Adenine deaminase, base editing system comprising same, and use thereof

Abstract

The application discloses adenine deaminase, a base editing system comprising the same, and application of the base editing system. The adenine deaminase has one or more amino acid differences selected from the 27th position, the 28th position, the 30th position, the 142nd position and the 144th position compared with the amino acid sequence shown in SEQ ID NO: 2. The adenine deaminase of the application can realize the original functions of adenine deaminase and cytosine deaminase at the same time, has a double-base mutation effect, provides more abundant editing product types, reduces the size of the construct, effectively solves the problem of delivery limitation caused by the oversize of the construct, and greatly promotes the application of the adenine deaminase in gene editing, clinical gene therapy, genetic screening, lineage tracing, molecular evolution, crop genetic breeding and the like.

Description

Technical Field

[0001] This invention belongs to the field of gene editing, specifically relating to an adenine deaminase, a base editing system containing the adenine deaminase, and their applications. Background Technology

[0002] Classical single-base editors are mainly divided into cytosine base editors (CBE, Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016, 533:420-424) and adenine base editors (ABE, Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of a*t to g*c in genomic DNA without DNA cleavage. Nature, 2017, 551:464-471). The former is derived from Streptococcus pyogenes, whose activity is impaired. The cytosine base editor BE3 consists of spCas9n (a pyogenes-derived cytosine deaminase), rAPOBEC1 (a rat-derived cytosine deaminase), and a uracil glycosidase inhibitor. The latter, ABE7.10, is an adenine base editor that can act on single-stranded DNA, resulting from the fusion of bacterial TadA with spCas9 and, with the aid of directed evolution and protein engineering, underwent seven rounds of evolution. Both base editors can deaminate cytosine or adenine within a specific range at the target site using either cytosine deaminase or a modified adenine deaminase, without causing DNA double-strand breaks (DSBs). This leads to precise C>T or A>G substitutions via DNA repair or replication.

[0003] However, ABE and CBE can only catalyze the conversion of a single type of base, which greatly limits their widespread application. Therefore, developing new base editors that can simultaneously generate two different base mutations will greatly enrich base editing tools and has important implications for gene therapy, species improvement, and molecular evolution. Inventors (Zhang X, Zhu B, Chen L, et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat Biotechnol, 2020, 38:856-860) and (Grunewald J, Zhou R, Lareau CA, et al. A dual-deaminase crispr base editor enables concurrent adenine and cytosine editing. Nat Biotechnol, 2020, 38:861-864) and (Sakata RC, Ishiguro S, Mori H, et al. Base editors for simultaneous introduction of c-to-t and a-to-g mutations. Nat Biotechnol, 2020, 38:861-864) from the United States and Japan respectively. (Biotechnol, 2020, 38:865-869) Three laboratories independently developed dual-base editors. The technologies they developed all used Cas9 protein as a backbone and then fused cytosine deaminase, adenine deaminase, and uracil glycoside inhibitor (UGI). However, the single product and the large volume and complex construction system limited their widespread application.

[0004] Currently, the dual-base editor can only achieve simultaneous mutations of A>G and C>T. Furthermore, the mutations of the two bases, A and C, must rely on two deaminases to function. This large and complex construction system will hinder future applications.

[0005] Beta-hemoglobinopathies, such as beta-thalassemia and sickle cell disease (SCD), are caused by mutations in the HBB gene, which encodes beta-hemoglobin. In rare cases, hereditary fetal hemoglobinopathies (HPFH) are benign genetic disorders. Some adult patients exhibit high expression of hemoglobin F (HbF, composed of γ-globin and α-globin), which can alleviate the disease phenotype caused by beta-hemoglobin deficiency. These patients experience milder complications and lower mortality rates. γ-globin is a globin expressed during fetal development that functions similarly to beta-globin. The gene HBG, which encodes beta-globin, is intact in beta-hemoglobin patients, but its expression is silenced in adulthood. Therefore, reactivating the silenced γ-globin in patients to compensate for the missing beta-globin, thereby alleviating or treating beta-hemoglobinopathies, is an effective treatment strategy that has emerged in recent years. In previous studies, the applicant used the cytosine editing editor CBE, which is safer and does not cause double-strand breaks, to target the BCL11A erythroid enhancer (this region is mainly responsible for inhibiting HbF in adults) and disrupt its GATA1 binding site (TTATCA). Because C in TTATCA was edited and disrupted, it caused an increase in fetal hemoglobin (Zeng J, Wu Y, Ren C, et al. Therapeutic base editing of human hematopoietic stem cells. Nat Med, 2020, 26:535-541). Furthermore, it has been reported that in a certain group of HPFH patients, mutations at the -114C site (C>T) or -113 site (A>G) in the HBG1 / 2 promoter region can increase fetal hemoglobin (HbF) expression, thereby alleviating symptoms. The mechanism is that mutations at -114C>T or -115C>T disrupt the binding site of the transcriptional repressor BCL11A, while -113A>G does not disrupt the BCL11A site but instead creates a GATA1 transcription factor binding site (which has been shown to activate HBG1 transcription). Building on this, the applicant further demonstrated that the dual-base editor A&C-BEmax, which simultaneously induces mutations in A / C at this site, can enhance γ-globin expression (Zhang X, Zhu B, Chen L, et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat Biotechnol, 2020, 38:856-860). Therefore, dual-base editing can more effectively increase fetal hemoglobin (HbF) expression, providing a safe and effective treatment strategy for patients with β-hemoglobin. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies, which require the simultaneous presence of two deaminases to achieve simultaneous A>G&C>T mutations, and whose base editors for achieving simultaneous A>G&C>T mutations are large and restrict application. This invention provides an adenine deaminase, a base editing system containing it, and their applications. The adenine deaminase of this invention simultaneously possesses the functions of both cytosine deaminase and adenine deaminase, providing a richer variety of products. Furthermore, the base editing system containing it is small in size, which is beneficial for its widespread application.

[0007] In the inventors' earlier research, the previously developed adenine and cytosine base editor, A&C-BEmax, used a Cas9 protein as a backbone, fused with cytosine deaminase, adenine deaminase, and a uracil glycoside inhibitor. Mutations in both A and C bases required the simultaneous use of these two deaminases to function. Furthermore, the single product (only capable of simultaneous mutations of A>G&C>T) and the large, complex construction system would hinder future applications such as gene therapy. Based on these challenges, the inventors used the currently commonly used adenine deaminase... Using TadA-8e (only 167 amino acids) as a starting point, through crystal structure analysis and rational design, the amino acids that alter the substrate recognition of TadA-8e were identified. This improved the recognition of another substrate, cytosine (C), without affecting the editing of the original substrate adenine (A). Thus, TadA-8e simultaneously possesses the original functions of both cytosine deaminase and adenine deaminase. By utilizing only one adenine deaminase fusion to exert the mutagenic effect of both bases, a miniature and highly efficient dual-base editor, AMPLE (Aplenty Mutagensis and Miniature Programmable Largely Editors), was developed. This provides a richer variety of product types (simultaneously achieving A>G&C>G, A>G&C>T, A>G&C>A, etc.), significantly reducing the construction volume and effectively solving the multi-factor challenges faced by dual-base editors, such as delivery limitations.

[0008] Building on this, the inventors used the mini, highly efficient dual-base editor AMPLE to generate more efficient and precise simultaneous A / C editing at two therapeutic targets (BCL11A site and HBG site) in β-hemoglobinopathies compared to A&C-BEmax. Furthermore, AMPLE is smaller in size, which is more conducive to the efficient targeted therapy and clinical delivery of β-hemoglobinopathies.

[0009] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0010] A first aspect of the present invention provides an adenine deaminase having one or more amino acid differences from the amino acid sequence shown in SEQ ID NO:2, selected from positions 27, 28, 30, 142, and 144.

[0011] In this invention, "amino acid difference" refers to a result compared to the amino acid sequence of SEQ ID NO:2, without specifying the process by which it was obtained, and does not imply that it must be based on SEQ ID NO:2 through mutation. That is, the adenine deaminase can be obtained by mutating other amino acid sequences or by mutating SEQ ID NO:2 as the base sequence, thereby obtaining a specific amino acid difference compared to SEQ ID NO:2; those skilled in the art can also directly obtain the corresponding adenine deaminase through sequence synthesis without undergoing a mutation process.

[0012] In some embodiments of the present invention, the 27th amino acid residue E is replaced with Q or R.

[0013] In some embodiments of the present invention, the 28th amino acid residue V is replaced with A, F or G.

[0014] In some embodiments of the present invention, the 30th amino acid residue V is replaced with L.

[0015] In some embodiments of the present invention, amino acid residue A at position 142 is replaced with R, V or W.

[0016] In some embodiments of the present invention, the 144th amino acid residue L is replaced with A.

[0017] In some embodiments of the present invention, amino acid residue E at position 27 is replaced with Q or R, and amino acid residue V at position 28 is replaced with A, F or G.

[0018] In some embodiments of the present invention, amino acid residue E at position 27 is replaced with Q or R, and amino acid residue V at position 30 is replaced with L.

[0019] In some embodiments of the present invention, amino acid residue E at position 27 is replaced with Q or R, and amino acid residue A at position 142 is replaced with R, V or W.

[0020] In some embodiments of the present invention, amino acid residue E at position 27 is replaced with Q or R, and amino acid residue L at position 144 is replaced with A.

[0021] In some embodiments of the present invention, amino acid residue V at position 28 is replaced with A, F or G, and amino acid residue V at position 30 is replaced with L.

[0022] In some embodiments of the present invention, amino acid residue V at position 28 is replaced with A, F or G, and amino acid residue A at position 142 is replaced with R, V or W.

[0023] In some embodiments of the present invention, amino acid residue V at position 28 is replaced with A, F or G, and amino acid residue L at position 144 is replaced with A.

[0024] In some embodiments of the present invention, the adenine deaminase has an amino acid difference selected from positions 27 and 28 compared to the amino acid sequence shown in SEQ ID NO:2; preferably, amino acid residue V at position 28 is replaced with G.

[0025] Preferably, the adenine deaminase has an amino acid difference at position 30, 142, or 144 compared to the amino acid sequence shown in SEQ ID NO:2; preferably, amino acid residue A at position 142 is replaced with R.

[0026] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with Q, and amino acid residue V at position 28 is replaced with A.

[0027] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with Q, and amino acid residue V at position 28 is replaced with G.

[0028] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with R, and amino acid residue V at position 28 is replaced with A.

[0029] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with R, and amino acid residue V at position 28 is replaced with G.

[0030] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with Q, and amino acid residue V at position 30 is replaced with L.

[0031] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with R, and amino acid residue V at position 30 is replaced with L.

[0032] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with Q, and amino acid residue A at position 142 is replaced with R.

[0033] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with R, and amino acid residue A at position 142 is replaced with R.

[0034] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with Q, and amino acid residue L at position 144 is replaced with A.

[0035] In some specific embodiments of the present invention, amino acid residue E at position 27 is replaced with R, and amino acid residue L at position 144 is replaced with A.

[0036] In some specific embodiments of the present invention, amino acid residue V at position 28 is replaced with G, and amino acid residue L at position 144 is replaced with A.

[0037] A second aspect of the invention provides a base editor for editing cytosine and adenine, the base editor comprising a nuclease and an adenine deaminase as described in the first aspect.

[0038] Preferably, the nuclease is a Cas protein or a variant thereof.

[0039] More preferably, the Cas protein is selected from spCas9 of Saccharomyces cerevisiae, SaCas9 of Staphylococcus aureus, LbCas12a of Trichophytonceae bacteria and enAsCas12a of Acidococcus spp. bacteria; the Cas protein variant is selected from VQR-spCas9, VRER-spCas9, spRY, spNG, SaCas9-KKH and SaCas9-NG.

[0040] More preferably, the Cas protein is spCas9 derived from Saccharomyces cerevisiae, and the amino acid sequence of spCas9 is shown in SEQ ID NO:4.

[0041] In some embodiments of the present invention, the base editor further includes a UGI molecule.

[0042] The UGI molecule has at least one copy, preferably two, three, or four copies.

[0043] In some specific embodiments of the present invention, the UGI molecule is two copies.

[0044] In some specific embodiments of the present invention, the amino acid sequence of the UGI molecule is shown in SEQ ID NO:5.

[0045] In this invention, when the UGI molecule has at least two copies, the amino acid sequence of the UGI molecule is as shown in SEQ ID NO:6.

[0046] In this invention, when the UGI molecule has at least two copies, the copies are connected by a linker, and the amino acid sequence of the linker is preferably as shown in SEQ ID NO:7.

[0047] In this invention, the base editor may further include functional molecules, which help to improve the efficiency of simultaneous A and C mutation, expand the product types of simultaneous A and C mutation, and adjust the editing range of simultaneous A and C mutation.

[0048] A third aspect of the present invention provides a fusion protein comprising a nuclease and an adenine deaminase as described in the first aspect; the nuclease being as described in the second aspect; and the nuclease and / or the adenine deaminase being linked to the UGI molecule via a first linker.

[0049] In some embodiments of the present invention, the nuclease, the adenine deaminase, and the UGI molecule are arranged in the following order from the N-terminus to the C-terminus: adenine deaminase-nuclease-UGI molecule; the UGI molecule and the nuclease are connected by a first linker.

[0050] In some embodiments of the present invention, the nuclease, the adenine deaminase, and the UGI molecule are arranged in the following order from the N-terminus to the C-terminus: nuclease-adenine deaminase-UGI molecule; the UGI molecule and the adenine deaminase are connected by a first linker.

[0051] In some embodiments of the present invention, the nuclease, the adenine deaminase, and the UGI molecule are arranged in the following order from the N-terminus to the C-terminus: adenine deaminase-UGI molecule-nuclease; the nuclease and / or the adenine deaminase are connected to the UGI molecule via a first linker.

[0052] In some embodiments of the present invention, the nuclease, the adenine deaminase, and the UGI molecule are arranged in the following order from the N-terminus to the C-terminus: nuclease-UGI molecule-adenine deaminase; the nuclease and / or the adenine deaminase are connected to the UGI molecule via a first linker.

[0053] In some embodiments of the present invention, the nuclease, the adenine deaminase, and the UGI molecule are arranged in the following order from the N-terminus to the C-terminus: UGI molecule-adenine deaminase-nuclease; the UGI molecule and the adenine deaminase are connected by a first linker.

[0054] In some embodiments of the present invention, the nuclease, the adenine deaminase, and the UGI molecule are arranged in the following order from the N-terminus to the C-terminus: UGI molecule-nuclease-adenine deaminase; the UGI molecule and the nuclease are connected by a first linker.

[0055] In some specific embodiments of the present invention, the amino acid sequence of the first linker is shown in SEQ ID NO:8.

[0056] In some embodiments of the present invention, when the UGI molecule is located at the N-terminus or C-terminus of the fusion protein, the adenine deaminase and the nuclease are connected by a second linker.

[0057] In some specific embodiments of the present invention, the amino acid sequence of the second linker is shown in SEQ ID NO:3.

[0058] In some embodiments of the present invention, the N-terminus and / or C-terminus of the fusion protein are connected to a nuclear localization signal sequence or a polyA signal sequence.

[0059] In this invention, the amino acid sequence of the nuclear localization signal sequence is shown in SEQ ID NO:1; the polyA signal sequence is preferably BGH polyA.

[0060] In this invention, the polyA signal sequence can be a polyadenylation signal sequence conventionally derived from eukaryotic or prokaryotic cells.

[0061] The BGH polyA is a bovine growth hormone polyadenylation signal.

[0062] In this invention, the lengths of the first and second connectors can be adjusted according to what is known in the art to improve the targeted editing efficiency of adenine and cytosine in the fusion protein, regulate the editing window, or improve the precision editing efficiency.

[0063] In some specific embodiments of the present invention, the N-terminus and C-terminus of the fusion protein are connected to nuclear localization signal sequences.

[0064] In some specific embodiments of the present invention, the C-terminus of the fusion protein is attached with a polyA signal sequence.

[0065] In this invention, the fusion protein may further include a functional molecular protein, which helps to improve the efficiency of simultaneous mutation of A and C, expand the product types of simultaneous mutation of A and C, and adjust the editing range of simultaneous mutation of A and C.

[0066] A fourth aspect of the present invention provides an isolated nucleic acid that encodes an adenine deaminase as described in the first aspect, a base editor as described in the second aspect, or a fusion protein as described in the third aspect.

[0067] A fifth aspect of the present invention provides a base editing system for editing cytosine and adenine, the base editing system comprising: sgRNA and a base editor as described in the second aspect.

[0068] Preferably, the target sequence of the sgRNA has a nucleotide sequence as shown in SEQ ID NO:9, 12 or 15.

[0069] In this invention, the sgRNA can be designed and constructed according to conventional techniques in the art.

[0070] A sixth aspect of the present invention provides a pharmaceutical composition comprising an adenine deaminase as described in the first aspect, a base editor as described in the second aspect, a fusion protein as described in the third aspect, or a base editing system as described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0071] A seventh aspect of the present invention provides a base editing method for non-therapeutic purposes, the base editing method comprising:

[0072] Base editing is performed on target cells by expressing adenine deaminase as described in the first aspect, a base editor as described in the second aspect, a fusion protein as described in the third aspect, or a base editing system as described in the fifth aspect.

[0073] Preferably, the target cells are derived from isolated cell lines.

[0074] More preferably, the isolated cell line is 293T cells, HELA cells, U2OS cells, NIH3T3 cells, or N2A cells.

[0075] In this invention, the non-therapeutic purpose can be base editing in the construction of animal models of diseases, crop breeding, etc., as well as gene expression regulation research in vitro.

[0076] The eighth aspect of the present invention provides the use of adenine deaminase as described in the first aspect, base editor as described in the second aspect, fusion protein as described in the third aspect, or base editing system as described in the fifth aspect in the preparation of base-edited drugs, the construction of animal models, gene expression regulation, crop breeding, the preparation of drugs for gene therapy, or the preparation of base editing tools.

[0077] In some embodiments of the present invention, the gene therapy includes: base editing of the regulatory elements of a target gene in target cells to change the expression level of the target gene, thereby treating the disease.

[0078] In some preferred embodiments of the present invention, the target cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, erythroid progenitor cells and erythroid precursor cells; preferably selected from hematopoietic stem cells.

[0079] In some preferred embodiments of the present invention, the target gene is selected from genes that express human globin and genes that regulate human globin expression; preferably selected from BCLA11, HBG1 and HBG2.

[0080] In some preferred embodiments of the present invention, the regulatory sequence is selected from enhancers and promoters.

[0081] In some preferred embodiments of the present invention, the disease is β-hemoglobinopathies, preferably selected from β-thalassemia and sickle cell disease.

[0082] In some specific embodiments of the present invention, the regulatory element of the target gene is an enhancer of BCLA11.

[0083] In some specific embodiments of the present invention, the regulatory element of the target gene is the promoter of HBG1.

[0084] In some specific embodiments of the present invention, the regulatory element of the target gene is the promoter of HBG2.

[0085] A ninth aspect of the present invention provides a pharmaceutical composition for treating and / or preventing β-hemoglobinopathies, characterized in that the pharmaceutical composition comprises an adenine deaminase as described in the first aspect, a base editor as described in the second aspect, a fusion protein as described in the third aspect, or a base editing system as described in the fifth aspect, and a pharmaceutically acceptable carrier; the pharmaceutical composition performs base editing on genes expressing human globin and / or regulatory elements of genes regulating human globin expression in target cells.

[0086] Preferably, the β-hemoglobinopathies, the target cells, the gene expressing human globin, and / or the regulatory elements of the gene regulating human globin expression are as described in the eighth aspect.

[0087] A tenth aspect of the present invention provides a gene expression cassette, the gene expression cassette comprising a promoter element and a target gene element;

[0088] The target gene element is selected from genes encoding adenine deaminase as described in the first aspect, a base editor as described in the second aspect, or a fusion protein as described in the third aspect.

[0089] Preferably, the promoter element is selected from CMV, CAG, PGK, EF1α, Ctsk and Lp1.

[0090] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0091] The reagents and raw materials used in this invention are all commercially available.

[0092] The positive and progressive effects of this invention are as follows:

[0093] The adenine deaminase of this invention enhances and recognizes cytosine without affecting adenine editing, enabling it to simultaneously perform the original functions of both adenine and cytosine deaminases, exerting a double-base mutation effect, providing a richer variety of editing products, reducing the size of the construct, and effectively solving the delivery limitation problem caused by the excessive size of the construct. This will greatly promote its application in gene editing, clinical gene therapy, genetic screening, lineage tracing, molecular evolution, and crop genetic breeding. Furthermore, it has already been able to generate more efficient and precise simultaneous A / C editing at gene loci related to β-hemoglobinopathy, which is more conducive to the efficient targeted therapy and clinical delivery of β-hemoglobinopathy. Attached Figure Description

[0094] Figure 1 A schematic diagram showing the comparison of C4, C6, and A7 base editing results achieved at the PPP1R12C site3 site on 293T for 10 ABE8e mutants obtained from Sanger sequencing.

[0095] Figure 2 A schematic diagram showing the comparison of C4, C6, and A7 base editing results achieved at the PPP1R12C site3 site on 293T for 10 ABE8e mutants obtained from high-throughput sequencing.

[0096] Figure 3 A schematic diagram showing the proportion of simultaneous A and C mutations achieved by constructing a PPP1R12C site3 site on 293T for 10 ABE8e mutants.

[0097] Figure 4 This is a schematic diagram showing the comparison results of C4, C6, and A7 base editing achieved at the PPP1R12C site3 on 293T using AMPLE-11 to 18 obtained from Sanger sequencing.

[0098] Figure 5 This is a schematic diagram showing the comparison results of C4, C6, and A7 base editing achieved at the PPP1R12C site3 on AMPLE-19~42 obtained by Sanger sequencing.

[0099] Figure 6 This is a schematic diagram showing the comparison results of C4, C6, and A7 base editing achieved at the PPP1R12C site3 on AMPLE-43~66 obtained by Sanger sequencing.

[0100] Figure 7 This is a partial schematic diagram of the HBG1 / 2 promoter region; the triangular arrows point to the -115C, -114C, and -113A sites from left to right, and the "-" indicates that they are located upstream of the HBG transcription origin.

[0101] Figure 8 This diagram illustrates the comparison of C4, C6, and A7 base editing results achieved at the BCL11A and HBG sites on 293T at AMPLE-43, 45, 49, and 51 obtained from Sanger sequencing. Detailed Implementation

[0102] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0103] In the embodiment, the order of the functional blocks of ABE8e is: bNLS+TadA8e+Linker+spCas9n(D10A)+bNLS;

[0104] The order of functional blocks in AMPLE-1~10 and AMPLE19~42 is: bNLS+TadA8e (mutation)+Linker+spCas9n(D10A)+bNLS;

[0105] The order of functional blocks in AMPLE-11~18 and AMPLE-43~66 is: bNLS+TadA8e (mutation)+Linker+spCas9n(D10A)+P2A+2×UGI+bNLS.

[0106] The amino acid sequence of bNLS is shown in SEQ ID NO:1:

[0107] MKRTADGSEFESPKKKRKV.

[0108] The amino acid sequence of TadA-8e is shown in SEQ ID NO:2:

[0109] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN.

[0110] The amino acid sequence of the linker (second linker) is shown in SEQ ID NO:3:

[0111] SGGSSGGSSGSETPGTSESATPESSGGSSGGS.

[0112] The amino acid sequence of spCas9n(D10A)(1-1368) is shown in SEQ ID NO:4.

[0113]

[0114] The amino acid sequence of UGI is as follows:

[0115] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 5).

[0116] The amino acid sequence of 2×UGI is as follows:

[0117] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 6).

[0118] The amino acid sequence of the linker Linker1 between 2×UGI is: SGGSGGSGGS (SEQ ID NO:7).

[0119] The amino acid sequence of P2A (first linker) is: TNFSLLKQAGDVEENPGP (SEQ ID NO:8).

[0120] In the embodiments, fusion proteins containing the above sequences can be synthesized according to conventional techniques in the art, and the retention of the starting amino acid M can be determined based on the position of the fragment corresponding to the above sequence in the fusion protein.

[0121] Example 1

[0122] 1.1 Plasmid Design and Construction

[0123] 1.1.1 Based on the crystal structure of TadA-8e binding to substrate DNA captured by cryo-electron microscopy, it was speculated that 6 amino acids may affect the main product of ABE8e, adenine editing, and the low-frequency byproduct, cytosine editing. A series of ABE8e mutants were designed by changing the hydrophobicity or polarity of the amino acids, as shown in Table 1, resulting in 10 constructs AMPLE-1 to 10. At the same time, an endogenous test target of human gene (PPP1R12C) PPP1R12C site3 (as shown in Table 2) was designed for screening and evaluation.

[0124] 1.1.2 ABE8e single-site and combined mutant sequences were synthesized according to Table 1, using ABE8e as a vector (Addgene#138489), followed by seamless cloning and assembly (using the Vazyme ClonExpress MultiS One StepCloning Kit, C113-01). The target site was synthesized as shown in Table 2, with CACC added to the positive strand and AAAC added to the reverse strand, which were then ligated to the U6-sgRNA-EF1α-GFP that had been digested with BbsI.

[0125] 1.1.3 The plasmids constructed in 1.1.1 and 1.1.2 were sequenced by Sanger sequencing to ensure they were completely correct.

[0126] Table 1 TadA-8e mutant sequences

[0127]

[0128]

[0129] Table 2 Targets and Sequences Used

[0130]

[0131] In the table, Oligo-up is the forward primer and Oligo-dn is the reverse primer.

[0132] 1.2 Cell transfection

[0133] On day 1, HEK293T cells (HEK293T cells are the ATCC CRL-3216 cell line) were seeded in 24-well plates:

[0134] (1) Digest HEK293T cells at a rate of 2×10⁻⁶. 5 Cells / wells are seeded into 24-well plates.

[0135] Note: After cell resuscitation, cells generally need to be passaged twice before they can be used for transfection experiments.

[0136] Day 2 transfection:

[0137] (2) Observe the state of cells in each well.

[0138] Note: The cell density should be 70%-90% before transfection and the cells should be in normal condition.

[0139] (3) Plasmid transfection is performed as follows:

[0140] Using the newly constructed ABE8e mutant plasmids from step 1.1 above, with a plasmid ratio of U6-sgRNA-EF1α-GFP = 750 ng: 250 ng, and PEI as the transfection reagent (3 μL PEI per 1 μg plasmid), co-transfect the HEK293T host, with ABE8e / A&C-BEmax as the control; set up n = 3 wells / group.

[0141] 1.3 Genome extraction and preparation of amplicon libraries

[0142] 72 hours after transfection, genomic DNA was extracted from the cells using the Tiangen Cell Genome Extraction Kit (DP304). Then, using the Hi-Tom Gene Editing Detection Kit (Novogene), corresponding identification primers were designed (as shown in Table 3). Specifically, a bridging sequence 5'-ggagtgagtacggtgtgc-3' (SEQ ID NO:8) was added to the 5' end of the forward identification primer, and a bridging sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO:9) was added to the 5' end of the reverse identification primer, resulting in a first-round PCR product. This first-round PCR product was then used as a template for a second-round PCR. The resulting products were then mixed, gel-cleaved, purified, and sent to the company for sequencing (the sequencing service provider was Suzhou Kingwise Biotechnology Co., Ltd.).

[0143] Table 3. Primers used for target identification

[0144]

[0145] In the table, F represents the forward identification primer and R represents the reverse identification primer.

[0146] 1.4 Analysis and Statistics of Deep Sequencing Results

[0147] The deep sequencing results were analyzed using the BE-analyzer website (http: / / www.rgenome.net / be-analyzer / #!), specifically by calculating the ratios of A to G, C to T, C to G, and C to A. Statistical graphs were then created using GraphPad Prism 9.1.0. The results are shown below. Figures 1-3 As shown in Tables 4 and 5.

[0148] like Figure 1 As shown, Sanger sequencing results revealed that, compared to ABE8e, AMPLE-1, AMPLE-2, and AMPLE-5 all exhibited C6 editing; for example... Figure 2As shown, according to the high-throughput sequencing results, AMPLE-1 to 10 all retain the original A>G function of ABE8e with an editing efficiency of 51.8% to 61%, and produce C>G, C>T, and C>A mutations of varying degrees at the C6 position. In contrast, the first-generation dual-base editor A&C-BEmax has an A>G mutation efficiency of only 8.5% and produces only one C>T mutation product at the C6 position.

[0149] like Figure 3 As shown, statistical analysis of the proportion of products containing both A and C mutations revealed that, compared to A&C-BEmax, AMPLE-1, AMPLE-2, AMPLE-3, AMPLE-5, and AMPLE-6 produced effective double mutations of three types: A>G&C>G / T / A. Taking AMPLE-5 as an example, the efficiency of the A>G&C>G double mutation was 20.18%, the efficiency of the A>G&C>T double mutation was 17.07%, and the efficiency of the A>G&C>A double mutation was 2.01%. In contrast, A&C-BEmax produced only one type of A>G&C>T double mutation, with an efficiency of only 5.19%.

[0150] Table 4. High-throughput sequencing: C4, C6, and A7 base editing efficiency results achieved at the PPP1R12C site3 site on 293T using 10 ABE8e mutants (unit, %).

[0151] A&CBEmax 0.4 53.0 0 0.0 27.4 0 8.5 ABE8e 0.2 0.2 0 3.7 2.2 0.2 57.4 AMPLE-1 1.2 0.4 0 19.3 11.8 1.1 53.0 AMPLE-2 1.6 0.7 0.1 23.0 15.0 1.8 55.5 AMPLE-3 0.4 0.1 0 14.4 7.8 0.8 61.0 AMPLE-4 1.0 0.4 0 2.0 1.0 0.1 61.0 AMPLE-5 1.8 0.5 0.1 28.1 18.2 2.3 51.8 AMPLE-6 2.9 1.0 0.1 9.2 5.3 0.4 58.9 AMPLE-7 0.3 0.1 0 3.4 1.8 0.2 57.1 AMPLE-8 0.1 0.1 0 4.2 2.5 0.2 55.9 AMPLE-9 0.1 0.1 0 1.4 0.9 0.1 51.0 AMPLE-10 0.2 0.1 0 3.4 2.1 0.1 61.0

[0152] Table 5. High-throughput sequencing: Proportion of simultaneous A & C mutations achieved at the PPP1R12C site3 site on 293T in 10 ABE8e mutants (unit, %)

[0153] A&CBEmax 0 5.19 0 ABE8e 4.00 2.32 0.24 AMPLE-1 15.71 11.32 0.99 AMPLE-2 20.17 14.44 1.65 AMPLE-3 14.68 7.74 0.76 AMPLE-4 3.13 1.39 0.08 AMPLE-5 20.18 17.07 2.01 AMPLE-6 11.99 5.97 0.42 AMPLE-7 3.71 1.86 0.20 AMPLE-8 4.17 2.53 0.22 AMPLE-9 1.51 1.03 0.11 AMPLE-10 3.73 2.23 0.1

[0154] Example 2: The TadA-8e mutant fused with P2A-2×UGI exhibits precise simultaneous mutations in A and C without causing mutations in other A or C molecules within the window, and controls the mutation products to be A>G&C>T.

[0155] To further improve the efficiency and editing range of A&C combination mutations, a second round of 24 double-mutant constructs were obtained based on the 10 constructs in the first round of Example 1, resulting in constructs AMPLE-19 to 42. These constructs were also screened and evaluated using PPP1R12Csite3.

[0156] 2.1 Plasmid Design and Construction

[0157] 2.1.1 Merge the partial constructs from the first step into P2A-2×UGI, using the same construction method as 1.1.2.

[0158] 2.1.2 The plasmid constructed in 2.1.1 was sequenced by Sanger sequencing to ensure it was completely correct.

[0159] 2.2 Cell transfection

[0160] On day 1, HEK293T cells (HEK293T cells are the ATCC CRL-3216 cell line) were seeded in 24-well plates:

[0161] (1) Digest HEK293T cells at a rate of 2×10⁻⁶. 5 Cells / wells are seeded into 24-well plates.

[0162] Note: After cell resuscitation, cells generally need to be passaged twice before they can be used for transfection experiments.

[0163] Day 2 transfection:

[0164] (2) Observe the state of cells in each well.

[0165] Note: The cell density should be 70%-90% before transfection and the cells should be in normal condition.

[0166] (3) Plasmid transfection levels are as follows:

[0167] The newly constructed plasmid in section 2.1 was U6-sgRNA-EF1α-GFP = 750 ng: 250 ng. The transfection reagent was PEI (3 μL of PEI per 1 μg plasmid). The plasmid was co-transfected into the HEK293T host, with ABE8e / A&C-BEmax as the control. The number of wells was set to n = 3.

[0168] 2.3 Genome extraction and preparation of amplicon libraries

[0169] 72 hours after transfection, genomic DNA was extracted from the cells using the Tiangen Cell Genome Extraction Kit (DP304). Then, using the Hi-Tom Gene Editing Detection Kit (Novogene), corresponding identification primers were designed (as shown in Table 3). Specifically, a bridging sequence 5'-ggagtgagtacggtgtgc-3' (SEQ ID NO:8) was added to the 5' end of the forward identification primer, and a bridging sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO:9) was added to the 5' end of the reverse identification primer, resulting in a first-round PCR product. This first-round PCR product was then used as a template for a second-round PCR. The resulting products were then mixed, gel-cleaved, purified, and sent to the company for sequencing (the sequencing service provider was Suzhou Kingwise Biotechnology Co., Ltd.).

[0170] 2.4 Analysis and Statistics of Deep Sequencing Results

[0171] The deep sequencing results were analyzed using the BE-analyzer website, specifically by calculating the ratios of A to G, C to T, C to G, and C to A, and then plotted using GraphPad Prism 9.1.0. The results are shown below. Figure 5 As shown.

[0172] like Figure 5 As shown, Sanger sequencing results revealed that constructs such as AMPLE-19, AMPLE-21, and AMPLE-27 produced effective mutations at C4, C6, and A7 sites. In particular, C4 editing was not observed in the first-round constructs. In summary, constructs such as AMPLE-1, AMPLE-2, AMPLE-3, AMPLE-5, AMPLE-6, AMPLE-19, AMPLE-21, and AMPLE-27 exhibited higher efficiency in two-base mutations, more combinations of mutation product types, and a wider editing range.

[0173] To precisely control the product type and editing scope for specific application scenarios, partial builds from the first and second rounds were merged into P2A-UGI, resulting in 32 builds: AMPLE-11–18 and AMPLE-43–66. These were then evaluated using PPP1R12Csite3, and the results are as follows: Figure 4 and Figure 6 As shown.

[0174] like Figure 4 As shown, in the single-point mutation fusion P2A-2×UGI construct, except for AMPLE-14, AMPLE-17, and AMPLE-18, the other five fusions have a better ability to simultaneously and accurately edit C6 and A7 than A&C-BEmax, and the mutation products are mainly A>G&C>T.

[0175] like Figure 6As shown, according to the Sanger sequencing results, compared with the single-point mutation fusion P2A-2×UGI construction, the combined mutation fusion P2A-2×UGI construction of AMPLE-43~66 further improved the editing performance. For example, AMPLE-43, AMPLE-45, AMPLE-46, AMPLE-47, AMPLE-48, AMPLE-49, AMPLE-51, AMPLE-52, AMPLE-53, AMPLE-54, AMPLE-63 all significantly increased the proportion of A>G&C>T mutations in C6 and A7. The editing performance of A&C-BEmax for C6 and A7 was still relatively low. In summary, the constructs AMPLE-11, AMPLE-12, AMPLE-13, AMPLE-15, AMPLE-16, AMPLE-43, AMPLE-45, AMPLE-46, AMPLE-47, AMPLE-48, AMPLE-49, AMPLE-51, AMPLE-52, AMPLE-53, AMPLE-54, and AMPLE-63 exhibited precise simultaneous mutations of A and C without causing mutations in other A or C components within the window, and controlled the mutation products to be A>G&C>T.

[0176] The above experiments show that the TadA-8e mutant of the present invention exhibits more efficient double-base mutations, more combinations of mutant product types, and a wider editing range.

[0177] Example 3: The TadA-8e mutant fusion with P2A-2×UGI induced highly efficient and precise simultaneous mutations of A and C at two therapeutic targets in β-hemoglobinopathies (BCL11Asite and HBG site), demonstrating a more promising therapeutic technology for clinical application.

[0178] 3.1 Plasmid Design and Construction

[0179] 3.1.1 Construct BCL11A site and HBG site targets (Table 2), using the same method as 1.1.2.

[0180] 3.1.2 Perform Sanger sequencing on the plasmid constructed in 3.1.1 to ensure it is completely correct.

[0181] 2.2 Cell transfection

[0182] On day 1, HEK293T cells (HEK293T cells are the ATCC CRL-3216 cell line) were seeded in 24-well plates:

[0183] (1) Digest HEK293T cells at a rate of 2×10⁻⁶. 5 Cells / well inoculated into 24-well plates.

[0184] Note: After cell resuscitation, cells generally need to be passaged twice before they can be used for transfection experiments.

[0185] Day 2 transfection:

[0186] (2) Observe the state of cells in each well.

[0187] Note: The cell density should be 70%-90% before transfection and the cells should be in normal condition.

[0188] (3) Plasmid transfection levels are as follows:

[0189] The newly constructed plasmid in section 2.1 was U6-sgRNA-EF1α-GFP = 750 ng: 250 ng. The transfection reagent was PEI (3 μL of PEI per 1 μg plasmid). The plasmid was co-transfected into the HEK293T host, with A&C-BEmax as a control. The number of wells was set to n = 3 per group.

[0190] 2.3 Genome extraction and Sanger sequencing

[0191] 72 hours after transfection, genomic DNA was extracted from the cells using the Tiangen Cell Genome Extraction Kit (DP304). Corresponding identification primers were then designed (as shown in Table 3): a bridging sequence 5'-ggagtgagtacggtgtgc-3' (SEQ ID NO:8) was added to the 5' end of the forward identification primer, and a bridging sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO:9) was added to the 5' end of the reverse identification primer. One round of PCR products was obtained, and after gel extraction, purification, and delivery to the company for Sanger sequencing (the Sanger sequencing service provider was Qingke Biotechnology Co., Ltd.).

[0192] 2.4 Analysis of Deep Sequencing Results

[0193] For the BCL11A site, the GATA binding region to be disrupted is TTATCA, where C and A are located at positions 6 and 7 respectively within the target window. Figure 8 As shown, Sanger sequencing results revealed that the A&C-BEmax treatment group produced inefficient C6 and A7 mutations, while the AMPLE-43 and AMPLE-49 constructs, which are independent of cytosine deaminase, produced precise and efficient C6 and A7 mutation capabilities; Figure 7As shown, for the HBG site, the -114 and -113 regions that need to be edited are C6 and A7, respectively. A&C-BEmax still shows low-frequency editing of C6 and A7, while AMPLE-43, AMPLE-45, AMPLE-49, and AMPLE-51 demonstrate precise and efficient simultaneous editing of C6>T and A7>G. The AMPLE series of mini dual-base editors demonstrates efficient and precise simultaneous A&C editing in both of the aforementioned β-hemoglobinopathies targeting strategies, thereby effectively activating the expression level of fetal hemoglobin HbF. In summary, efficient and precise A&C targeted editing, smaller delivery volume, and the high safety of the base editor will promote the clinical application of the AMPLE series of mini dual-base editors in the treatment of β-hemoglobinopathies. SEQUENCE LISTING <110> East China Normal University Shanghai Bangyao Biotechnology Co., Ltd. <120> Adenine deaminase, base editing systems containing it, and their applications <130> P210110708C <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> bNLS <400> 1 Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys 1 5 10 15 Arg Lys Val <210> 2 <211> 167 <212> PRT <213> Artificial Sequence <220> <223> TadA-8e <400> 2 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly 100 105 110 Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Ile Asn 165 <210> 3 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Second linker <400> 3 Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr 1 5 10 15 Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser 20 25 30 <210> 4 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> spCas9nD10A1-1368 <400> 4 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 5 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> UGI <400> 5 Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val 1 5 10 15 Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile 20 25 30 Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu 35 40 45 Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr 50 55 60 Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile 65 70 75 80 Lys Met Leu <210> 6 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> 2×UGI <400> 6 Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val 1 5 10 15 Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile 20 25 30 Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu 35 40 45 Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr 50 55 60 Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile 65 70 75 80 Lys Met Leu Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Thr Asn Leu 85 90 95 Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu 100 105 110 Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys 115 120 125 Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp 130 135 140 Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp 145 150 155 160 Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu 165 170 175 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker1 <400> 7 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10 <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> First Linker <400> 8 Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PPP1R12C site3 <400> 9 gagctcactg aacgctggca tgg 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligo-up-PPP1R12C site3 <400> 10 caccgagctc actgaacgct ggca 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligo-dn-PPP1R12C site3 <400> 11 aaactgccag cgttcagtga gctc 24 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HBG website <400> 12 ttgaccaata gccttgacaa gg 22 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligo-up-HBG website <400> 13 caccgttgac caatagcctt gaca 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligo-dn-HBG website <400> 14 aaactgtcaa ggctattggt caac 24 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Site BCL11A <400> 15 tttatcacag gctccaggaa ggg 23 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligo-up-BCL11A website <400> 16 caccgtttat cacaggctcc aggaa 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Site Oligo-dn-BCL11A <400> 17 aaacttcctg gagcctgtga taaac 25 <210> 18 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> F-PPP1R12C site3 <400> 18 ggagtgagta cggtgtgcgc tcctgccaac ctttcaaaag agg 43 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> R-PPP1R12C site3 <400> 19 gagttggatg ctggatgggt catgcgtgac acgtggattg tg 42 <210> 20 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> F-HBG site <400> 20 ggagtgagta cggtgtgccc ccttccccac actatctcaa tgc 43 <210> 21 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> R-HBG site <400> 21 gagttggatg ctggatggtt attcttcatc cctagccagc cgc 43 <210> 22 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> F-BCL11A site <400> 22 ggagtgagta cggtgtgctc cagtgcaaag tccatacagg t 41 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R-BCL11A site <400> 23 gagttggatg ctggatgggt ctgccagtcc tcttctaccc 40

Claims

1. An adenine deaminase, characterized in that, The amino acid differences between the adenine deaminase and the amino acid sequence shown in SEQ ID NO: 2 are selected from any of the following groups: (1) The 27th amino acid residue E is replaced with Q, and the 28th amino acid residue V is replaced with A; (2) The 27th amino acid residue E is replaced with Q, and the 28th amino acid residue V is replaced with G; (3) The 27th amino acid residue E is replaced with R, and the 28th amino acid residue V is replaced with A; (4) The 27th amino acid residue E is replaced with R, and the 28th amino acid residue V is replaced with G; (5) The 27th amino acid residue E is replaced with Q, and the 30th amino acid residue V is replaced with L; (6) The 27th amino acid residue E is replaced with R, and the 30th amino acid residue V is replaced with L; (7) The 27th amino acid residue E is replaced with Q, and the 142nd amino acid residue A is replaced with R; (8) The 27th amino acid residue E is replaced with R, and the 142nd amino acid residue A is replaced with R; (9) The 27th amino acid residue E is replaced with Q, and the 144th amino acid residue L is replaced with A; and, (10) The 27th amino acid residue E is replaced with R, and the 144th amino acid residue L is replaced with A.

2. A base editor for editing cytosine and adenine, characterized in that, The base editor includes: (i) nucleases and adenine deaminase as described in claim 1; or (ii) a nuclease, adenine deaminase and UGI molecule, wherein the adenine deaminase is an adenine deaminase that differs from the amino acid sequence shown in SEQ ID NO: 2 in that the amino acid residue E at position 27 is replaced with Q or R.

3. The base editor as described in claim 2, characterized in that, The nuclease is a Cas protein or a variant thereof; and / or, the base editor in (i) also includes a UGI molecule.

4. The base editor as described in claim 3, characterized in that, The Cas protein is selected from spCas9 of Saccharomyces cerevisiae, SaCas9 of Staphylococcus aureus, LbCas12a of Trichophytonceae bacteria and enAsCas12a of Acidococcus spp. bacteria; the Cas protein variant is selected from VQR-spCas9, VRER-spCas9, spRY, spNG, SaCas9-KKH and SaCas9-NG.

5. The base editor as described in claim 2 or 3, characterized in that, The UGI molecule is at least one copy.

6. The base editor as described in claim 2 or 3, characterized in that, The UGI molecule has two, three, or four copies; and / or, the amino acid sequence of the UGI molecule is as shown in SEQ ID NO:

5.

7. The base editor as described in claim 3, characterized in that, The Cas protein is spCas9 derived from Saccharomyces cerevisiae, and the amino acid sequence of spCas9 is shown in SEQ ID NO:

4.

8. The base editor as described in claim 2 or 3, characterized in that, The UGI molecule consists of at least two copies; the copies are connected by connectors.

9. The base editor as described in claim 8, characterized in that, The amino acid sequence of the UGI molecule is shown in SEQ ID NO: 6; and / or, the amino acid sequence of the linker is shown in SEQ ID NO:

7.

10. A fusion protein, characterized in that, The fusion protein comprises a nuclease and an adenine deaminase; wherein the adenine deaminase is the adenine deaminase as described in claim 1, or an adenine deaminase differing from the amino acid sequence shown in SEQ ID NO: 2 by replacing the 27th amino acid residue E with Q or R; the nuclease is the nuclease defined in the base editor as described in any one of claims 2-7; the nuclease and / or the adenine deaminase are linked to a UGI molecule via a first linker; the UGI molecule is the UGI molecule defined in the base editor as described in any one of claims 5, 6, 8, and 9.

11. The fusion protein of claim 10, characterized in that, The nuclease, the adenine deaminase, and the UGI molecule are arranged in the following order from N-terminus to C-terminus: adenine deaminase-nuclease-UGI molecule, or nuclease-adenine deaminase-UGI molecule, or adenine deaminase-UGI molecule-nuclease, or nuclease-UGI molecule-adenine deaminase, or UGI molecule-adenine deaminase-nuclease, or UGI molecule-nuclease-adenine deaminase; and / or, when the UGI molecule is located at the N-terminus or C-terminus of the fusion protein, the adenine deaminase and the nuclease are connected by a second linker; and / or, the N-terminus and / or C-terminus of the fusion protein are connected to a nuclear localization signal sequence or a polyA signal sequence.

12. The fusion protein as described in claim 11, characterized in that, The amino acid sequence of the first linker is shown in SEQ ID NO: 8; and / or, the amino acid sequence of the second linker is shown in SEQ ID NO: 3; and / or, the amino acid sequence of the nuclear localization signal sequence is shown in SEQ ID NO: 1; the polyA signal sequence is BGH polyA.

13. An isolated nucleic acid, characterized in that, The nucleic acid encodes the adenine deaminase as described in claim 1, the base editor as described in any one of claims 2-9, or the fusion protein as described in any one of claims 10-12.

14. A base editing system for editing cytosine and adenine, characterized in that, The base editing system includes: sgRNA and a base editor as described in any one of claims 2-9.

15. The base editing system as described in claim 14, characterized in that, The target sequence of the sgRNA is a nucleotide sequence as shown in SEQ ID NO: 9, 12 or 15.

16. A base editing method for non-therapeutic purposes, characterized in that, The base editing method includes: Base editing is performed on target cells by expressing the adenine deaminase as described in claim 1, the base editor as described in any one of claims 2-9, the fusion protein as described in any one of claims 10-12, or the base editing system as described in claim 14 or 15.

17. The base editing method as described in claim 16, characterized in that, The target cells are derived from isolated cell lines.

18. The base editing method as described in claim 17, characterized in that, The isolated cell lines are 293T cells, HELA cells, U2OS cells, NIH3T3 cells, or N2A cells.

19. The use of the adenine deaminase as described in claim 1, the base editor as described in any one of claims 2-9, the fusion protein as described in any one of claims 10-12, or the base editing system as described in claim 14 or 15 in the preparation of base-edited drugs, the construction of animal models, gene expression regulation, crop breeding, the preparation of drugs for gene therapy, or the preparation of base editing tools; wherein the application is for non-therapeutic purposes.

20. The application as described in claim 19, characterized in that, The gene therapy includes: base editing of the regulatory elements of a target gene in target cells to change the expression level of the target gene, thereby treating the disease.

21. The application as described in claim 20, characterized in that, The target cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, erythroid progenitor cells, and erythroid precursor cells; and / or, the target gene is selected from genes expressing human globin and genes regulating human globin expression; and / or, the regulatory sequence is selected from enhancers and promoters; and / or, the disease is β-hemoglobinopathies.

22. The application as described in claim 21, characterized in that, The target cells are hematopoietic stem cells; and / or, the target gene is selected from BCLA11, HBG1, and HBG2; and / or, the β-hemoglobinopathies are selected from β-thalassemia and sickle cell disease.

23. The application as described in any one of claims 20-22, characterized in that, The regulatory elements of the target gene are selected from the enhancer of BCLA11, the promoter of HBG1, and the promoter of HBG2.

24. A pharmaceutical composition for treating and / or preventing β-hemoglobinopathies, characterized in that, The pharmaceutical composition comprises the adenine deaminase as described in claim 1, the base editor as described in any one of claims 2-9, the fusion protein as described in any one of claims 10-12, or the base editing system as described in claim 14 or 15, and a pharmaceutically acceptable vector; the pharmaceutical composition performs base editing on genes expressing human globin and / or regulatory elements of genes regulating human globin expression in target cells.

25. The pharmaceutical composition according to claim 24, characterized in that, The β-hemoglobinopathies are selected from β-thalassemia and sickle cell disease; and / or, the target cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, erythroid progenitor cells, and erythroid precursor cells; and / or, the gene expressing human globin is the HBG1 and / or HBG2 gene; and / or, the gene regulating human globin expression is the BCLA11 gene; and / or, the regulatory elements are selected from enhancers and promoters.

26. The pharmaceutical composition according to claim 24 or 25, characterized in that, The regulatory element of the gene expressing human globin is the promoter of the HBG1 gene and / or the HBG2 gene; the regulatory element of the gene regulating human globin expression is the enhancer of the BCLA11 gene.

27. A gene expression cassette, characterized in that, The gene expression cassette includes a promoter element and a target gene element; The target gene element is selected from genes encoding adenine deaminase as described in claim 1, base editor as described in any one of claims 2-9, or fusion protein as described in any one of claims 10-12.

28. The gene expression cassette as claimed in claim 27, characterized in that, The promoter element is selected from CMV, CAG, PGK, EF1α, Ctsk and Lp1.

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