi, insects, plants, or animals. In some embodiments, the target sequence is a sequence in the human genome. In some embodiments, the 3' end of the target sequence is adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 3 or 5'-NAA-3'). In some embodiments, a guide nucleic acid (e.g., a guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder). Some aspects of the present disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of the present disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein and at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 consecutive nucleotides that are complementary to the target sequence. The domains of the base editors disclosed herein can be arranged in any order. The defined target region can be a deamination window. The deamination window can be a defined region in which a base editor acts on a target nucleotide and deaminates the target nucleotide. In some embodiments, the deamination window is within a region of 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM. The base editors of the present disclosure may comprise any domain, feature, or amino acid sequence that facilitates editing of a target polynucleotide sequence. Methods using fusion proteins or complexes comprising cytidine or adenosine deaminase and Cas9 domains Some aspects of the present disclosure provide methods of using the fusion proteins or complexes provided herein.For example, some aspects of the present disclosure provide methods comprising contacting a DNA molecule with any fusion protein or complex provided herein and at least one guide RNA described herein. In some embodiments, the fusion proteins or complexes disclosed herein are used to edit a target gene of interest. Specifically, the cytidine deaminase or adenosine deaminase nucleobase editors described herein are capable of making multiple mutations within a target sequence. These mutations can affect the function of the target. For example, when a cytidine deaminase or adenosine deaminase nucleobase editor is used to target a regulatory region, the function of the regulatory region is altered and the expression of downstream proteins is reduced or eliminated. Base editor efficiency In some embodiments, the purpose of the method provided herein is to change genes and / or gene products via gene editing. The core base editing proteins provided herein can be used for human treatment based on gene editing in vitro or in vivo. It will be understood by technicians that the core base editing proteins provided herein (e.g., fusion proteins or complexes comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a core base editing domain (e.g., adenosine deaminase domain or cytidine deaminase domain)) can be used to edit nucleotides from A to G or C to T. Advantageously, the base editing system as provided herein provides genome editing without generating double-stranded DNA breaks, does not require a donor DNA template, and does not induce excessive random insertions and deletions as is possible with CRISPR. In some embodiments, the disclosure provides base editors that effectively generate expected mutations (such as stop codons) in nucleic acids (e.g., nucleic acids within a subject's genome) without generating a large number of unexpected mutations (such as unexpected point mutations). The base editors of the present disclosure advantageously modify specific nucleotide bases encoding proteins without generating a significant proportion of indels (i.e., insertions or deletions). Such indels can lead to frameshift mutations within the coding region of a gene. In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels (i.e., intended point mutations: unintended point mutations) greater than 1:1. In some embodiments, the base editors provided herein are capable of generating a ratio of expected mutations to indels of at least 1.5: 1, at least 2: 1, at least 2.5: 1, at least 3: 1, at least 3.5: 1, at least 4: 1, at least 4.5: 1, at least 5: 1, at least 5.5: 1, at least 6: 1, at least 6.5: 1, at least 7: 1, at least 7.5: 1, at least 8: 1, at least 10: 1, at least 12: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 100: 1, at least 200: 1, at least 300: 1, at least 400: 1, at least 500: 1, at least 600: 1, at least 700: 1, at least 800: 1, at least 900: 1, or at least 1000: 1 or greater. The number of expected mutations and indels can be determined using any suitable method. In some embodiments, the base editors provided herein can limit the formation of indels in a nucleic acid region. In some embodiments, the region is located at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any base editor provided herein can limit the formation of indels at a nucleic acid region to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%.

[1398] Base editing is often referred to as a "modification," such as genetic modification, gene modification, and modification of a nucleic acid sequence, and is clearly understood based on the context that the modification is a base editing modification. Thus, a base editing modification is a modification at the nucleotide base level (e.g., due to deaminase activity as discussed throughout the disclosure), which then results in a change in the gene sequence and may affect the gene product.

[1399] In some embodiments, the modification (e.g., single base editing) results in a decrease in targeted gene expression by about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or to undetectable levels.

[1400] The present disclosure provides adenosine deaminase variants (e.g., ABE8 variants) with increased efficiency and specificity. Specifically, the adenosine deaminase variants described herein are more likely to edit the desired base within a polynucleotide and less likely to edit unintended altered bases (e.g., "bystander").

[1401] In some embodiments, any base editing system described herein comprising one of the ABE8 base editor variants has at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less bystander editing or mutations compared to a base editor system comprising an ABE7 base editor (e.g., ABE7.10).

[1402] In some embodiments, any of the ABE8 base editor variants described herein have a higher base editing efficiency compared to the ABE7 base editor. In some embodiments, any of the ABE8 base editor variants described herein have a base editing efficiency that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 200%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 510%, 520%, 530%, 540%, 550%, 5%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 450% or 500% base editing efficiency. The ABE8 base editor variants described herein can be delivered to host cells via plasmids, vectors, LNP complexes, or mRNA. In some embodiments, any ABE8 base editor variant described herein is delivered to host cells as mRNA. In some embodiments, the methods described herein (e.g., base editing methods) have minimal to no off-target effects. In some embodiments, the methods described herein (e.g., base editing methods) have minimal to no chromosomal translocations. In some embodiments, the base editing methods described herein result in about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a cell population being successfully edited. In some embodiments, the percentage of live cells in the cell population after base editing intervention is greater than at least 60%, 70%, 80%, or 90% of the starting cell population at the time of the base editing event. In some embodiments, the percentage of live cells in the cell population after editing is about 70%. In some embodiments, the percentage of live cells in the cell population after editing is about 75%. In some embodiments, the percentage of live cells in the cell population after editing is about 80%. In some embodiments, the percentage of live cells in the cell population as described above is about 85%. In some embodiments, the percentage of live cells in the cell population as described above is about 90%, or about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the population at the time of the base editing event. In embodiments, the population of cells is a population of cells contacted with a base editor, complex, or base editor system of the present disclosure.

[1403] Any suitable method can be used to determine the number of expected mutations and indels, such as those described in International PCT Application Nos. PCT / US2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632); Komor, AC et al., “Programmable editing of a target base in genomic DNAwithout double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, AC et al., “Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product "purity" Science Advances 3:eaao4774 (2017); the entire contents of which are hereby incorporated by reference.

[1404] In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences flanking the window in which indels may occur. If no exact match is found, the read is excluded from the analysis. If the length of this indel window exactly matches the reference sequence, the read is classified as not containing indels. If the indel window is two or more bases longer or shorter than the reference sequence, the sequencing reads are classified as insertions or deletions, respectively. In some embodiments, the base editors provided herein can limit the formation of indels in nucleic acid regions. In some embodiments, the region is located at the nucleotide targeted by the base editor or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the base editor.

[1405] Multiple Editing

[1406] In some embodiments, the base editor system provided herein is capable of multiple editing of multiple nuclear base pairs in one or more genes or polynucleotide sequences. In some embodiments, multiple nuclear base pairs are located in the same gene or one or more genes, wherein at least one gene is located in a different locus. In some embodiments, multiple editing includes one or more guide polynucleotides. In some embodiments, multiple editing includes one or more base editor systems. In some embodiments, multiple editing includes one or more base editor systems, which have a single guide polynucleotide or multiple guide polynucleotides. In some embodiments, multiple editing includes one or more guide polynucleotides and a single base editor system. It should be understood that the features of multiple editing using any base editor as described herein can be applied to any combination of methods using any base editor provided herein. It should also be understood that multiple editing using any base editor as described herein can include continuous editing of multiple nuclear base pairs. In some embodiments, a base editor system capable of multiplexed editing of multiple nucleobase pairs in one or more genes comprises one of the ABE7, ABE8, and / or ABE9 base editors. Reduce the expression of target genes in cells In some embodiments, provided herein are cells (e.g., cells from the liver, eyes, and / or central nervous system, or components thereof) having at least one modification in an endogenous gene or one or more regulatory elements thereof. Methods, base editors, base editor systems, guide RNAs, and compositions for modifying cells are also provided herein. In some embodiments, cells may comprise further modifications in at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more endogenous genes or their regulatory elements. In some embodiments, at least one modification is a single nuclear base modification. In some embodiments, at least one modification is generated by base editing. Base editing may be located at any suitable position of a gene, or in a regulatory element of a gene. Therefore, it is understood that, for example, a single base editor at a start codon can completely eliminate gene expression. In some embodiments, base editing may be performed at a site within an exon. In some embodiments, base editing may be performed at a site exceeding one exon. In some embodiments, base editing may be performed at any exon of multiple exons in a gene. In some embodiments, base editing may introduce a premature stop codon into an exon, resulting in a lack of translation product or a truncation that may be misfolded and thus eliminated by degradation, or base editing may generate unstable mRNA that is easily degraded. In some embodiments, the cell is a hepatocyte, and / or a cell from the liver, eye, and / or central nervous system, or a component thereof. In some embodiments, the gene is a C3 polynucleotide. In some embodiments, the editing of an endogenous gene reduces the expression of a gene. In some embodiments, the editing of an endogenous gene reduces the expression of a gene by at least 50% compared to a control cell without modification. In some embodiments, the editing of an endogenous gene reduces the expression of a gene by at least 60% compared to a control cell without modification. In some embodiments, the editing of an endogenous gene reduces the expression of a gene by at least 70% compared to a control cell without modification. In some embodiments, the editing of an endogenous gene reduces the expression of a gene by at least 80% compared to a control cell without modification. In some embodiments, the editing of an endogenous gene reduces the expression of a gene by at least 90% compared to a control cell without modification. In some embodiments, the editing of an endogenous gene reduces the expression of a gene by at least 100% compared to a control cell without modification. In some embodiments, the editing of an endogenous gene eliminates gene expression. In some embodiments, base editing can be performed on an intron. For example, base editing can be performed on an intron. In some embodiments, base editing can be performed at a site within an intron. In some embodiments, base editing can be performed at a site in one or more introns. In some embodiments, base editing can be performed at any intron among multiple introns in a gene. In some embodiments, one or more base editing can be performed on an exon, an intron, or any combination of exons and introns. In some embodiments, the modification or base editing may be within a promoter site. In some embodiments, base editing may be introduced within an alternative promoter site. In some embodiments, base editing may be within a 5' regulatory element, such as an enhancer. In some embodiments, base editing may be introduced to disrupt the binding site of a nucleic acid binding protein. Exemplary nucleic acid binding proteins may be polymerases, nucleases, gyrases, topoisomerases, methylases or methyltransferases, transcription factors, enhancers, PABPs, zinc finger proteins, and the like. In some embodiments, base editing can be used for splicing disruption to silence target protein expression. In some embodiments, base editing can produce splice acceptor-splice donor (SA-SD) sites. Generating SA-SD or targeted base editing at SA-SD sites can result in reduced gene expression. In some embodiments, base editors (e.g., ABE, CBE, or CABE) are used to target the dinucleotide motifs constituting splice acceptor and splice donor sites, which are the first and last two nucleotides of each intron. In some embodiments, splicing disruption is achieved with adenosine base editors (ABE). In some embodiments, splicing disruption is achieved with cytidine base editors (CBE). In some embodiments, base editors (e.g., ABE, CBE, or CABE) are used to edit exons by creating stop codons. In some embodiments, the modification generates a premature stop codon in an endogenous gene. In some embodiments, the stop codon silences target protein expression. In some embodiments, the modification is a single base modification. In some embodiments, the modification is generated by base editing. Premature stop codons may be generated in exons, introns, or untranslated regions. In some embodiments, base editing can be used to introduce more than one stop codon in one or more alternative reading frames. In some embodiments, the stop codon is generated by an adenosine base editor (ABE). In some embodiments, the stop codon is generated by a cytidine base editor (CBE). In some embodiments, CBE generates any one of the following edits (shown in underlined font) to generate a stop codon: CAG→TAG; CAA→TAA; CGA→TGA; TGG → TGA ; TGG → TAG ;or TGG → TAA . In some embodiments, modifications / base editing can be introduced at the 3'-UTR, for example, in the polyadenylation (poly-A) site. In some embodiments, base editing can be performed on the 5'-UTR region. Delivery system Nucleic acid-based delivery of base editor systems Nucleic acid molecules encoding base editor systems according to the present disclosure can be administered to subjects or delivered to cells in vitro or in vivo by methods known in the art or as described herein. For example, a base editor system comprising a deaminase (e.g., cytidine or adenine deaminase) can be delivered by a vector (e.g., a viral or non-viral vector), or by naked DNA, DNA complexes, lipid nanoparticles, or a combination of the foregoing components. The base editor system can be delivered to cells using any method available in the art, including but not limited to physical methods (e.g., electroporation, particle guns, calcium phosphate transfection), viral methods, non-viral methods (e.g., liposomes, cationic methods, lipid nanoparticles, polymeric nanoparticles), or biological non-viral methods (e.g., attenuated bacteria, engineered phages, mammalian virus-like particles, biological liposomes, erythrocyte ghosts, exosomes). Nanoparticles (which may be organic or inorganic) can be used to deliver base editor systems or components thereof. Nanoparticles are well known in the art, and any suitable nanoparticles can be used to deliver base editor systems or components thereof, or nucleic acid molecules encoding such components. In one example, organic (e.g., lipid and / or polymer) nanoparticles are suitable for use as delivery vehicles in certain embodiments of the present disclosure. Non-limiting examples of lipid nanoparticles suitable for use in the disclosed methods include those described in International Patent Application Publication Nos. WO2022140239, WO2022140252, WO2022140238, WO2022159421, WO2022159472, WO2022159475, WO2022159463, WO2021113365, and WO2021141969, the disclosure of each of which is incorporated herein by reference in its entirety for all purposes. viral vectors The base editors described herein can be delivered with a viral vector. In some embodiments, the base editors disclosed herein can be encoded on a nucleic acid contained in a viral vector. In some embodiments, one or more components of the base editor system can be encoded on one or more viral vectors. Viral vectors can include lentiviruses (e.g., vectors based on HIV and FIV), adenoviruses (e.g., AD100), retroviruses (e.g., Moloney murine leukemia virus, MML-V), herpes virus vectors (e.g., HSV-2), and adeno-associated virus (AAV), or other plasmid or viral vector types, particularly using formulations and dosages from, for example, U.S. Pat. No. 8,454,972 (formulations, doses of adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses of AAV), and U.S. Pat. No. 5,846,946 (formulations, doses of DNA plasmids), as well as from clinical trials involving lentiviruses, AAV, and adenoviruses and publications on such clinical trials. For example, for AAV, the route of administration, formulation, and dosage can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV. For adenovirus, the route of administration, formulation, and dosage can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dosage can be as in U.S. Patent No. 5,846,946 and clinical studies involving plasmids. The dosage can be based on or extrapolated to an average of 70 kg of individuals (e.g., male adults) and can be adjusted for patients, subjects, and mammals of different weights and species. The frequency of administration is within the authority of a medical or veterinary practitioner (e.g., physician, veterinarian), depending on common factors, including the patient's or subject's age, sex, general health, other illnesses, and the specific illness or symptom addressed. The viral vector can be injected into the tissue of interest. For cell type-specific base editing, the expression of the base editor and the optional guide nucleic acid can be driven by a cell type-specific promoter. The viral vector can be selected based on the application. For example, AAV may be superior to other viral vectors for in vivo gene delivery. In some embodiments, AAV allows for low toxicity, which may be due to the purification method not requiring ultracentrifugation of cell pellets that can activate an immune response. In some embodiments, AAV allows for a low probability of causing insertional mutagenesis because it does not integrate into the host genome. Adenoviruses are often used as vaccines because they induce strong immunogenic responses. The packaging capacity of viral vectors can limit the size of base editors that can be packaged into the vector. AAV has a packaging capacity of about 4.5 Kb or 4.75 Kb, including two inverted terminal repeats (ITRs) of 145 bases. This means that the disclosed base editors, as well as promoters and transcription terminators, can be assembled into a single viral vector. Constructs larger than 4.5 or 4.75 Kb can result in a significant reduction in virus production. For example, SpCas9 is quite large, and the gene itself exceeds 4.1 Kb, which makes it difficult to package into AAV. Therefore, embodiments of the present disclosure include utilizing a disclosed base editor that is shorter than a conventional base editor. In some examples, the base editor is less than 4 kb. The disclosed base editors may be less than 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, 4 kb, 3.9 kb, 3.8 kb, 3.7 kb, 3.6 kb, 3.5 kb, 3.4 kb, 3.3 kb, 3.2 kb, 3.1 kb, 3 kb, 2.9 kb, 2.8 kb, 2.7 kb, 2.6 kb, 2.5 kb, 2 kb, or 1.5 kb. In some embodiments, the disclosed base editors are 4.5 kb or less in length.

[1407] The AAV can be AAV1, AAV2, AAV5, AAV6, or any combination thereof. The type of AAV can be selected based on the cells to be targeted; for example, AAV serotypes 1, 2, 5, or hybrid capsids AAV1, AAV2, AAV5, or any combination thereof can be selected for targeting brain or neuronal cells; and AAV4 can be selected for targeting cardiac tissue. AAV8 can be used for delivery to the liver. A list of certain AAV serotypes for these cells can be found in Grimm, D. et al., J. Virol. 82:5887-5911 (2008).

[1408] In some embodiments, a lentiviral vector is used to transduce cells of interest with a polynucleotide encoding a base editor or base editor system as provided herein. Lentiviruses are complex retroviruses that have the ability to infect and express their genes in mitotic and post-mitotic cells. The most common lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a wide range of cell types.

[1409] In another embodiment, minimal non-primate lentiviral vectors based on equine infectious anemia virus (EIAV) are also contemplated. is a lentiviral gene therapy vector based on equine infectious anemia virus that expresses the angiogenesis inhibitory proteins endostatin and angiostatin, which is intended to be delivered via subretinal injection. In another embodiment, the use of a self-inactivating lentiviral vector is contemplated.

[1410] Any RNA of the system, such as a guide RNA or base editor encoding mRNA, can be delivered in the form of RNA. Base editor encoding mRNA can be generated using in vitro transcription. For example, a nuclease mRNA can be synthesized using a PCR cassette containing the following elements: a T7 promoter, an optional kozak sequence (GCCACC), a nuclease sequence, and a 3'UTR, such as a 3'UTR from a β-globin-polyA tail. The cassette can be used for transcription of T7 polymerase. In vitro transcription can also be used to transcribe a guide polynucleotide (e.g., gRNA) from a cassette containing a T7 promoter, followed by the sequence "GG" and a guide polynucleotide sequence.

[1411] Non-viral platforms for gene transfer

[1412] Non-viral platforms for introducing heterologous polynucleotides into cells of interest are known in the art. For example, present disclosure provides the method that heterologous polynucleotide is inserted into the genome of cell using Cas9 or Cas12 (for example, Cas12b) ribonucleoprotein complex (RNP)-DNA template complex, wherein RNP comprises Cas9 or Cas12 nuclease domain and guide RNA, wherein the guide RNA is specifically hybridized with the target region of the genome of cell, and wherein Cas nuclease domain cuts target region to create insertion site in the genome of cell.Then heterologous polynucleotide is introduced using DNA template.In embodiments, DNA template is double-stranded or single-stranded DNA template, and wherein the size of DNA template is about 200 nucleotides or greater than about 200 nucleotides, and wherein 5' end and 3' end of DNA template comprise nucleotide sequence homologous to the genomic sequence of flanking insertion site.In some embodiments, DNA template is single-stranded circular DNA template.In embodiments, the molar ratio of RNP to DNA template in complex is about 3:1 to about 100:1. In some embodiments, the DNA template is a linear DNA template. In some instances, the DNA template is a single-stranded DNA template. In certain embodiments, the single-stranded DNA template is a pure single-stranded DNA template. In some embodiments, the single-stranded DNA template is a single-stranded oligodeoxynucleotide (ssODN). In other embodiments, single-stranded DNA (ssDNA) can produce efficient HDR with minimal off-target integration. In one embodiment, ssDNA phage is used to efficiently and cheaply grow circular ssDNA (cssDNA) donors. When used with Cas9 or Cas12 (e.g., Cas12a, Cas12b), these cssDNA donors serve as effective HDR templates with better integration frequencies than linear ssDNA (lssDNA) donors. Inteins Inteins (intermediate proteins) are autoprocessing domains present in a variety of different organisms that carry out a process known as protein splicing. Non-limiting examples of inteins include any intein or intein pair known in the art, including synthetic inteins based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pairs have been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference), and DnaE. Non-limiting examples of intein pairs that can be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and CnePrp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference). Exemplary nucleotide and amino acid sequences of inteins are provided in the sequence listing as SEQ ID NOs: 370-377 and 389-424. Inteins suitable for use in the disclosed embodiments and methods of use thereof are described in U.S. Patent No. 10,526,401, International Patent Application Publication No. WO2013 / 045632, and U.S. Patent Application Publication No. US 2020 / 0055900, the complete disclosures of which are incorporated herein by reference in their entirety for all purposes. Intein-N and intein-C can be fused to the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9, respectively, to connect the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split-Cas9, forming a structure of N--[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split-Cas9, forming a structure of N--[intein-C]--[C-terminal portion of split-Cas9]-C. In embodiments, the base editor is encoded by two polynucleotides, one of which encodes a base editor fragment fused to intein-N and the other polynucleotide encodes a base editor fragment fused to intein-C. Methods for designing and using inteins are known in the art and are described by, for example, WO2014004336, WO2017132580, WO2013045632A1, US20150344549, and US20180127780, each of which is herein incorporated by reference in its entirety. In some embodiments, the ABE is cleaved into N-terminal and C-terminal fragments at Ala, Ser, Thr, or Cys residues within selected regions of SpCas9. These regions correspond to loop regions identified by Cas9 crystal structure analysis. The N-terminus of each fragment was fused to intein-N, and the C-terminus of each fragment was fused to intein-C at amino acid positions S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, and S590 (referring to SEQ ID NO: 197). Pharmaceutical composition In some aspects, the present disclosure provides a pharmaceutical composition comprising any of the cells, polynucleotides, vectors, base editors, base editor systems, guide polynucleotides, fusion proteins, complexes, or fusion protein-guide polynucleotide complexes described herein. Pharmaceutical compositions of the present disclosure can be prepared according to known techniques. See, for example, Remington, The Science And Practice of Pharmacy (21st edition 2005). In general, cells or their colonies are mixed with a suitable carrier before administration or storage, and in some embodiments, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers generally comprise inert substances, which help to administer the pharmaceutical composition to the subject, help to process the pharmaceutical composition into a deliverable formulation, or help to store the pharmaceutical composition before administration. Pharmaceutically acceptable carriers can include agents that can stabilize, optimize or otherwise change the form, consistency, viscosity, pH, pharmacokinetics, solubility of the preparation. Such agents include buffers, wetting agents, emulsifiers, diluents, encapsulating agents and skin penetration enhancers. For example, carriers can include but are not limited to saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethylcellulose and combinations thereof. In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. Suitable routes of administration of the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered topically to the site of disease (e.g., the liver, eyes, or central nervous system). In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, by catheter, by suppository, or by implant, wherein the implant is a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes or fibers. In some embodiments, any fusion protein, gRNA and / or complex described herein is provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any fusion protein or complex provided herein. In some embodiments, the pharmaceutical composition comprises gRNA, nucleic acid programmable DNA binding protein, cationic lipid and a pharmaceutically acceptable excipient. In embodiments, the pharmaceutical composition comprises lipid nanoparticles and a pharmaceutically acceptable excipient. In embodiments, the lipid nanoparticles contain gRNA, base editor, complex, base editor system or components thereof disclosed herein, and / or one or more polynucleotides encoding them. The pharmaceutical composition may optionally comprise one or more additional therapeutically active substances. The compositions described above can be administered in an effective amount. The effective amount will depend on the mode of administration, the specific condition being treated, and the desired outcome. It may also depend on the stage of the condition, the age and physical condition of the subject, the nature of concurrent treatment (if any), and similar factors well known to medical practitioners. For therapeutic applications, the amount is sufficient to achieve the medically desired outcome. In some embodiments, compositions according to the present disclosure may be used to treat any of a variety of diseases, disorders, and / or conditions. Treatment Some aspects of the present disclosure provide methods for treating a subject in need thereof, comprising administering to a subject in need thereof an effective therapeutic amount of a pharmaceutical composition as described herein. In other embodiments, the methods of the present disclosure include expressing a base editor polypeptide and one or more guide RNAs that can target a nucleic acid molecule encoding at least one polypeptide, or introducing them into a cell. Those of ordinary skill in the art will recognize that multiple administrations of the pharmaceutical compositions contemplated in a particular embodiment may be required to achieve the desired treatment. For example, the composition may be administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or longer. The administration of the pharmaceutical compositions contemplated herein can be carried out using conventional techniques, including but not limited to infusion, transfusion or parenteral. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid and intrasternal infusion or injection. In various embodiments, the methods of the present disclosure are associated with a reduction in complement activation in a subject. In some cases, the methods are associated with a reduction in inflammation in a subject. Combination therapy In various embodiments, the methods of the present disclosure comprise administering an inhibitor of complement component C3. In embodiments, the pharmaceutical compositions of the present disclosure contain an inhibitor of complement component C3. In some embodiments, the complement inhibitor is compstatin or a compstatin analog or mimetic. Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation. US Patent No. 6,319,897 describes a peptide having the sequence I[CVVQDWGHHRC]T (SEQ ID NO: 853), wherein the disulfide bond between the two cysteines is indicated in brackets. Compstatins are also described by Morikis et al., Protein Sci., 7(3):619-27, 1998. In some cases, compstatins are amidated at the C-terminus. Compstatin analogs, mimetics, derivatives thereof, and / or compositions containing the same suitable for use in the methods and compositions of the present disclosure include those described in WO2021007111 (PCT / US2020 / 040741); WO2021011927 (PCT / US2020 / 042676); WO2004026328 (PCT / US2003 / 029653); Morikis, D. et al., Biochem Soc Trans. 32(Pt 1):28-32, 2004, Mallik, B. et al., J. Med. Chem., 274-286, 2005; Katragadda, M. et al., J. Med. Chem., 49: 4616-4622, 2006; WO2007062249 (PCT / US2006 / 045539); WO2007044668 (PCT / US2006 / 039397); WO2009046198 (PCT / US2008 / 078593); WO2010127336 (PCT / US2010 / 033345); WO2012155107; WO2014078731; WO2019166411; WO2009121065; WO2021163654; WO2021142171; WO2017062879; WO2014152391; WO2014028861; WO2018187813; WO2019089653; WO2016049385; W O2018075373; WO2019118938; WO2022061304; WO2012006599; WO2011163394; WO2012178083; US9291622; US10407466; US8580735; and Hillmen et al. versus Eculizumab in Paroxysmal Nocturnal Hemoglobinuria,” N Engl J Med. 2021 Mar 18;384(11):1028-1037; the disclosures of all of which are incorporated herein by reference in their entirety for all purposes. In certain embodiments, the compstatin analog is pegcetacoplan (“APL-2”), which has Figure 19The structure of a compound wherein n is from about 800 to about 1100 and / or the average molecular weight of PEG is about 40 kD. Pecitabolom is also known as poly(oxy-1,2-ethanediyl), α-hydro-ω-hydroxy-, 15,15'-diester with N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophanyl-L-glutaminyl-L-α-aspartyl-L-tryptophanylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-N 6 -carboxyl-L-lysinamide cyclic (2-->12)-(disulfide); or O,O'-bis[(S 2 ,S 12 -cyclo{N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophanyl-L-glutaminyl-L-α-aspartyl-L-tryptophanylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-L-lysinamide})-N 6.15 -carbonyl] polyethylene glycol (n = 800-1100). In some embodiments, the complement inhibitor is an antibody, such as an anti-C3 antibody or a fragment thereof. In some embodiments, the antibody fragment can be used to inhibit C3 activation. The antibody fragment can be a Fab', Fab'(2), Fv or single-chain Fv. In some embodiments, the anti-C3 antibody is monoclonal. In some embodiments, the anti-C3 antibody is polyclonal. In some embodiments, the anti-C3 antibody is deimmunized. In some embodiments, the anti-C3 antibody is a fully human monoclonal antibody. In some cases, the complement inhibitor is an inhibitory polynucleotide (e.g., siRNA), such as those described in WO2021163654, the disclosure of which is incorporated herein in its entirety for all purposes. In some embodiments, the complement inhibitor is a polypeptide inhibitor and / or a nucleic acid aptamer (see, eg, US Publication No. 20030191084). Exemplary polypeptide inhibitors include enzymes that degrade C3 or C3b (see, eg, US Patent No. 6,676,943). Reagent test kit The present disclosure provides a kit for treating a subject to reduce complement activation. In some embodiments, the kit also includes a base editor system or a polynucleotide encoding a base editor system, wherein the base editor polypeptide system includes a nucleic acid programmable DNA binding protein (napDNAbp), a deaminase, and a guide RNA. In some embodiments, napDNAbp is Cas9 or Cas12. In some embodiments, the polynucleotide encoding the base editor is an mRNA sequence. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the kit includes a guide RNA and / or a base editor system and instructions for using the guide RNA and / or base editor system. The kit may also include written instructions for using the base editor and / or base editor system as described herein. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or affixed to the container as a label, or as a separate paper, brochure, card, or container or a folder provided with the container. In a further embodiment, the kit includes instructions in the form of a label or a separate insert (package insert) for suitable operating parameters. In yet another embodiment, the kit includes one or more containers having appropriate positive and negative controls or control samples for use as standards for detection, calibration, or normalization. The kit also includes a second container comprising a pharmaceutically acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The practice of the disclosed embodiments employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the scope of the skilled artisan. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present disclosure and can therefore be considered in making and practicing embodiments of the present disclosure. Particularly useful techniques for specific embodiments are discussed in the following sections. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed assays, screening and treatment methods and are not intended to limit the scope of what the inventors regard as their invention. Example Example 1: Complement component 3 (C3) knockout (KO) guide screening in Hek293T cells Experiments were performed to identify guide RNA sequences suitable for knocking out complement component 3 (C3) expression in cells. The 162 guides listed in Table 1A were designed to knock down C3 protein expression (listed in Table 1A). These guides were used to destroy splice sites using ABE deaminases, or to generate stop codons or destroy splice sites using CBE deaminases. 26 guides were screened for splice site destruction using ABE and CBE deaminases. All 188 (i.e., 162+26) guides-editor combinations were screened for editing the C3 gene. Editor mRNA+ sgRNA was transfected in triplicate in Hek293T cells, and the total RNA dose for all samples was 800 ng (200 ng guides+ 600 ng editor mRNA). In addition to the guide-editor pairs of interest (ie, base editor systems), a positive control guide-editor pair ABE8.8_sgRNA_088 was tested, which contains the spacer sequence CAGGAUCCGCACAGACUCCA (SEQ ID NO: 792) and is known to effectively edit sites outside the C3 gene. An untreated condition was included as a negative control. Genomic DNA was harvested from cells 3 days after transfection. A wide range of editing rates was observed among the 188 guide-editor combinations screened, with a subset of guides exhibiting favorable editing efficiencies at the targeted sites ( Figure 1 and 2 These included 29 guides that were screened with the ABE8.8 deaminase and achieved an editing rate ≥ 40% ( Figure 1 ), and 26 guides that were screened with BE4 deaminase and achieved an editing rate > 50% ( Figure 2 ). Example 2: Complement Component 3 (C3) Knockout (KO) Guide Screening and Functional Knockdown Assessment in Hepatocytes Extracted from Humanized Mouse Liver (PXB Cells) 22 guide + editor combinations that achieved favorable editing in Hek293T cells were selected for screening in human primary hepatocytes to evaluate the ability of editing efficiency and functional knockdown of C3 protein expression. After 3 days of cell incubation, editor mRNA + sgRNA was transfected in human hepatocytes (PXB cells, PhoenixBio) extracted from humanized mouse livers. In addition to the 22 guide-editor pairs of interest, the positive control base editor system sgRNA_088_ABE8.8_SpCas9 was also transfected. Untreated conditions were included as negative controls. All conditions were performed in triplicate. In order to assess functional C3 knockdown, cell supernatants were collected and stored at -80 ° C. Such collections were performed 2 days before transfection (3 days of incubation) and 4, 7, 10, 13 and 16 days after transfection. Another medium exchange was performed 1 day after transfection, but the supernatant was discarded. Genomic DNA was harvested from cells 16 days after transfection and the editing efficiency was assessed by next-generation sequencing (NGS). A human C3 ELISA assay was used to assess C3 protein concentration in cell supernatants before transfection and 7, 13, and 16 days after transfection. Before transfection, no significant differences in C3 protein concentrations were observed between samples ( Figure 3 ). 7 days after transfection, a significant increase in C3 levels was observed in all conditions, but again no significant differences were observed between samples ( Figure 4 By 13 days post-transfection, a roughly 50% reduction in C3 levels was observed for gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 compared to sgRNA_088_ABE8.8_SpCas9 that did not edit within the C3 gene ( Figure 5 A similar trend was observed 16 days after transfection ( Figure 6 ). C3 protein knockdown was positively correlated with editing rate across samples ( Figure 5 and 6 ). Example 3: ABE deaminase optimization ABE8.8 was used in Examples 1 and 2 to screen for complement component 3 (C3) knockout (KO) guides in Hek293T cells and PXB cells. Other 8th generation ABE deaminase variants include ABE8.13, ABE8.17, and ABE8.20. It was also found that when the V82T mutation was incorporated into ABE8.20 (ABE9.51), there was a potential editing advantage. To optimize the editing performance of C3 knockout (KO) guides, CD KO guides (i.e., gRNA676, gRNA696, gRNA701, gRNA662, gRNA661, gRNA695, and gRNA715) that exhibited potential C3 protein knockdown in PXB cells were screened in Hek293T cells using five 8th generation ABE editor variants (i.e., ABE8.8, ABE8.13, ABE8.17, ABE8.20, and ABE8.20_V82T (ABE9.51). All samples were transfected in triplicate with 400 ng (100 ng guide + 300 ng editor mRNA) of total RNA. sgRNA_088_ABE8.8_SpCas9 was transfected as a positive control, and an untreated condition was also included as a negative control. The editing efficiency of ABE8.8 was as good as or better than that of the other ABE8 variants tested for gRNA676, gRNA696, gRNA701, gRNA662, gRNA661, and gRNA695. For gRNA715, ABE8.13 achieved the highest editing efficiency ( Figure 7 ). Example 4: Spacer Length Optimization of Complement Component 3 (C3) Knockout (KO) Guides To further optimize the editing performance of the C3 KO guides, different spacer lengths (19, 20, 21, 22, and 23 bp) were screened for guides that showed potential C3 protein knockdown (see Table 1C). All samples were transfected in triplicate with a dose of 400 ng (100 ng guide + 300 ng editor mRNA) of total RNA. sgRNA_088_ABE8.8_SpCas9 was transfected as a positive control. An untreated condition was also included as a negative control. The editing efficiency of the standard 20 bp spacer performed as well or better than the other spacer lengths tested for gRNA676, gRNA696, gRNA701, gRNA662, gRNA661, and gRNA695. For gRNA715, the 21 bp prototype spacer achieved the highest editing efficiency ( Figure 8 ). Example 5: Repetitive Editing and Functional Knockdown Assessment of Complement Component 3 (C3) Guides in PXB Cells PXB cells were repeatedly transfected with the following guides showing potential for C3 knockdown: gRNA676_ABE8.8_SpCas9, gRNA696_ABE8.8_SpCas9, gRNA701_ABE8.8_SpCas9, gRNA662_ABE8.8_SpCas9, gRNA661_ABE8.8_SpCas9, gRNA695_ABE8.8_SpCas9, and gRNA715_ABE8.13_SpCas9-VRQR ( Figure 5 and 6 ). After 3 days of cell incubation, editor mRNA + sgRNA (i.e., base editor system) was transfected in triplicate in PXB cells. In addition to the 7 guide-editor pairs of interest, two positive control guide-editor pairs were also transfected. These include sgRNA_088_ABE8.8_SpCas9, which produces high editing efficiency at sites outside the C3 gene; and gRNA1688_SpCas9, which has the spacer sequence CAACAAGUUCGUGACCGUGC (SEQ ID NO: 793) and induces functional C3 knockdown. Most samples were transfected with 800 ng (200 ng guide + 600 ng editor mRNA) and 1200 ng (300 ng guide + 900 ng editor mRNA) doses. Only 800 ng doses were tested for gRNA1688_SpCas9, gRNA715_ABE8.13_SpCas9-VRQR, and gRNA695_ABE8.8_SpCas9. Untreated conditions were also included as negative controls. All conditions were performed in triplicate. To assess functional C3 knockdown, cell supernatants were collected and stored at -80 ° C. Such collections were performed immediately before transfection (3 days of incubation) and 4, 7, 10, and 13 days after transfection. An additional medium exchange was performed 1 day after transfection, but the supernatant was discarded. Genomic DNA was harvested from cells 13 days after transfection, and editing efficiency was assessed by next-generation sequencing (NGS). Human C3 ELISA was used to assess C3 protein concentrations in cell supernatants before and 13 days after transfection. Before transfection, no significant differences in C3 concentrations were observed between samples ( Figure 9 By 13 days post-transfection, a >55% reduction in C3 levels was observed for gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 compared to sgRNA_088_ABE8.8_SpCas9 at doses of 800 ng and 1200 ng ( Figure 10 Most guides had high editing efficiencies of >60% ( Figure 10 ). Example 6: Complement Component 3 (C3) Knockout (KO) Guide Screening and Functional Knockdown Assessment in Primary Human Hepatocyte (PHH) Co-cultures After 3 days of cell incubation, editor mRNA + sgRNA (i.e., base editor system) was transfected in triplicate in primary human hepatocyte (PHH) co-cultures. The following guides (sgRNAs) that showed potential C3 knockdown were used in the initial PXB cell experiments (see above): gRNA676_ABE8.8_SpCas9, gRNA696_ABE8.8_SpCas9, gRNA701_ABE8.8_SpCas9, gRNA662_ABE8.8_SpCas9, gRNA661_ABE8.8_SpCas9, and gRNA715_ABE8.13_SpCas9-VRQR. In addition to the 6 guide-editor pairs (i.e., base editor systems) of interest, two positive control guide-editor pairs were also transfected. These included sgRNA_088_ABE8.8_SpCas9, which produces high editing efficiency at sites outside the C3 gene; and gRNA1688_SpCas9, which contains the spacer sequence CAACAAGTTCGTGACCGTGC (SEQ ID NO: 793) and uses a nuclease-based strategy to induce functional C3 knockdown. Most samples were transfected with separate 800ng (200ng guide + 600ng editor mRNA) and 1200ng (300ng guide + 900ng editor mRNA) doses, but only the 800ng dose was tested for gRNA1688_SpCas9 and gRNA715_ABE8.13_SpCas9-VRQR. Untreated conditions were also included as negative controls. All conditions were performed in duplicate or triplicate. To assess functional C3 protein knockdown, cell supernatants were collected and stored at -80°C. Such collection was performed immediately before transfection (3 days of incubation) and 4, 7, 10, and 13 days after transfection. Another culture medium was replaced 1 day after transfection, but the supernatant was discarded. Genomic DNA was harvested from cells 13 days after transfection, and editing efficiency was assessed by next generation sequencing (NGS). Human C3 ELISA was used to assess C3 protein concentration in cell supernatants before and 13 days after transfection. Before transfection, no significant differences in C3 protein concentrations were observed between samples ( Figure 11By 13 days post-transfection, a >55% reduction in C3 protein levels was observed for gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 compared to sgRNA_088_ABE8.8_SpCas9 at doses of 800 ng and 1200 ng ( Figure 12 The editing efficiency of most guides was high, >60% under most conditions ( Figure 12 ). Example 7: Evaluation of the editing performance of complement component 3 (C3) knockout (KO) guides in primary cynomolgus monkey hepatocyte (PCH) monolayers Editor mRNA + sgRNA were transfected in triplicate in primary cynomolgus monkey (Macaca fascicularis) hepatocyte (PCH) monolayers to obtain guides that showed potent C3 knockdown in initial PXB cell experiments (see above): gRNA676_ABE8.8_SpCas9, gRNA696_ABE8.8_SpCas9, gRNA701_ABE8.8_SpCas9, gRNA662_ABE8.8_SpCas9, gRNA661_ABE8.8_SpCas9, and gRNA715_ABE8.13_SpCas9-VRQR. For gRNA676 and gRNA696, alternative cynomolgus guides were transfected, gRNA1793 and gRNA1798, respectively (see Table 1B for alternative spacer sequences). In addition to the 6 guide-editor pairs of interest (i.e., base editor systems), sgRNA_088_ABE8.8_SpCas9 was also transfected, and an untreated condition was also included as a negative control. All conditions were performed in triplicate. Genomic DNA was harvested from cells 3 days after transfection, and editing efficiency was assessed by next-generation sequencing (NGS). The editing efficiency of all guides was >35% ( Figure 13 These editing rates are comparable to those of the positive control sgRNA_088_ABE8.8_SpCas9. Example 8: Evaluation of Editing and Protein Knockdown (KD) of Complement Component 3 (C3) Knockout Guides in Primary Cynomolgus Hepatocyte (PCH) Co-cultures. The base editor systems gRNA1793_ABE8.8_SpCas9 and gRNA1798_ABE8.8_SpCas9 (human versions of which (i.e., gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9) exhibit high on-target base editing and functional C3 protein knockdown in PXB cells and PHH co-cultures) were transfected in triplicate in primary cynomolgus macaque (Macaca fascicularis) hepatocyte (PCH) co-cultures. sgRNA_088_ABE8.8_SpCas9 was transfected as a positive control, and an untreated condition was included as a negative control. All conditions were performed in triplicate. To assess functional C3 knockdown, cell supernatants were collected and stored at -80°C. Such collections were performed immediately before transfection (3 days of incubation) and 4, 7, 10, and 13 days after transfection. An additional medium change was performed 1 day after transfection, but the supernatant was discarded. Genomic DNA was harvested from cells 13 days after transfection, and editing efficiency was assessed by next-generation sequencing (NGS). A modified C3 ELISA assay was used to assess the concentration of cynomolgus monkey C3 protein in cell supernatants before transfection and 7 and 13 days after transfection. Before transfection, no significant differences in cynomolgus monkey C3 concentrations were observed between samples ( Figure 14 By 7 days post-transfection, a roughly 70% reduction in cynomolgus monkey C3 levels was observed for gRNA1793_ABE8.8_SpCas9 and gRNA1798_ABE8.8_SpCas9 compared to sgRNA_088_ABE8.8_SpCas9 that did not edit within the C3 gene ( Figure 15 A similar trend was observed 13 days after transfection ( Figure 16 The editing efficiency of gRNA1793_ABE8.8_SpCas9 and gRNA1798_ABE8.8_SpCas9 was high, about 70% ( Figure 15 and 16 This is comparable to the positive control sgRNA_088_ABE8.8_SpCas9 ( Figure 15 and 16 ). Example 9: Reduction of complement component 3 (C3) protein and RNA in FRG liver humanized mice after on-target editing using a base editor system Experiments were conducted to evaluate on-target editing and complement component 3 (C3) protein and RNA knockdown using a base editor system in FRG liver humanized mice. Because the base editor system containing the guide polynucleotide gRNA676 and the base editor ABE8.8 containing the SpCas9 nucleic acid programmable DNA binding protein domain (i.e., gRNA676_ABE8.8_SpCas9) showed high editing efficiency and high C3 protein knockdown in primary hepatocyte experiments in vitro, the base editor system was further evaluated in mice. The base editor system was formulated with lipid nanoparticles (BL4 LNP) and administered via intravenous retroorbital (RO) tail vein injection to liver humanized mice Fah- / -Rag2- / -Il2rg- / - (FRG) mice, in which human hepatocyte repopulation (Yecuris; donor: HHF04030) was ≥70% at doses of 2.0 and 0.3 mg / kg. Tris-buffered saline (TBS) alone was also delivered to control animals. Serum samples were collected before administration and 7 and 14 days after administration. Rapidly frozen liver fragments were collected for next generation sequencing (NGS) at the end of the study, and frozen liver fragments in RNAlater were collected for C3 RNA level analysis. NGS was used to assess the editing level at the target locus, and serum protein levels were measured using mid-scale discovery (MSD) assays. RT-qPCR was used to measure C3 RNA levels in the liver. Fourteen days after administration, serum C3 protein levels were observed to be reduced by approximately 90% relative to pre-administration C3 protein levels in animals dosed with 2.0 mg / kg ( Figure 20 This reduction in C3 protein levels corresponded to approximately 90% A>G editing at the target site (splice site) in harvested liver tissue and an approximately 90% reduction in liver C3 RNA levels compared to unedited control animals that were administered tris-buffered saline (TBS) instead of the base editor system ( Figure 20 and 21 Approximately 45-75% on-target editing was observed in animals dosed at 0.3 mg / kg ( Figure 20 In animals dosed at 0.3 mg / kg, editing rates correlated well with reductions in C3 protein and RNA levels, with animals that showed higher editing also showing higher reductions in C3 protein and RNA levels ( Figure 20 and 21 The measured reduction in C3 RNA was confirmed using four different Taqman probes spanning the length of the C3 gene, including Hs01100912_m1, which binds upstream of the gRNA676 target splice site (exon 8 splice site donor). Figure 21 ). Example 10: Effects of Heavy Chemical Modification on On-Target Editing and Complement Component 3 (C3) Protein Knockdown in FRG Liver Humanized Mice Using a Base Editor System Experiments were performed to determine the effect of heavy chemical modification of the guide polynucleotide on on-target editing and reduction of C3 protein levels in FRG liver humanized mice using the base editing system. Since the base editor systems gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 both showed high editing and C3 protein knockdown in primary hepatocyte experiments in vitro, and gRNA676_ABE8.8_SpCas9 also showed high efficacy in FRG liver humanized mice (i.e., the level of base editing at a specific dose of the base editor system), experiments were performed to determine the in vivo editing efficacy of the gRNA696_ABE8.8_SpCas9 base editor system and to determine the in vivo effect of heavy chemical modification of the guide polynucleotide on the two base editor systems. To evaluate the in vivo performance of gRNA696_ABE8.8_SpCas9 and the improvement of base editor system efficacy associated with various guide heavy chemical modification schemes, base editor systems gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 (i.e., guide gRNA2202-gRNA2209) with and without guide polynucleotide heavy chemical modification were formulated with lipid nanoparticles (BL4 LNPs) and administered via intravenous retroorbital (RO) tail vein injection in liver humanized mice Fah- / -Rag2- / -Il2rg- / - (FRG) mice, where human hepatocyte repopulation was ≥70% at a dose of 0.3 mg / kg (Yecuris; donor: HHF04030). sgRNA_088_ABE8.8_SpCas9 was formulated and administered as a control. Serum samples were collected before administration and 7 and 14 days after administration. At study termination, snap-frozen liver fragments were collected for next-generation sequencing (NGS) to assess editing levels at the target locus. Serum C3 protein levels were measured using the human C3 midscale discovery (MSD) assay. Fourteen days after administration, approximately 65-80% A>G on-target editing was observed at the target splice site in harvested liver tissue for gRNA676_ABE8.8_SpCas9 and base editor system variants containing guide polynucleotides with heavy chemical modifications (gRNA2202-gRNA2205), with gRNA2203_ABE8.8_SpCas9 associated with an increased editing rate relative to gRNA676_ABE8.8_SpCas9 ( Figure 22This on-target editing corresponded to a reduction of approximately 55-65% in serum C3 protein levels 14 days after administration compared to pre-administration C3 protein levels, with gRNA2203_ABE8.8_SpCas9 also associated with a greater reduction in C3 protein levels relative to those associated with gRNA676_ABE8.8_SpCas9 ( Figure 22 The base editor system gRNA696_ABE8.8_SpCas9 or its variants containing guide polynucleotides with heavy chemical modifications (gRNA2206-gRNA2209) were associated with on-target editing levels at target splice sites lower than approximately 40% ( Figure 22 ). Example 11: Differences in base editing rates at human and cynomolgus monkey target sites in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) Experiments were performed in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) to determine the difference in editing efficiency between a base editor system containing gRNA2202 (targeting the human C3 gene for base editing) and a base editor system containing gRNA2200 (targeting the cynomolgus monkey C3 gene for base editing). In previous experiments, gRNA2202_ABE8.8_SpCas9 produced approximately 70% editing in FRG liver humanized mice dosed at 0.3 mg / kg, and both C3 protein and RNA levels were reduced. The human sequence targeted by gRNA2202 for base editing is not conserved in cynomolgus monkeys. In previous experiments, the base editor system gRNA2200_ABE8.8_SpCas9 (containing gRNA2200 targeting the cynomolgus monkey C3 gene) was associated with a base editing rate of approximately 20% at a dose of 1.5 mg / kg in cynomolgus monkeys. To determine the potential efficacy differences between the cynomolgus monkey C3 gene targeting guide and the human C3 gene targeting guide, primary cynomolgus monkey hepatocytes (PCH) were transfected with the base editor system gRNA2200_ABE8.8_SpCas9, and primary human hepatocytes (PHH) were transfected with the base editor system gRNA2202_ABE8.8_SpCas9. Base editing was performed in both cell types using the base editor system sgRNA_088_ABE8.8_SpCas9 as a control because the sequence targeted by this base editor system is conserved between humans and cynomolgus monkeys. Each base editor system was transfected into cells at a 3:1 editor to sgRNA ratio with a base editor system dose of 1000, 100, 50, 10, 5, 1, 0.5, 0.1, and 0.01 ng (mass of the combined guide polynucleotide and mRNA encoding the base editor). All wells were normalized to a 1000 ng total mRNA dose using universal human reference RNA (Life Technologies, QS0639). All conditions were evaluated in duplicate. Genomic DNA was harvested from cells 3 days after transfection, and editing efficiency was assessed by next-generation sequencing (NGS). The data were fitted to a variable slope (four-parameter) curve, and the EC50 of all base editor systems was calculated. The on-target editing data were well fitted to a variable slope (four-parameter) curve, where R for all base editor systems evaluated was 2 A value of 0.99 or higher ( Figure 23 Comparison of EC50 revealed that the EC50 of gRNA2200_ABE8.8_SpCas9 in PCH cells was 13.63-fold higher than the EC50 observed for gRNA2202_ABE8.8_SpCas9 in PHH cells (65.44 ng vs. 4.80 ng). Figure 23Comparison of the EC50 of the base editor system sgRNA_088_ABE8.8_SpCas9 revealed that although the sequence targeted for base editing was conserved between PCH and PHH, the EC50 was 9.71-fold higher in PCH cells compared to PHH cells (33.12 ng vs. 3.41 ng) ( Figure 23 ). Example 12: Differences in base editing rates between human and cynomolgus monkey target sites in engineered Hek293T cell lines Given the differences in base editing rates observed between PCH cells and PHH cells using similar base editing systems, experiments were performed to determine the cause of this difference in base editing rates. To this end, an engineered HEK293T cell line was generated that contains the protospacer + PAM sequence of a human sequence targeted by the gRNA2202 guide and a cynomolgus macaque sequence targeted by the gRNA2200 guide, with the target sequences separated by 50 bp. This cell line was transfected with the base editor systems gRNA2202_ABE8.8_SpCas9 and gRNA2200_ABE8.8_SpCas9, respectively, to assess the differences in base editing between the two base editor systems. Cells were transfected with the base editor system at a 1:3 serial dilution series from 300 ng to 0.15 ng total base editing dose (2:1 editor to sgRNA ratio), where "total base editing dose" represents the combined mass of the guide polynucleotide and the mRNA encoding the base editor. All wells were normalized to a total RNA dose of 300-400 ng using non-translated ABE8.8 encoding mRNA. All conditions were evaluated in triplicate. Genomic DNA was harvested from cells 3 days after transfection, and editing efficiency was assessed by next-generation sequencing (NGS). The data were fitted to a variable slope (four-parameter) curve, and the EC50 of the two base editor systems was calculated. The on-target editing data were well fitted to a variable slope (four-parameter) curve, where the R 2 The value is 0.98-0.99 ( Figure 24 Comparison of EC50 revealed that the EC50 of gRNA2200_ABE8.8_SpCas9 was approximately 3.5-fold higher than the EC50 observed for gRNA2202_ABE8.8_SpCas9 (2.128 ng vs. 0.6132 ng). Figure 24 ). Example 13: ABE editor optimization for gRNA676, gRNA696, gRNA661 and gRNA715 The base editor system containing guides gRNA676, gRNA696, gRNA661 and gRNA715 targets the C3 gene for base editing, showing high editing and C3 protein knockdown in PXB cell and PHH co-culture studies. Therefore, experiments were performed to optimize the base editor used in the adenosine deaminase base editor (ABE) system containing these guides. As described in Example 3 above, these guides were initially ABE optimized to optimize editing performance. In order to further optimize the editing performance of the base editor system containing guides gRNA676, gRNA696 or gRNA661, the base editor system containing the guide and one of 16 base editors containing 8th and 9th generation TadA* deaminase domain variants and / or Cas9 variants were screened in HepG2 cells (Tables 8 to 10). To further optimize the editing performance of the base editor system containing the guide gRNA715, the base editor system containing the guide and one of the five base editors containing the 8th and 9th generation TadA* deaminase domain variants were screened in HepG2 cells (Table 11). The base editor system sgRNA_088_ABE8.8_SpCas9 was used as a positive control for base editing, and an untreated condition was also included as a negative control. The base editor system was transfected with a 1:3 serial dilution series from 300 ng to 0.02 ng total base editing dose (2:1 editor to sgRNA ratio), where "total base editing dose" refers to the combined total mass of the guide polynucleotide and the mRNA encoding the base editor. All wells were standardized to a total RNA dose of 300-400 ng using non-translated ABE8.8 encoding mRNA or universal human reference RNA (Life Technologies, QS0639). All conditions were evaluated in triplicate. Genomic DNA was harvested from cells 3 days after transfection and editing efficiency was assessed by next-generation sequencing (NGS). Data were fitted to a variable slope (four-parameter) curve and the total guide + editor efficiency was calculated.

[1413] EC50 of the combination.

[1414] Table 8. Summary of base editor system data.

[1415]

[1416] TadA*9.47 corresponds to TadA*8.8 with the change V82T; TadA*9.48 corresponds to TadA*8.8 with the changes V82T, Y147T and Q154S; TadA*9.49 corresponds to TadA*8.17 with the change V82T; TadA*9.50 corresponds to TadA*8.17 with the changes V82T, Y147T and Q154S; TadA*9.51 corresponds to TadA*8.20 with the change V82T; TadA*9.52 corresponds to TadA*8.20 with the changes V82T, Y147T and Q154S.

[1417] Table 9. Summary of base editor system data.

[1418]

[1419] TadA*9.47 corresponds to TadA*8.8 with the change V82T; TadA*9.48 corresponds to TadA*8.8 with the changes V82T, Y147T and Q154S; TadA*9.49 corresponds to TadA*8.17 with the change V82T; TadA*9.50 corresponds to TadA*8.17 with the changes V82T, Y147T and Q154S; TadA*9.51 corresponds to TadA*8.20 with the change V82T; TadA*9.52 corresponds to TadA*8.20 with the changes V82T, Y147T and Q154S.

[1420] Table 10. Summary of base editor system data.

[1421]

[1422] TadA*9.47 corresponds to TadA*8.8 with the change V82T; TadA*9.48 corresponds to TadA*8.8 with the changes V82T, Y147T and Q154S; TadA*9.49 corresponds to TadA*8.17 with the change V82T; TadA*9.50 corresponds to TadA*8.17 with the changes V82T, Y147T and Q154S; TadA*9.51 corresponds to TadA*8.20 with the change V82T; TadA*9.52 corresponds to TadA*8.20 with the changes V82T, Y147T and Q154S.

[1423] Table 11. Summary of base editor system data.

[1424]

[1425] Target editing 5A refers to the bases shown in bold in the following protospacer sequence CCGCAGATCTGGGA CGTGGT (SEQ ID NO: 483)

[1426] TadA*9.51 corresponds to TadA*8.20 with the V82T change.

[1427] When paired with gRNA676, five editor variants (ABE8.13_SpCas9, ABE9.48_SpCas9, ABE9.50_SpCas9, ABE9.52_SpCas9) exhibited similar potency and maximum editing to ABE8.8_SpCas9, where "potency" indicates the level of base editing achieved at a specific dose. (Table 8). When paired with gRNA696, five editor variants (ABE8.13_SpCas9, ABE8.20_SpCas9, ABE9.51_SpCas9, ABE9.52_SpCas9, ABE8.13_SpCas9_A1283D_E1250K [KGPKPKKEE SEK (SEQ ID NO: 940) linker]) exhibited similar or higher potency and maximum editing to ABE8.8_SpCas9 (Table 9). Seven editor variants (ABE8.13_SpCas9, ABE8.20_SpCas9, ABE9.47_SpCas9, ABE9.48_SpCas9, ABE9.51_SpCas9, ABE9.52_SpCas9, ABE8.13_SpCas9_A1283D_E1250K [KGPKPKKEESEK (SEQ ID NO: 940) linker]) showed similar or higher potency and maximal editing than ABE8.8_SpCas9 when paired with gRNA661 (Table 10). ABE9.51_SpCas9-VRQR showed higher potency than ABE8.13_SpCas9-VRQR when paired with gRNA715 (Table 11).

[1428] Editor variants defined above that showed good editing efficiency when combined with each guide were further screened in primary human hepatocyte (PHH) monolayers at low, subsaturating doses.

[1429] For the base editor system containing gRNA676, PHH monolayers were transfected with 2.5 and 10 ng total base editing doses, respectively. Conditions were normalized to a 300 ng total RNA dose using universal human reference RNA (Life Technologies, QS0639). Guide sgRNA_088 was used as a positive editing control, and untransfected conditions were used as negative controls. All conditions were evaluated in triplicate. Genomic DNA was harvested from all cells 3 days after transfection, and editing efficiency was assessed by next-generation sequencing (NGS) For the base editor system containing gRNA696, gRNA661, and gRNA715 guides, PHH monolayers were transfected with 5 and 20 ng total base editing doses, respectively. Conditions were normalized to a 300 ng total RNA dose using non-translated ABE8.8 encoding mRNA. Guide sgRNA_088 was used as a positive editing control, and an untransfected condition was used as a negative control. All conditions were evaluated in triplicate. Genomic DNA was harvested from all cells 3 days after transfection, and editing efficiency was assessed by next-generation sequencing (NGS) We found that the base editor ABE9.48_SpCas9 slightly improved on-target editing efficiency compared to ABE8.8_SpCas9 when paired with gRNA676 at 2.5 ng and 10 ng total base editing doses ( Figure 25 ). It was found that when paired with gRNA696 at 5 ng and 20 ng base editing doses, the base editor ABE8.13_SpCas9 slightly improved the on-target editing efficiency compared to ABE8.8_SpCas9 ( Figure 26 and 27 ). It was found that the base editor ABE9.52_SpCas9 slightly improved the on-target editing efficiency compared with ABE8.8_SpCas9 when paired with gRNA661 at 5 ng and 20 ng base editing doses ( Figure 26 and 27 When paired with gRNA715, the base editor ABE9.51_SpCas9-VRQR did not improve on-target editing efficiency compared to ABE8.13_SpCas9-VRQR ( Figure 27 ). Example 14: Effects of guided polynucleotide rechemical modification and optimized adenosine deaminase base editor (ABE) on on-target editing and reduction of complement component 3 (C3) protein and RNA levels using a base editor system in FRG liver humanized mice As described above, it was determined that the base editor system gRNA676_ABE8.8_SpCas9 was associated with high and medium on-target editing and functional complement component 3 (C3) protein knockdown in FRG liver humanized mice. It was also found that the base editor system gRNA2203_ABE8.8_SpCas9 containing the guide gRNA2203 with a heavy chemical modification scheme showed improvements in on-target editing and functional complement component 3 (C3) in FRG liver humanized mice. In addition, base editor optimization studies in HepG2 cell lines and primary human hepatocytes showed that the editor ABE9.48_SpCas9 used in combination with gRNA676 had improved performance over ABE8.8_SpCas9. Therefore, in view of these results, the following experiments were performed to further optimize the base editor system containing the guide gRNA6...

Claims

1. A method of changing the nucleobase of a complement component 3 (C3) polynucleotide, the method comprising contacting the C3 polynucleotide with a base editor system comprising one or more guide RNA or one or more polynucleotides encoding the one or more guide RNA, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain or one or more polynucleotides encoding the base editor, in: (a) The one or more guide RNAs to target the base editor to achieve alteration of the nucleobase of the C3 polynucleotide that breaks the splicing site in the C3 polynucleotide; (b) The deaminase domain is a TadA variant (TadA*) comprising a combination of alterations listed in Tables 5A, 5B, 5C, 5D, 5E, 6A, 6B, 6C, 6D, 6E, 6F or 7, wherein the TadA* is not TadA*7.9 or TadA*7.10, and / or wherein the TadA* variant is a TadA*8.8, TadA*8.17 or TadA*8.20 variant containing one or more of V82T, Y147T and Q154S; (c) The one or more guide RNAs comprises a nucleic acid sequence containing at least 10-23 consecutive nucleotides of the spacer nucleic acid sequence listed in any one of Tables 1A, 1B, 1C, 1D, 1E, 1F and 2; (d) The one or more guide RNAs target the base editor to achieve changes in the nucleobase in the codon encoding amino acid residues selected from the group consisting of A741, S742, H743, A747, L746, R748, S749, I1125, H1126, Q1127 and E1128 relative to the following reference sequence: C3 amino acid sequence MGPTSGPSLLLLLLTHLPLALGSPMYSIITPNILRLESEETMVLEAHDAQGDVPVTVTVHDFPGKKLVLSSEKTVLTPATNHMGNVTFTIPANREFKSEKGRNKFVTVQATFGTQVVEKVVLVSLQSGYLFIQTDKTIYTPGSTVLYRIFTVNHKLLPVGRTVMVNIENPEGIPVKQDSLSSQNQLGVLPLSWDIPELVNMGQWKIRA YYENSPQQVFSTEFEVKEYVLPSFEVIVEPTEKFYYIYNEKGLEVTITARFLYGKVEGTAFVIFGIQDGEQRISLPESLKRIPIEDGSGEVVLSRKVLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAERSGIPIVTSPYQIHFTKTPKYFKPGMPFDLMVFVTNPDGSPAYRVPVAVQGEDTVQSLTQGDGVAKLSINTHPSQ KPLSITVRTKKQELSEAEQATRTMQALPYSTVGNSNNYLHLSVLRTELRPGETLNVNFLLRMDRAHEAKIRYYTYLIMNKGRLLKAGRQVREPGQDLVVLPLSITTDFIPSFRLVAYYTLIGASGQREVVADSVWVDVKDSCVGSLVVKSGQSEDRQPVPGQQMTLKIEGDHGARVVLVAVDKGVFVLNKKNKLTQSKIWDVVEKADI GCTPGSGKDYAGVFSDAGLTFTSSSGQQTAQRAELQCPQPAARRRRSVQLTEKRMDKVGKYPKELRKCCEDGMRENPMRFSCQRRTRFISLGEACKKVFLDCCNYITELRRQHARASHLGLARSNLDEDIIAEENIVSRSEFPESWLWNVEDLKEPPKNGISTKLMNIFLKDSITTWEILAVSMSDKKGICVADPFEVTVMQDFFIDLR LPYSVVRNEQVEIRAVLYNYRQNQELKVRVELLHNPAFCSLATTKRRHQQTVTIPPKSSLSVPYVIVPLKTGLQEVEVKAAVYHHFISDGVRKSLKVVPEGIRMNKTVAVRTLDPERLGREGVQKEDIPPADLSDQVPDTESETRILLQGTPVAQMTEDAERLKHLIVTPSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKR QGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPP VVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSRSSKITHRIHWESASLLRSEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLA FKVHQYFNVELIQPGAVKVYAYYNLEESCTRFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEHWPEEDECQDEENQKQCQDLGAFTESMVVFGCPN(SEQID NO:796), or to achieve a change in the corresponding position in another C3 polypeptide sequence; or (e) The one or more guide RNAs target the base editor to achieve changes in the nucleobase in the starting codon of the C3 polynucleotide; Thus, the nucleobase of the C3 polynucleotide is changed.

2. The method of claim 1, wherein the splicing site corresponds to any of the prototype spacers listed in Tables 1A, 1B, or 1C.

3. The method of claim 1, wherein the deaminase is an adenosine deaminase selected from the group consisting of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T and TadA*8.20_V82T_Y147T_Q154S.

4. The method of claim 1, wherein the base editor is selected from the group consisting of ABE8.8, ABE8.13, ABE8.17, ABE8.20, ABE8.8_V82T, ABE8.8_V82T_Y147T_Q154S, ABE8.17_V82T, ABE8.17_V82T_Y147T_Q154S, ABE8.20_V82T, ABE8.20_V82T_Y147T_Q154S, BE4 and those base editors listed in Tables 1A, 1B, 1C, 1D, 1E, 1F, 2 and 8 to 15.

5. The method of claim 1, wherein the one or more guide RNAs comprises a spacer consisting of about 19 to about 23 nucleotides.

6. The method of claim 5, wherein the spacer is composed of 21 nucleotides.

7. The method of claim 1, wherein the one or more guide RNAs comprises a spacer corresponding to a guide polynucleotide selected from the group consisting of gRNA676, gRNA661, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA3342, gRNA3343 and gRNA3345.

8. A method of changing the nucleobase of a complement component 3 (C3) polynucleotide, the method comprising contacting the C3 polynucleotide with one or more guide RNA or one or more polynucleotides encoding the one or more guide RNA and with a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain or one or more polynucleotides encoding the base editor, wherein (a) The deaminase domain is selected from the group consisting of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, rAPOBEC1 and ppAPOBEC; and (b) The one or more guide RNAs include spacers corresponding to guide polynucleotides selected from the group consisting of gRNA661, gRNA662, gRNA676, gRNA695, gRNA696, gRNA701, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA1793, gRNA1798, gRNA3342, gRNA3343 and gRNA3345; Thus, the nucleobase of the complement component 3 (C3) polynucleotide is changed.

9. The method of any of claims 1-8, wherein the napDNAbp domain comprises a Cas9 variant.

10. The method of claim 9, wherein the Cas9 variant comprises one or more of A1283D and E1250K relative to the SpCas9 reference amino acid sequence.

11. The method of claim 9, wherein the Cas9 variant comprises one or more of the following amino acid alterations relative to the spCas9 reference amino acid sequence: I322V, S409I, E427G, R654L, R753G and R1114G; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y and R1337K; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K and A1283D; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R220A and R221A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A and Q768A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A, Q768A, K772A and K775A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, R1337K, A1283D and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, A1283D and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y and R1337K; and I322V, S409I, E427G, R654L, R753G, R1114G and R1337K.

12. The method of any of claims 9-11, wherein the Cas9 variant is SaCas9-KHH, SpCas9-MQKFRAER or SpCas9-VRQR.

13. The method of any of claims 1-8, wherein the one or more guide RNAs include nucleic acid analogs.

14. The method of claim 13, wherein the one or more guide RNAs comprises one or more of 2'-OMe and phosphorothioate.

15. The method of claim 14, wherein the one or more guide RNAs comprises one of the following nucleotide sequences: End-mod SpCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNNNNGUUUAGAGCUAG AAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGCACCGAGUCGGUGCUsmUsmUsmUsmUsmU (SEQ ID NO: 943); End-mod SaCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAAAAGGCAAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmUsmUsmU (SEQ ID NO: 944); H M01: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmAmAmAmUmAmGmAmAmUmAmGmCAAGUUAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmCmUmGmAmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmGmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 855); H M07: mNsmNsmNsmNmNmNmNmNmNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUUUAGmAmGmCmUmAmAmAmUmAmUmAmAmAmUAmGmGmCmUmAmAAmUAmAmGmGmCmUmAmAAmUAmAmGmGmCmUmAGUmAmUmAmAmAmGmGmCmUmGmCmUmGmUsmUsmUsmU (SEQ ID NO: 856); NLS(bpsv 40): mNsmNsmNsNNNNNNNNNNNNNNNNNNNNNNNGUUUAGAGCUAG AAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGCACCGAGUCGGUGCmUsmUsmUsmUsmUsmUsmUsmUsmUsmUsmU (SEQ ID NO: 857); LONG EST: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmGmCAAGUUAAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmCmUmGmAmAmAmCmUmGmAmAmAmCmUmGmCmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 859); NLS+LONG ST: mNsmNsmNNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmCmGmGmCAAGUUAAAAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmCmUmGmAmAmAmCmUmGmAmAmAmCmUmGmGmCmUsmUsmUsmUsmU (SEQ ID NO: 860); or LONGEST+G OLD: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmAmAmAmCmGmCmGmGmCAAGUUAAAAAAAGGCUAGUCCGUUAmUmCAAmCmUmUmCAAmCmUmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 861); Where "mN" represents a 2'-OMe modification of nucleotide "N" and "Ns" represents a nucleotide "N" linked to the following nucleotide by phosphorothioate (PS).

16. The method of claim 15, wherein the one or more guide RNAs are covalently linked to a peptide having the following amino acid sequence at the 5' end: CKRTADGSEFESPK KKRKV (SEQ ID NO: 858).

17. The method of any of claims 1-8, wherein the base editor comprises a linker peptide between the deaminase domain and the napDNAbp domain, wherein the linker peptide comprises the amino acid sequence KGPKPKKEESEK (SEQID NO: 940).

18. The method of any one of claims 1-8, wherein the nucleobase change enables a change of the encoded amino acid residue, wherein the change disrupts the conditioning effect of C3.

19. The method of any one of claims 1-8, wherein the nucleobase alteration disrupts splicing of the C3 transcript.

20. The method of any of claims 1-8, wherein the nucleobase change enables an alteration of the encoded amino acid residue, wherein the change disrupts the cleavage of the C3 polypeptide by the C3 convertase.

21. The method of any of claims 1-8, wherein the one or more guide RNAs target the base editor to achieve changes in the nucleobase in the codon encoding amino acid residues selected from the group consisting of A741, S742, H743, A747, L746, R748, S749, I1125, H1126, Q1127, and E1128 relative to the following reference sequence: C3 amino acid sequence MGPTSGPSLLLLLLTHLPLALGSPMYSIITPNILRLESEETMVLEAHDAQGDVPVTVTVHDFPGKKLVLSSEKTVLTPATNHMGNVTFTIPANREFKSEKGRNKFVTVQATFGTQVVEKVVLVSLQSGYLFIQTDKTIYTPGSTVLYRIFTVNHKLLPVGRTVMVNIENPEGIPVKQDSLSSQNQLGVLPLSWDIPELVNMGQWKIRA YYENSPQQVFSTEFEVKEYVLPSFEVIVEPTEKFYYIYNEKGLEVTITARFLYGKVEGTAFVIFGIQDGEQRISLPESLKRIPIEDGSGEVVLSRKVLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAERSGIPIVTSPYQIHFTKTPKYFKPGMPFDLMVFVTNPDGSPAYRVPVAVQGEDTVQSLTQGDGVAKLSINTHPSQ KPLSITVRTKKQELSEAEQATRTMQALPYSTVGNSNNYLHLSVLRTELRPGETLNVNFLLRMDRAHEAKIRYYTYLIMNKGRLLKAGRQVREPGQDLVVLPLSITTDFIPSFRLVAYYTLIGASGQREVVADSVWVDVKDSCVGSLVVKSGQSEDRQPVPGQQMTLKIEGDHGARVVLVAVDKGVFVLNKKNKLTQSKIWDVVEKADI GCTPGSGKDYAGVFSDAGLTFTSSSGQQTAQRAELQCPQPAARRRRSVQLTEKRMDKVGKYPKELRKCCEDGMRENPMRFSCQRRTRFISLGEACKKVFLDCCNYITELRRQHARASHLGLARSNLDEDIIAEENIVSRSEFPESWLWNVEDLKEPPKNGISTKLMNIFLKDSITTWEILAVSMSDKKGICVADPFEVTVMQDFFIDLR LPYSVVRNEQVEIRAVLYNYRQNQELKVRVELLHNPAFCSLATTKRRHQQTVTIPPKSSLSVPYVIVPLKTGLQEVEVKAAVYHHFISDGVRKSLKVVPEGIRMNKTVAVRTLDPERLGREGVQKEDIPPADLSDQVPDTESETRILLQGTPVAQMTEDAERLKHLIVTPSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKR QGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPP VVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSRSSKITHRIHWESASLLRSEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLA FKVHQYFNVELIQPGAVKVYAYYNLEESCTRFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEHWPEEDECQDEENQKQCQDLGAFTESMVVFGCPN(SEQID NO:796).

22. The method of claim 21, wherein the nucleobase change achieves alteration of encoded amino acid residues selected from the group consisting of A741V, S742G, H743R, H743Y, A747V, A747T, L746P, R748G, R748K, S749G, C1010R, C1010Y, Q1013R, I1125M, H1126R, H1126Y, Q1127R, E1128G and E1128K.

23. The method of any of claims 1-8, wherein the one or more guide RNAs target the base editor to achieve alteration of nucleobases in the start codon of the C3 polynucleotide.

24. The method of any one of claims 1-8, wherein the base editor system has an editing rate greater than 35%.

25. The method of any one of claims 1-8, wherein the C3 polynucleotide is in a mammalian cell.

26. The method of claim 25, wherein the cell is a retinal cell or other cell of the eye, a CNS cell or a hepatocyte.

27. A method of treating a disease or disorder associated with inappropriate activation of a complement system in a subject in need thereof, the method comprising changing the nucleobase of a complement component 3 (C3) polynucleotide in the subject by administering to the subject one or more guide RNA or one or more polynucleotides encoding the one or more guide RNA, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain or one or more polynucleotides encoding the base editor, in: (a) The one or more guide RNAs to target the base editor to achieve alteration of the nucleobase of the C3 polynucleotide that breaks the splicing site in the C3 polynucleotide; (b) The deaminase domain is a TadA variant (TadA*) containing a combination of alterations listed in Tables 5A, 5B, 5C, 5D, 5E, 6A, 6B, 6C, 6D, 6E, 6F or 7, wherein the TadA* is not TadA*7.9 or TadA*7.10, (c) The one or more guide RNAs comprises a nucleic acid sequence containing at least 10-23 consecutive nucleotides of the spacer nucleic acid sequence listed in any one of Tables 1A, 1B, 1C, 1D, 1E, 1F and 2; or (d) The one or more guide RNAs target the base editor to achieve changes in the nucleobase in the codon encoding amino acid residues selected from the group consisting of A741, S742, H743, A747, L746, R748, S749, I1125, H1126, Q1127 and E1128 relative to the following reference sequence: C3 amino acid sequence MGPTSGPSLLLLLLTHLPLALGSPMYSIITPNILRLESEETMVLEAHDAQGDVPVTVTVHDFPGKKLVLSSEKTVLTPATNHMGNVTFTIPANREFKSEKGRNKFVTVQATFGTQVVEKVVLVSLQSGYLFIQTDKTIYTPGSTVLYRIFTVNHKLLPVGRTVMVNIENPEGIPVKQDSLSSQNQLGVLPLSWDIPELVNMGQWKIRA YYENSPQQVFSTEFEVKEYVLPSFEVIVEPTEKFYYIYNEKGLEVTITARFLYGKVEGTAFVIFGIQDGEQRISLPESLKRIPIEDGSGEVVLSRKVLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAERSGIPIVTSPYQIHFTKTPKYFKPGMPFDLMVFVTNPDGSPAYRVPVAVQGEDTVQSLTQGDGVAKLSINTHPSQ KPLSITVRTKKQELSEAEQATRTMQALPYSTVGNSNNYLHLSVLRTELRPGETLNVNFLLRMDRAHEAKIRYYTYLIMNKGRLLKAGRQVREPGQDLVVLPLSITTDFIPSFRLVAYYTLIGASGQREVVADSVWVDVKDSCVGSLVVKSGQSEDRQPVPGQQMTLKIEGDHGARVVLVAVDKGVFVLNKKNKLTQSKIWDVVEKADI GCTPGSGKDYAGVFSDAGLTFTSSSGQQTAQRAELQCPQPAARRRRSVQLTEKRMDKVGKYPKELRKCCEDGMRENPMRFSCQRRTRFISLGEACKKVFLDCCNYITELRRQHARASHLGLARSNLDEDIIAEENIVSRSEFPESWLWNVEDLKEPPKNGISTKLMNIFLKDSITTWEILAVSMSDKKGICVADPFEVTVMQDFFIDLR LPYSVVRNEQVEIRAVLYNYRQNQELKVRVELLHNPAFCSLATTKRRHQQTVTIPPKSSLSVPYVIVPLKTGLQEVEVKAAVYHHFISDGVRKSLKVVPEGIRMNKTVAVRTLDPERLGREGVQKEDIPPADLSDQVPDTESETRILLQGTPVAQMTEDAERLKHLIVTPSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKR QGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPP VVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSRSSKITHRIHWESASLLRSEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLA FKVHQYFNVELIQPGAVKVYAYYNLEESCTRFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEHWPEEDECQDEENQKQCQDLGAFTESMVVFGCPN(SEQID NO:796), or to achieve a change in the corresponding position in another C3 polypeptide sequence; or (e) The one or more guide RNAs target the base editor to achieve changes in the nucleobase in the starting codon of the C3 polynucleotide; Thus, the disease or condition is treated.

28. A method of treating a disease or disorder associated with inappropriate activation of a complement system in a subject in need thereof, the method comprising changing the nucleobase of a complement component 3 (C3) polynucleotide in the subject by administering to the subject one or more guide RNA or one or more polynucleotides encoding the one or more guide RNA, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain or one or more polynucleotides encoding the base editor, in: (a) The deaminase domain is selected from the group consisting of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, rAPOBEC1 and ppAPOBEC; and (b) The one or more guide RNAs include spacers corresponding to guide polynucleotides selected from the group consisting of gRNA661, gRNA662, gRNA676, gRNA695, gRNA696, gRNA701, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA1793, gRNA1798, gRNA3342, gRNA3343 and gRNA3345; Thus, the disease or condition is treated.

29. The method of claim 27 or claim 28, wherein the one or more guide RNAs target the base editor to achieve an alteration of the nucleobase of the C3 polynucleotide that destroys the splicing site in the C3 polynucleotide, wherein the splicing site is selected from the group consisting of those splicing sites corresponding to any of the prototype spacers listed in Tables 1A, 1B, or 1C.

30. The method of claim 27 or claim 28, wherein the deaminase is an adenosine deaminase or a cytidine deaminase.

31. The method of claim 27 or claim 28, wherein the deaminase is an adenosine deaminase selected from the group consisting of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T and TadA*8.20_V82T_Y147T_Q154S.

32. The method of claim 27 or claim 28, wherein the base editor is selected from the group consisting of ABE8.8, ABE8.13, ABE8.17, ABE8.20, ABE8.8_V82T, ABE8.8_V82T_Y147T_Q154S, ABE8.17_V82T, ABE8.17_V82T_Y147T_Q154S, BE4 and those base editors listed in Tables 1A, 1B, 1C, 1D, 1E, 1F, 2 and 8 to 15.

33. The method of claim 27 or claim 28, wherein the napDNAbp domain comprises a Cas9 variant.

34. The method of claim 33, wherein the Cas9 variant comprises one or more of A1283D and E1250K relative to the SpCas9 reference amino acid sequence.

35. The method of claim 33, wherein the Cas9 variant comprises one or more of the following amino acid alterations relative to the spCas9 reference amino acid sequence: I322V, S409I, E427G, R654L, R753G and R1114G; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y and R1337K; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K and A1283D; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R220A and R221A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A and Q768A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A, Q768A, K772A and K775A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, R1337K, A1283D and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, A1283D and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y and R1337K; and I322V, S409I, E427G, R654L, R753G, R1114G and R1337K.

36. The method of any of claims 33-35, wherein the Cas9 variant is SaCas9-KHH, SpCas9-MQKFRAER or SpCas9-VRQR.

37. The method of claim 27 or claim 28, wherein the one or more guide RNAs target the base editor to achieve changes in the nucleobase in the codon encoding amino acid residues selected from the group consisting of A741, S742, H743, A747, L746, R748, S749, I1125, H1126, Q1127, and E1128 relative to the following reference sequence: C3 amino acid sequence MGPTSGPSLLLLLLTHLPLALGSPMYSIITPNILRLESEETMVLEAHDAQGDVPVTVTVHDFPGKKLVLSSEKTVLTPATNHMGNVTFTIPANREFKSEKGRNKFVTVQATFGTQVVEKVVLVSLQSGYLFIQTDKTIYTPGSTVLYRIFTVNHKLLPVGRTVMVNIENPEGIPVKQDSLSSQNQLGVLPLSWDIPELVNMGQWKIRA YYENSPQQVFSTEFEVKEYVLPSFEVIVEPTEKFYYIYNEKGLEVTITARFLYGKVEGTAFVIFGIQDGEQRISLPESLKRIPIEDGSGEVVLSRKVLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAERSGIPIVTSPYQIHFTKTPKYFKPGMPFDLMVFVTNPDGSPAYRVPVAVQGEDTVQSLTQGDGVAKLSINTHPSQ KPLSITVRTKKQELSEAEQATRTMQALPYSTVGNSNNYLHLSVLRTELRPGETLNVNFLLRMDRAHEAKIRYYTYLIMNKGRLLKAGRQVREPGQDLVVLPLSITTDFIPSFRLVAYYTLIGASGQREVVADSVWVDVKDSCVGSLVVKSGQSEDRQPVPGQQMTLKIEGDHGARVVLVAVDKGVFVLNKKNKLTQSKIWDVVEKADI GCTPGSGKDYAGVFSDAGLTFTSSSGQQTAQRAELQCPQPAARRRRSVQLTEKRMDKVGKYPKELRKCCEDGMRENPMRFSCQRRTRFISLGEACKKVFLDCCNYITELRRQHARASHLGLARSNLDEDIIAEENIVSRSEFPESWLWNVEDLKEPPKNGISTKLMNIFLKDSITTWEILAVSMSDKKGICVADPFEVTVMQDFFIDLR LPYSVVRNEQVEIRAVLYNYRQNQELKVRVELLHNPAFCSLATTKRRHQQTVTIPPKSSLSVPYVIVPLKTGLQEVEVKAAVYHHFISDGVRKSLKVVPEGIRMNKTVAVRTLDPERLGREGVQKEDIPPADLSDQVPDTESETRILLQGTPVAQMTEDAERLKHLIVTPSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKR QGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPP VVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSRSSKITHRIHWESASLLRSEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLA FKVHQYFNVELIQPGAVKVYAYYNLEESCTRFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEHWPEEDECQDEENQKQCQDLGAFTESMVVFGCPN(SEQID NO:796).

38. The method of claim 37, wherein the nucleobase change achieves alteration of encoded amino acid residues selected from the group consisting of A741V, S742G, H743R, H743Y, A747V, A747T, L746P, R748G, R748K, S749G, C1010R, C1010Y, Q1013R, I1125M, H1126R, H1126Y, Q1127R, E1128G and E1128K.

39. The method of claim 27 or claim 28, wherein the one or more guide RNAs include nucleic acid analogs.

40. The method of claim 39, wherein the one or more guide RNAs comprises one or more of 2'-OMe and phosphorothioate.

41. The method of claim 40, wherein the one or more guide RNAs comprises one of the following nucleotide sequences: End-mod SpCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNNNNGUUUAGAGCUAG AAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGCACCGAGUCGGUGCUsmUsmUsmUsmUsmU (SEQ ID NO: 943); End-mod SaCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAAAAGGCAAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmUsmUsmU (SEQ ID NO: 944); HM01: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmAmAmAmUmAmGmAmAmUmAmGmCAAGUUAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmCmUmGmAmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmGmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 855); HM07: mNsmNsmNsmNmNmNmNmNmNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUUUAGmAmGmCmUmAmAmAmUmAmUmAmAmAmUAmGmGmCmUmAmAAmUAmAmGmGmCmUmAmAAmUAmAmGmGmCmUmAGUmAmUmAmAmAmGmGmCmUmGmCmUmGmUsmUsmUsmU (SEQ ID NO: 856); NLS (bpsv40): mNsmNsmNsNNNNNNNNNNNNNNNNNNNNNNNGUUUAGAGCUAG AAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGCACCGAGUCGGUGCmUsmUsmUsmUsmUsmUsmUsmUsmUsmUsmU (SEQ ID NO: 857); LONGEST: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmGmCAAGUUAAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmCmUmGmAmAmAmCmUmGmAmAmAmCmUmGmCmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 859); NLS+LONGEST: mNsmNsmNNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmCmGmGmCAAGUUAAAAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmCmUmGmAmAmAmCmUmGmAmAmAmCmUmGmGmCmUsmUsmUsmUsmU (SEQ ID NO: 860); or LONGEST+GOLD: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmAmAmAmCmGmCmGmGmCAAGUUAAAAAAAGGCUAGUCCGUUAmUmCAAmCmUmUmCAAmCmUmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 861); Where "mN" represents a 2'-OMe modification of nucleotide "N" and "Ns" represents a nucleotide "N" linked to the following nucleotide by phosphorothioate (PS).

42. The method of claim 41, wherein the one or more guide RNAs are covalently linked to a peptide having the following amino acid sequence at the 5' end: CKRTADGSEFESP KKKRKV (SEQ ID NO: 858).

43. The method of claim 27 or claim 28, wherein the base editor comprises a linker peptide between the deaminase domain and the napDNAbp domain, wherein the linker peptide comprises the amino acid sequence KGPKPKKEESEK (SEQ ID NO: 940).

44. The method of claim 27 or claim 28, wherein the editing rate is greater than 35%.

45. The method of claim 44, wherein the editing rate is greater than 50%.

46. ​​The method of claim 27 or claim 28, wherein the one or more guide RNAs comprises a spacer consisting of about 19 to about 23 nucleotides.

47. The method of claim 46, wherein the spacer is composed of 21 nucleotides.

48. The method of claim 27 or claim 28, wherein the method comprises administering to the subject a lipid nanoparticle comprising the one or more guide RNA and an mRNA molecule encoding the base editor.

49. The method of claim 27 or claim 28, wherein the nucleobase change enables a change in the encoded amino acid residue, wherein the change disrupts the conditioning effect of C3.

50. The method of claim 27 or claim 28, wherein the nucleobase alteration disrupts splicing of the C3 transcript.

51. The method of claim 27 or claim 28, wherein the nucleobase change enables an alteration of the encoded amino acid residue, wherein the change disrupts cleavage of the C3 polypeptide by the C3 convertase.

52. The method of claim 27 or claim 28, wherein the one or more guide RNAs target the base editor to achieve alteration of nucleobases in the start codon of the C3 polynucleotide.

53. The method of claim 27 or claim 28, wherein C3 activity and / or expression is reduced by at least about 50% or 60% compared to control subjects without the alteration.

54. The method of claim 27 or claim 28, wherein inappropriate activation of the complement system is associated with an elevated level of one or more of inflammation, presence of autoantibodies, neurodegeneration, and microthrombosis.

55. The method of claim 27 or claim 28, wherein inappropriate activation of the complement system is associated with damage to the central nervous system (CNS), the eyes, the gastrointestinal system, the lung system, the musculoskeletal system, the circulatory system, the skin system, the blood cells, the thyroid, the kidney, the joint, the gastrointestinal system, or the transplanted organ.

56. The method of claim 27 or claim 28, wherein the disease or condition is selected from the group consisting of acute antibody-mediated rejection, age-related macular degeneration, allergic bronchiopulmonary aspergillosis, allergic neuritis, allergic rhinitis, amyotrophic lateral sclerosis, allergic reactions and scleritis, atopic dermatitis, atypical hemolytic syndrome (aHUS), autoimmune hemolytic anemia, Bechchet disease, bronchiolithitis, C3 glomerular disease, inflammatory disorders of the central nervous system (CNS), choroidal neovascularization (CNV), veins Membranitis, chronic allograft vascular lesions, chronic hepatitis, chronic muscle inflammation, chronic pain, chronic pancreatitis, chronic urticaria, Chag-Straus syndrome, conjunctivitis, ciliary body inflammation, demyelinating diseases, dermatitis, dermatomyositis, diabetic retinopathy, encephalitis, eosinophilic pneumonia, map atrophy, giant cell arteritis, glaucoma, glomerulonephritis, graft or transplant rejection or failure, HELLP syndrome, allergic purpura, allergic pneumonia, idiopathic pulmonary fibrosis (IPF), IgA nephropathy (IgAN), inflammatory bowel disease , Inflammatory arthritis, inflammatory skin diseases, infusion reactions, interstitial pneumonia, iridocyclitis, iridocyclitis, ischemia / reperfusion injury, Kawasaki disease, keratitis, lupus nephritis, membranous proliferative glomerulonephritis (MPGN), meningitis, microscopic polyvascular inflammation, myasthenia gravis, myasthenia, nasal polyposis, neuromyelitis optic, neuropathic pain, eye inflammation, osteoarthritis, pancreatitis, lipidomitis, paroxysmal sleep hemoglobinuria (PNH), ciliary flat inflammation, pemphigoid, pemphigus, polyarthritis nodular, multiple myositis, primary membranous nephropathy , Proliferative vitreous retinopathy, proteinuria, psoriasis, pulmonary fibrosis, nephropathy, respiratory distress syndrome, retinal neovascularization (RNV), premature infant retinopathy, rheumatoid arthritis (RA), sinusitis, sarcoma, sarcoma, sclerodermatitis, sclerodermatitis, sclerodermatitis, sclerodermatitis, sclerodermatitis, sclerodermatitis, Scholar syndrome, systemic lupus erythematosus, systemic sclerodermatitis, large arteritis, thyroiditis, thyroiditis, thyroiditis, thyroidoisis, ulcerative colitis, uveitis, vasculitis and Wegener granuloma.

57. The method of claim 27 or claim 28, wherein the administration is topical administration.

58. The method of claim 27 or claim 28, wherein the C3 polynucleotide is in contact with two or more guide RNAs, and wherein each guide RNA binds to a different position within the C3 polynucleotide.

59. The method of claim 27 or claim 28, wherein the nucleobase change results in premature stop codons.

60. The method of claim 27 or claim 28, wherein the subject is a mammal.

61. The method of claim 27 or claim 28, further comprising a second administration to the subject of the one or more guide RNA or one or more polynucleotides encoding the one or more guide RNA and the base editor or one or more polynucleotides encoding the base editor, wherein the second administration is about or at least about 1 month, 6 months or one year after the first administration.

62. A cell produced by the method of claim 25.

63. The cell of claim 62, wherein the cell is a retinal cell or other cell of the eye, a cell of CNS or a hepatocyte.

64. A base editor system comprising a base editor or one or more polynucleotides encoding the base editor, including a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, and one or more guide polynucleotides or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive nucleobases of the spacer listed in Tables 1A, 1B, 1C, 1D, 1E, 1F or 2.

65. The base editor system of claim 64, wherein the deaminase is a deaminase selected from the group consisting of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, rAPOBEC1 and ppAPOBEC.

66. The base editor system of claim 64, wherein the base editor is selected from the group consisting of ABE8.8, ABE8.13, ABE8.17, ABE8.20, ABE8.8_V82T, ABE8.8_V82T_Y147T_Q154S, ABE8.17_V82T, ABE8.17_V82T_Y147T_Q154S, ABE8.20_V82T, ABE8.20_V82T_Y147T_Q154S, BE4 and those base editors listed in Tables 1A, 1B, 1C, 1D, 1E, 1F, 2 and 8 to 15.

67. The base editor system of claim 64, wherein the guiding polynucleotide comprises a spacer consisting of about 19 to about 23 nucleotides.

68. The base editor system of claim 67, wherein the spacer is composed of 21 nucleotides.

69. The base editor system of claim 64, wherein the one or more guide polynucleotides comprises a spacer corresponding to a guide polynucleotide selected from the group consisting of gRNA676, gRNA661, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA3342, gRNA3343 and gRNA3345.

70. The base editor system of claim 64, wherein the napDNAbp domain comprises a Cas9 variant.

71. The base editor system of claim 70, wherein the Cas9 variant comprises one or more of A1283D and E1250K relative to the SpCas9 reference amino acid sequence.

72. The base editor system of claim 70, wherein the Cas9 variant comprises one or more of the following amino acid alterations relative to the spCas9 reference amino acid sequence: I322V, S409I, E427G, R654L, R753G and R1114G; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y and R1337K; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K and A1283D; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R220A and R221A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A and Q768A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A, Q768A, K772A and K775A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, R1337K, A1283D and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, A1283D and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y and R1337K; and I322V, S409I, E427G, R654L, R753G, R1114G and R1337K.

73. The base editor system of any of claims 70-72, wherein the Cas9 variant is SaCas9-KHH, SpCas9-MQKFRAER or SpCas9-VRQR.

74. The base editor system of claim 64 or claim 69, wherein the one or more guiding polynucleotides include nucleic acid analogs.

75. The base editor system of claim 74, wherein the one or more guiding polynucleotides comprises one or more of 2'-OMe and phosphorothioate.

76. The base editor system of claim 75, wherein the one or more guiding polynucleotides comprises one of the following nucleotide sequences: End-mod SpCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNNNNGUUUAGAGCUAG AAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGCACCGAGUCGGUGCUsmUsmUsmUsmUsmU (SEQ ID NO: 943); End-mod SaCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAAAAGGCAAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmUsmUsmU (SEQ ID NO: 944); HM01: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmAmAmAmUmAmGmAmAmUmAmGmCAAGUUAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmCmUmGmAmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmGmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 855); HM07: mNsmNsmNsmNmNmNmNmNmNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUUUAGmAmGmCmUmAmAmAmUmAmUmAmAmAmUAmGmGmCmUmAmAAmUAmAmGmGmCmUmAmAAmUAmAmGmGmCmUmAGUmAmUmAmAmAmGmGmCmUmGmCmUmGmUsmUsmUsmU (SEQ ID NO: 856); NLS (bpsv40): mNsmNsmNsNNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsmUsmUsmUsmUsmUsmU (SEQ ID NO: 857); LONGEST: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmGmCAAGUUAAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmCmUmGmAmAmAmCmUmGmAmAmAmCmUmGmCmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 859); NLS+LONGEST: mNsmNsmNNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmCmGmGmCAAGUUAAAAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmCmUmGmAmAmAmCmUmGmAmAmAmCmUmGmGmCmUsmUsmUsmUsmU (SEQ ID NO: 860); or LONGEST+GOLD: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmAmAmAmCmGmCmGmGmCAAGUUAAAAAAAGGCUAGUCCGUUAmUmCAAmCmUmUmCAAmCmUmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 861); Where "mN" represents a 2'-OMe modification of nucleotide "N" and "Ns" represents a nucleotide "N" linked to the following nucleotide by phosphorothioate (PS).

77. The base editor system of claim 76, wherein the one or more guiding polynucleotides comprises a scaffold covalently linked to a peptide having the following amino acid sequence at the 5' end: CKRTADGSEFESPKKKRKV (SEQ ID NO: 858).

78. The base editor system of claim 64, wherein the base editor comprises a linker between the deaminase domain and the napDNAbp domain, wherein the linker comprises the amino acid sequence KGPKPKKEESEK (SEQID NO: 940).

79. A polynucleotide or a group of polynucleotides encoding a base editor system as claimed in any of claims 64-78, or components thereof.

80. A kit comprising a base editor system as claimed in any one of claims 64-78 for the method of any one of claims 1-61.

81. The kit of claim 80, further comprising a written description of the use of the kit to treat a disease or condition associated with inappropriate activation of the complement system in the subject.

82. A pharmaceutical composition comprising an effective amount of a base editor system as claimed in any one of claims 64-78.

83. A guide polynucleotide comprising the spacer sequences listed in Tables 1A, 1B, 1C, 1D, 1E, 1F or 2.

84. A base editor system comprising a base editor or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides or one or more polynucleotides encoding the one or more guide polynucleotides encoding the one or more guide polynucleotides, wherein a) The guide polynucleotide comprises a spacer corresponding to gRNA676, the napDNAbp domain is SpCas9, and the deaminase domain is selected from the group consisting of TadA*8.8 with amino acid altered V82T, Y147T and Q154S, TadA*8.20 with amino acid altered V82T, Y147T and Q154S, TadA*8.8, TadA*8.13 and TadA*8.20 with amino acid altered V82T; b) The guide polynucleotide comprises a spacer corresponding to gRNA661, the napDNAbp domain is SpCas9 or SpCas9 with amino acid alteration A1283D and E1250K, and the deaminase domain is selected from the group consisting of TadA*8.20, TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.8 with amino acid alteration V82T, Y147T and Q154S, and TadA*8.20 with amino acid alteration V82T; c) The guide polynucleotide comprises a spacer corresponding to gRNA696, the napDNAbp domain is SpCas9 or SpCas9 with amino acid alteration A1283D and E1250K, and the deaminase domain is selected from the group consisting of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.20 with amino acid alteration V82T, and TadA*8.20 with amino acid alteration V82T, Y147T and Q154S; d) The guide polynucleotide comprises a spacer corresponding to gRNA715, the napDNAbp domain is SpCas9 or SpCas9-VRQR, and the deaminase domain is selected from the group consisting of TadA*8.8, TadA*8.13 and TadA*8.20 with amino acid alteration V82T; e) The guide polynucleotide comprises a spacer corresponding to gRNA821, the napDNAbp is SpCas9, the deaminase domain is a cytidine deaminase domain, and the base editor also contains two uracil glycosylase inhibitors; f) The guide polynucleotide comprises a spacer corresponding to gRNA827, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8; g) The guide polynucleotide comprises a spacer corresponding to gRNA828, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8; h) The guide polynucleotide comprises a spacer corresponding to gRNA829, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8; i) The guide polynucleotide comprises a spacer corresponding to gRNA837, the napDNAbp is SpCas9-VRQR, and the deaminase domain is TadA*8.8; j) The guide polynucleotide comprises a spacer corresponding to gRNA838, the napDNAbp is SpCas9-VRQR, and the deaminase domain is TadA*8.8; k) The guide polynucleotide comprises a spacer corresponding to gRNA3342, the napDNAbp is SpCas9, and the deaminase is selected from the group consisting of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.8 with amino acid alteration V82T, Y147T and Q154S, TadA*8.17 with amino acid alteration V82T, Y147T and Q154S, TadA*8.20 with amino acid alteration V82T, Y147T and Q154S; 1) The guide polynucleotide comprises a spacer corresponding to gRNA3343, the napDNAbp selected from the group consisting of: i) SpCas9-MQKFRAER; ii) SpCas9-MQKFRAER with combinations of amino acid alteration I322V, S409I, E427G, R654L, R753G, R1114G and R1337K; iii) SpCas9-MQKFRAER with a combination of amino acid alteration I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, A1283D and E1250K; and iv) SpCas9-MQKFRAER with a combination of amino acid alteration I322V, S409I, E427G, R654L, R753G and R1114G; and The deaminase domain is TadA*8.20 or TadA*7.10; or m) The guide polynucleotide comprises a spacer corresponding to gRNA3345, the napDNAbp domain is SaCas9-KHH, the deaminase domain is a cytidine deaminase domain, and the base editor also contains two uracil glycosylase inhibitors.

85. The base editor system of claim 84, wherein the napDNAbp domain is a nick enzyme.

86. The base editor system of claim 84, wherein the deaminase domain and the napDNAbp domain are separated by a linker comprising the amino acid sequence KGPKPKKEESEK (SEQ ID NO:940).

87. The base editor system of claim 84, wherein the polynucleotide encoding the base editor is codon-optimized.

88. The base editor system of claim 84, wherein the one or more guiding polynucleotides comprises a sequence selected from the group consisting of: HM07: mNsmNsmNsmNmNmNmNmNmNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUUUAGmAmGmCmUmAmAmAmUmAmUmAmAmAmAmGmGmCmUmAmAAmUAmAmGmGmCmUmAmAAmUAmAmGmGmCmUmAGUmAmUmAmAmAmGmGmCmUmGmCmUmGmUsmUsmUsmU (SEQ ID NO: 856); End-mod SpCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNNNNGUUUAGAGCUAG AAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGCACCGAGUCGGUGCUsmUsmUsmUsmUsmU (SEQ ID NO: 943); End-mod SaCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAAAAGGCAAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmUsmUsmU (SEQ ID NO: 944); HM01: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmAmAmAmUmAmGmAmAmUmAmGmCAAGUUAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmCmUmGmAmAmAmAmAmGmUmGmGmCmAmCmGmAmGmUmGmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 855); NLS (bpsv40): mNsmNsmNsNNNNNNNNNNNNNNNNNNNNNNNGUUUAGAGCUAG AAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGCACCGAGUCGGUGCmUsmUsmUsmUsmUsmUsmUsmUsmUsmUsmU (SEQ ID NO: 857); LONGEST: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmGmCAAGUUAAAAAAAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmCmUmGmAmAmAmCmUmGmAmAmAmCmUmGmCmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 859); NLS+LONGEST: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmCmGmGmCAAGUUAAAAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmGmAmAmAmCmUmGmAmAmAmCmUmGmGmCmUsmUsmUsmUsmU (SEQ ID NO: 860); and LONGEST+GOLD: mNsmNsmNNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmGmGmAmAmAmCmGmCmCmGmGmAmAmAmCmGmCmGmGmCAAGUUAAAAAAAGGCUAGUCCGUUAmUmCAAmCmUmUmCAAmCmUmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmGmGmUmGmCmUsmUsmUsmUsmU (SEQ ID NO: 861); Where "mN" represents a 2'-OMe modification of nucleotide "N" and "Ns" represents a nucleotide "N" linked to the following nucleotide by phosphorothioate (PS).

89. The base editor system of claim 88, wherein the one or more guiding polynucleotides comprises a scaffold covalently linked to a peptide having the following amino acid sequence at the 5' end: CKRTADGSEFESPKKKRKV (SEQ ID NO: 858).

90. The base editor system of claim 84, wherein the one or more polynucleotides are selected from the group consisting of gRNA661, gRNA676, gRNA2203, and gRNA4232.

91. A polynucleotide or a group of polynucleotides encoding the base editor system of claim 84.

92. A cell comprising the base editor system of claim 84.

93. A lipid nanoparticle comprising the base editor system of claim 84.

94. A method for changing the nucleobase of a complement component 3 (C3) polynucleotide, the method comprising contacting the C3 polynucleotide with the base editor system of claim 84.

95. A method of treating a disease or condition associated with inappropriate activation of a complement system in a subject in need thereof, the method comprising altering a C3 polynucleotide in the subject by administering to the subject a base editor system as claimed in claim 84.

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