Multifunctional RNA base editor with flexible editing window

Abstract

The invention provides an RNA base editor (such as an adenine base editor, a cytosine base editor and an adenine / cytosine double-base editor) with a controllable editing window and application of the RNA base editor. In addition, the invention also provides a series of novel TadA mutants for RNA base editing, and a base editing tool based on the TadA mutants.

Description

Technical Field

[0001] This application relates to the field of base editing, specifically to an RNA base editor (e.g., an adenine base editor, a cytosine base editor, an adenine / cytosine dual base editor) with a controllable editing window and its applications. Furthermore, this application also provides a series of novel TadA mutants for RNA base editing, and base editing tools based on said TadA mutants. Background Technology

[0002] I. RNA base editing.

[0003] Base editing of DNA or RNA is a powerful strategy for both basic research and clinical applications. Programmable RNA base editing can reversibly recode genetic information without altering the genomic DNA sequence, offering a safer and more flexible option compared to DNA base editing. Furthermore, RNA editing demonstrates unique advantages in applications requiring short-term and dynamic editing effects, such as signal transduction pathway modulation, pain relief, and viral infection treatment.

[0004] Given this potential, several different types of RNA base editors have been developed, each capable of enabling A-to-I pairing in mammalian cells. 1-5 C-to-U 5,6 and U-to-Ψ 7-9 RNA base editing is a process of base conversion. To date, editors based on exogenous or endogenous expression of RNA adenosine deaminase (ADAR) dominate the field of RNA base editing. Building upon this, editing tools with higher editing activity and better specificity have been developed, and their effectiveness has been validated in primary cell lines and animal models, offering great promise for clinical applications. However, we still need novel RNA base editors to expand the scope of RNA base editing applications.

[0005] II. The concept of a base editing window.

[0006] A specific base editor can function within a given region of RNA or DNA and efficiently introduce point mutations; this region is referred to as the "editing window" or "active window" of the RNA or DNA base editor. 10-12 In DNA base editing, the Cas9 complex unwinds double-stranded DNA (dsDNA) and forms a single-stranded DNA (ssDNA) region, which can be used by APOBECs. 13 And the evolved tRNA adenosine deaminase (TadA) 14The ssDNA deaminase is then used for editing. In this case, the editing window is typically 4-5 nucleotides wide, but it can also be designed to be only 1-2 nucleotides wide. Different applications may require editing different numbers of nucleotides: while narrow editing windows are more suitable for correcting pathogenic SNPs, wide editing windows are needed when multiple nucleotides need to be edited to salvage gene function. For example, in the treatment of sickle cell disease and β-thalassemia, disrupting the binding site of the transcriptional repressor LFR in the HBG promoter (CCCCTTCCCC) is an effective strategy to reactivate fetal hemoglobin (HbF) expression. 15 It has been demonstrated that editing all eight cytosines using a DNA cytosine base editor (CBE) results in a significant reduction in LRF binding and higher HbF expression levels compared to editing only the tetrad cytosine at the 3' position. 16 This is consistent with structural evidence that all eight cytosines are important for LRF binding. 17 In the second example, we need to disrupt two consecutive oxidative activation sites (Met281 and Met282) on the CaMKIIδ protein to prevent the oxidation and overactivation of CaMKIIδ and provide additional cardioprotection. 18-20 To achieve this goal, we need to use the DNA adenine base editor (ABE) to simultaneously edit the A841 and A844 sites of the CaMKIIδ gene. 21 Therefore, the editing window is a very important parameter for DNA base editors.

[0007] In contrast, there is little discussion about the concept of an editing window in the field of RNA base editing. This is because ADAR-based editing tools dominate this field, recognizing double-stranded RNA rather than single-stranded RNA substrates. ADAR-based RNA editors preferentially act on single mismatched nucleotides at a given target site, thus ADAR is suitable for editing single bases; however, consecutive base mismatches significantly reduce the editing efficiency of ADAR. Therefore, an RNA base editor with a flexible, variable editing window is currently lacking.

[0008] III. Single-stranded deaminase TadA and TadA-based DNA base editors.

[0009] TadA (tRNA adenosine deaminase A) is a deaminase that catalyzes the deamination of adenine (A) at a specific position on tRNA into hypoxanthine (I). 22 .exist Escherichia coli In this process, TadA can specifically catalyze tRNA. Arg2At position 34, adenine is deaminated to hypoxanthine, which corresponds to the third position of the codon. Since hypoxanthine can pair with A, C, and U bases, the anticodon of the same tRNA molecule after deamination can recognize three codons. Structural biology studies have shown that the activation of TadA deamination activity requires TadA to recognize the stem-loop structure on the anticodon loop of the tRNA molecule and specific nucleotides. Only tRNAs conforming to specific structures can be deaminated by TadA, ensuring the precision of TadA-catalyzed deamination.

[0010] David R. Liu's research group first used TadA to develop adenine base editors (ABEs) that can catalyze the deamination of adenine at specific sites on DNA into hypoxanthine. 14 Since naturally occurring deaminases capable of deaminating single-stranded DNA do not exist in nature, researchers used wild-type DNA... Escherichia coli The derived TadA (EcTadA) is fused with the Cas9 protein to form a fusion protein. Through bacterial selection, EcTadA evolved the ability to deaminate single-stranded DNA after introducing several amino acid mutations. The optimized version ABE7.10 consists of the Cas9 protein, the evolved TadA*, and wild-type wt-EcTadA, enabling highly efficient and precise editing of adenine bases in DNA.

[0011] Building upon ABE7.10, David R. Liu's research group developed the ABE8e tool, which significantly improves editing efficiency compared to ABE7.10, through phage-assisted non-continuous and continuous evolution (PANCE and PACE) technology. 23 The study also noted that with increased editing efficiency, ABE8e caused relatively more off-target editing of adenine on DNA and RNA transcripts. Subsequent structural biology studies revealed the structure by which ABE8e captures single-stranded DNA substrates and confirmed that both ABE8e and miniABE-max possess catalytic activity in deamination of adenine on single-stranded RNA. 24 Subsequent work has also focused on reducing the off-target editing of adenine in DNA and RNA transcripts by introducing amino acid mutations at TadAmax and TadA8e. 25-28Researchers at Beam Therapeutics further optimized the bacterial selection strategy employed during the evolution of ABE7.10, and through continued directed molecular evolution of ABE7.10, they obtained a series of ABE8s tools, among which ABE8.20 performed the best. 29 Compared to ABE7.10, ABE8.20 has a wider editing window and higher editing efficiency (~1.5-4.2 times improvement), but it also causes more off-target editing on the genome and transcriptome.

[0012] IV. A-to-I base editing tools for TadA-based RNA.

[0013] In July 2024, Weixin Tang's research group published a study online demonstrating the use of TadA deaminase to achieve programmable A-to-I base editing on RNA (DECOR). 30 This is the first RNA A-to-I base editor that does not rely on ADAR deaminase. DECOR is developed by using a highly active TadA8e (this TadA variant has been reported in base editors on DNA). 23 DECOR is obtained by linking dRfxCas13d to form a fusion protein. DECOR can achieve an A-to-G conversion efficiency of about 20% to 50% on endogenous transcripts, but at the same time it will also cause a large number of bystander edits on the target transcripts and 840 to 880 off-target edits across the whole transcriptome. Summary of the Invention

[0014] Based on in-depth research, the inventors of this application have developed a novel RNA base editing toolkit, named AIM, which enables controllable number and location of RNA base edits within a user-defined editing window. Specifically, AIM induces a circular structure on the target RNA using a specially designed guide RNA (this guide RNA is named a circularizable guide RNA or "LF-gRNA"), thereby allowing precise control of the AIM editing window by adjusting the size of the loop. Figure 1 (A and 1B).

[0015] Based on this, in order to further expand the application scope of AIM tools, the inventors of this application have rationally designed the single-chain deaminase TadA and developed a series of novel mutant-based AIM tools that can achieve precise and efficient A-to-I, C-to-U or simultaneous A&C editing.

[0016] I. Base editing system based on gRNA that can be guided to circularize.

[0017] Therefore, in a first aspect, this application provides a composition comprising: (a) A first component, selected from: (i) a guide RNA molecule comprising a backbone sequence and a guide sequence, the guide sequence being capable of hybridizing with a target RNA sequence comprising bases to be edited; (ii) a nucleotide sequence encoding the guide RNA molecule; (iii) any combination of (i) and (ii); as well as, (b) A second component selected from: (i) a polypeptide construct comprising a base deaminase and a guide RNA molecule binding domain linked to the base deaminase, the guide RNA molecule binding domain being capable of binding to the backbone sequence of the guide RNA molecule; (ii) a nucleotide sequence encoding the polypeptide construct; (iii) any combination of (i) and (ii); The guide sequence of the guide RNA molecule is designed such that, after hybridization with the target RNA sequence, it causes the target RNA sequence to form a single-stranded RNA loop region containing the bases to be edited.

[0018] Based on the disclosure of this application, those skilled in the art will readily understand that in an embodiment where the first component is a nucleotide sequence encoding the guide RNA molecule and the second component is a nucleotide sequence encoding the polypeptide construct, the nucleotide sequences of the first component and the second component may exist in different nucleic acid molecules or vectors, or they may exist in the same nucleic acid molecule or vector.

[0019] In some embodiments, the target RNA sequence comprises: a target circularizable region containing the bases to be edited, a first region located at the 5' end of the target circularizable region, and a second region located at the 3' end of the target circularizable region; The guide sequence of the guide RNA molecule is designed to include a first region, a second region, and an optional third region located between the first and second regions, the second region being upstream of the first region; and the first region is complementary to a first region of the target RNA sequence, the second region is complementary to a second region of the target RNA sequence, and the third region is absent or cannot be complementary to a target circularizable region of the target RNA sequence. Therefore, after the guide RNA molecule hybridizes with the target RNA sequence, the target loopable region located between region I and region II of the target RNA sequence exists as a single strand, thereby forming a single-stranded RNA loop region containing the bases to be edited.

[0020] Furthermore, since the polypeptide construct of this application includes a guide RNA binding domain that is linked to a base deaminase and can recognize and bind to a guide RNA molecule, the guide RNA molecule can guide the polypeptide construct containing the base deaminase to the target RNA, thereby causing deamination of the base to be edited in the single-stranded RNA loop region of the target RNA.

[0021] In some implementations, the third region of the guide RNA molecule is absent.

[0022] In some embodiments, the third region of the guide RNA molecule has fewer nucleotide residues than the target circularizable region of the target RNA sequence.

[0023] In some embodiments, the first and second regions of the guide RNA molecule have the same or different numbers of nucleotide residues.

[0024] Based on the disclosure of this application, those skilled in the art will readily understand that the nucleotide residue composition of the single-stranded RNA loop region formed by the hybridized target RNA sequence can be controlled through the design of the guide RNA molecule (e.g., the number of nucleotide residues in the single-stranded RNA loop region and the position of the base to be edited in the single-stranded RNA loop region). Specifically, according to the disclosure of this application, the target loopable region located between region I and region II in the target RNA sequence corresponds to the single-stranded loop region formed after the target RNA sequence hybridizes with the guide RNA. Thus, according to specific editing requirements, region I and region II can be selected on both sides of the target loopable region of the target RNA sequence (the target loopable region with the target nucleotide composition; for example, in the target loopable region, the base to be edited is located at the target position and / or the target loopable region has a selected number of nucleotide residues). Based on this design, a guide RNA molecule containing a first region and a second region that are complementary to region I and region II respectively can be designed, so that the target RNA sequence hybridizes with the guide RNA molecule to form a single-stranded RNA loop region with the target nucleotide composition. Therefore, the composition provided in this application can achieve adjustable base editing with a customizable editing window (that is, the number of residues in the editing window is adjustable, and the position of the base to be edited in the editing window is adjustable).

[0025] In some embodiments, the guide RNA molecule binding domain is selected from: Cas effector proteins of the CRISPR system lacking nuclease activity, MS2 bacteriophage coat protein (MCP), and bacteriophage Lambda N protein (λ N-peptide).

[0026] Based on the disclosure of this application, those skilled in the art will readily understand that the design of the backbone sequence of the guide RNA molecule in the composition of this application corresponds to the selection of the guide RNA molecule binding domain of the polypeptide construct. That is, the backbone sequence of the guide RNA molecule is designed to be recognized and bound by the guide RNA molecule binding domain contained in the polypeptide construct. For example, when the guide RNA molecule binding domain is a Cas effector protein of the CRISPR system lacking nuclease activity, the backbone sequence of the guide RNA molecule is selected from the backbone sequence of crRNA (or crRNA / tracrRNA complex or construct) that the Cas effector protein can recognize and bind.

[0027] In some embodiments, the binding domain of the guide RNA molecule is selected from CRISPR-Cas13 effector proteins lacking nuclease activity. In some embodiments, the backbone sequence of the guide RNA molecule is selected from the backbone sequence of crRNAs that the CRISPR-Cas13 effector protein can recognize and bind to.

[0028] In some embodiments, the binding domain of the guide RNA molecule is selected from the MS2 phage coat protein (MCP). In some embodiments, the backbone sequence of the guide RNA molecule is selected from an MS2 aptamer that the MS2 phage coat protein (MCP) can recognize and bind to.

[0029] As is commonly understood by those skilled in the art, the MS2 aptamer sequence is an RNA sequence derived from bacteriophage MS2, which can form a stem-loop structure through intramolecular base interactions that is specifically recognized and bound by the MS2 capsid protein (MCP). The sequences of various natural or artificially modified mutant MS2 aptamers and their corresponding MCP proteins are well known in the art. The present invention can be implemented using any MS2 aptamer and its corresponding MCP protein.

[0030] In some embodiments, the binding domain of the guide RNA molecule is selected from the phage Lambda N protein. In some embodiments, the backbone sequence of the guide RNA molecule is selected from the boxB sequence that the phage Lambda N protein can recognize and bind to.

[0031] As is generally understood by those skilled in the art, a boxB sequence (or boxB element) is a type of RNA motif located within the nut site (N utilization site) of the phage genome that can form a stem-loop structure. The stem-loop structure formed by the phage can be recognized and bound by the phage Lambda N protein.

[0032] In some embodiments, the guide RNA molecule binding domain is a PspCas13b lacking nuclease activity. In some embodiments, the guide RNA molecule binding domain comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the backbone sequence of the guide RNA molecule comprises the amino acid sequence shown in SEQ ID NO: 13.

[0033] This invention does not limit the number of bases to be edited in the target RNA sequence. Without theoretical limitations, for a given target RNA sequence and the composition provided in this application designed for it, theoretically all bases within the editing window of the target RNA sequence can be converted into another base through the catalysis of the base deaminase.

[0034] In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule contains one or more bases to be edited. In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases to be edited.

[0035] In some embodiments, the base to be edited is selected from adenine, cytosine, and any combination thereof.

[0036] In some embodiments, the first region of the guide RNA molecule consists of 3-50 (e.g., 5-50, 5-40, 5-30, 5-25, 5-20, 5-18, 5-15, 10-50, 10-40, 10-30, 10-25, 10-20, 10-18, 12-50, 12-40, 12-30, 12-25, 12-20, 12-18) nucleotide residues.

[0037] In some embodiments, the first region of the guide RNA molecule consists of 3-50 (e.g., 5-50, 5-40, 5-30, 5-25, 5-20, 5-18, 5-15, 10-50, 10-40, 10-30, 10-25, 10-20, 10-18, 12-50, 12-40, 12-30, 12-25, 12-20, 12-18) nucleotide residues.

[0038] In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of at least 3, at least 4, at least 5, or at least 6 nucleotide residues. In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of 3-30 (e.g., 6-30, 6-25, 6-20, 6-19, 6-18, or 6-17) nucleotide residues. In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotide residues.

[0039] In some embodiments, the base to be edited in the target RNA sequence is separated from the first region by at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotide residues, and / or is separated from the second region by at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotide residues.

[0040] In some embodiments, the base deaminase is adenosine deaminase or cytidine deaminase, or the base deaminase comprises both adenosine deaminase and cytidine deaminase, or the base deaminase has both adenosine deaminase and cytidine deaminase activities.

[0041] In some embodiments, the base to be edited is adenine, and the base deaminase is adenosine deaminase.

[0042] In some embodiments, the base to be edited is cytosine, and the base deaminase is cytidine deaminase.

[0043] In some embodiments, the base to be edited is adenine, cytosine, or a combination thereof, and the base deaminase comprises adenosine deaminase and cytidine deaminase, or the base deaminase has both adenosine deaminase and cytidine deaminase activities.

[0044] In some embodiments, in the polypeptide construct, the guide RNA molecule binding domain is linked to the N-terminus and / or C-terminus of the base deaminase.

[0045] In some embodiments, the peptide construct further comprises a nuclear localization signal (NLS) and / or a nuclear exocytosis signal (NES). In some embodiments, the peptide construct further comprises one or more NLS and / or one or more NES. In some embodiments, the NLS comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 22-23. In some embodiments, the NES comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0046] In some embodiments, the polypeptide construct is a fusion protein, for example, a fusion protein comprising the base deaminase and the guide RNA molecule binding domain.

[0047] In some embodiments, the polypeptide construct is a fusion protein, and adjacent domains of the fusion protein are independently linked by or without a linker (e.g., a peptide linker, such as a peptide linker containing one or more glycines and / or one or more serines). In some embodiments, the linker comprises the amino acid sequence GS or the amino acid sequence shown in SEQ ID NO: 21.

[0048] II. Base editing system based on guided circular gRNA - adenine base editor.

[0049] In some embodiments, the base to be edited is adenine, and the base deaminase contained in the polypeptide construct has adenosine deaminase activity. In some embodiments, the base deaminase does not have cytidine deaminase activity, or has significantly low cytidine deaminase activity (e.g., having up to 30%, up to 20%, up to 10%, up to 5%, up to 2%, or up to 1% of the cytidine deaminase activity of the base deaminase shown in SEQ ID NO: 6).

[0050] In some embodiments, the base deaminase is selected from: TadA, a TadA mutant comprising the sequence shown in SEQ ID NO: 1 or 2, and a functional fragment of TadA or the TadA mutant; The TadA mutant, compared to TadA containing the sequence shown in SEQ ID NO: 1, contains mutations selected from the following: (1) V28R, (2) V30A, (3) I76Y, (4) V82T, (5) F84H, F84V, F84N, F84S, F84I, F84T, F84C or F84A, (6) R98E, (7) N108H, (8) R111C or R111L, (9) Y147R, (10) Q154R, (11) Any combination of (1) to (10).

[0051] As used herein, the mutation “V28R” means that the residue in the TadA mutant that corresponds to the residue “V” at position 28 of SEQ ID NO: 1 is replaced with residue “R”; wherein, the residue that corresponds to the residue “V” at position 28 of SEQ ID NO: 1 refers to the amino acid site / residue in the sequence being compared with the amino acid residue “V” at position 28 of SEQ ID NO: 1 when the amino acid sequence of the TadA mutant is optimally aligned with SEQ ID NO: 1, that is, when the amino acid sequence of the TadA mutant is aligned with SEQ ID NO: 1 to obtain the highest percentage identity, the amino acid site / residue in the sequence being compared is located at the same position as the amino acid residue “V” at position 28 of SEQ ID NO: 1.

[0052] Unless otherwise specified or clearly contradicted by the context, the meaning of the rest of the similar expressions in this document shall be defined in a manner similar to that described above.

[0053] In some embodiments, the base deaminase is selected from the TadA mutant and its functional fragments.

[0054] In some embodiments, the functional fragment possesses the adenosine deaminase activity of the base deaminase from which it is derived.

[0055] In some embodiments, the TadA mutant comprises a mutation selected from the group consisting of TadA containing the sequence shown in SEQ ID NO: 1, compared to TadA containing the sequence shown in SEQ ID NO: 1: (i) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and / or, (e) Q154R; (ii) (a) F84H, F84V or F84I, and (b) V82T; (iii) (a) F84H, F84V or F84I, (b) V82T, and (c) Y147R; (iv) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and (e) Q154R; (v) F84I, V82T, Y147R, I76Y, and / or, Q154R; (vi) F84I and V82T; (vii) F84I, V82T and Y147R; (viii) F84I, V82T, Y147R, I76Y and Q154R.

[0056] In some embodiments, the TadA mutant has mutations F84I and V82T compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0057] In some embodiments, the TadA mutant has mutations F84I, V82T, and Y147R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0058] In some embodiments, the TadA mutant has mutations F84I, V82T, Y147R, I76Y and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0059] In some embodiments, the guide RNA molecule binding domain comprises an amino acid sequence as shown in SEQ ID NO: 10; and / or, the backbone sequence of the guide RNA molecule comprises a nucleotide sequence as shown in SEQ ID NO: 13.

[0060] In some embodiments, the base deaminase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-2, 4-5.

[0061] In some embodiments, the polypeptide construct is a fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NO: 14-16.

[0062] I-II. Base editing system based on guided circular gRNA - cytosine base editor.

[0063] In some embodiments, the base to be edited is cytosine, and the base deaminase contained in the polypeptide construct has cytidine deaminase activity. In some embodiments, the base deaminase does not have adenosine deaminase activity, or has significantly low adenosine deaminase activity (e.g., having up to 30%, up to 20%, up to 10%, up to 5%, up to 2%, or up to 1% of the adenosine deaminase activity of the base deaminase shown in SEQ ID NO: 4).

[0064] In some embodiments, the base deaminase is selected from TadA mutants and their functional fragments; wherein, compared to TadA containing the sequence shown in SEQ ID NO: 1, the TadA mutant contains mutations: (i) N46P or N46V, and (ii) A48G, and further contains mutations selected from: V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P H96N and any combination thereof.

[0065] In some embodiments, the functional fragment possesses the cytidine deaminase activity of the base deaminase from which it is derived.

[0066] In some embodiments, the TadA mutant comprises a mutation selected from the group consisting of TadA containing the sequence shown in SEQ ID NO: 1, compared to TadA containing the sequence shown in SEQ ID NO: 1: (i) (a) N46P and A48G, and (b) V82T, I76Y, Y147R and / or Q154R; (ii) (a) N46P and A48G, (b) V82T, I76Y, Y147R and / or Q154R, and (c) R26G, V28A, Y73P and / or H96N; (iii) N46P, A48G and V82T; (iv) N46P, A48G and I76Y; (v) N46P, A48G and Y147R; (vi) N46P, A48G and Q154R; (vii) N46P, A48G, V82T, I76Y, Y147R and Q154R; (viii) N46P, A48G, V82T, I76Y, Y147R, Q154R and R26G; (ix) N46P, A48G, V82T, I76Y, Y147R, Q154R and V28A; (x) N46P, A48G, V82T, I76Y, Y147R, Q154R and Y73P; (xi) N46P, A48G, V82T, I76Y, Y147R, Q154R and H96N; (xii) N46P, A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N; (xiii) (a) N46V and A48G, and (b) V82T, I76Y, Y147R and / or Q154R; (xiv) (a) N46V and A48G, (b) V82T, I76Y, Y147R and / or Q154R, and (c) R26G, V28A, Y73P and / or H96N; (xv) N46V, A48G and V82T; (xvi) N46V, A48G and I76Y; (xvii) N46V, A48G and Y147R; (xviii) N46V, A48G and Q154R; (xix) N46V, A48G, V82T, I76Y, Y147R and Q154R; (xx) N46V, A48G, V82T, I76Y, Y147R, Q154R and R26G; (xxi) N46V, A48G, V82T, I76Y, Y147R, Q154R and V28A; (xxii) N46V, A48G, V82T, I76Y, Y147R, Q154R and Y73P; (xxiii) N46V, A48G, V82T, I76Y, Y147R, Q154R and H96N; (xxiv) N46V, A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N.

[0067] In some embodiments, the TadA mutant has mutations N46P, A48G, and V82T compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0068] In some embodiments, the TadA mutant has mutations N46P, A48G, and I76Y compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0069] In some embodiments, the TadA mutant has mutations N46P, A48G, and Y147R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0070] In some embodiments, the TadA mutant has mutations N46P, A48G, and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0071] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0072] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and R26G compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0073] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and V28A compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0074] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and Y73P compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0075] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and H96N compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0076] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P, and H96N compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0077] In some embodiments, the guide RNA molecule binding domain comprises an amino acid sequence as shown in SEQ ID NO: 10; and / or, the backbone sequence of the guide RNA molecule comprises a nucleotide sequence as shown in SEQ ID NO: 13.

[0078] In some embodiments, the base deaminase comprises an amino acid sequence as shown in SEQ ID NO: 6 or 7.

[0079] In some embodiments, the polypeptide construct is a fusion protein containing an amino acid sequence as shown in SEQ ID NO: 17 or 18.

[0080] I-III. Base editing system based on guided circular gRNA - adenine / cytosine dual base editor.

[0081] In some embodiments, the base to be edited is cytosine, adenine, or a combination thereof, and the polypeptide construct contains adenosine deaminase and cytidine deaminase, or the base deaminase contained in the polypeptide construct has both adenosine deaminase and cytidine deaminase activities.

[0082] In some embodiments, the base deaminase is selected from TadA mutants and their functional fragments; wherein, compared to TadA containing the sequence shown in SEQ ID NO: 1, the TadA mutant contains mutations A48G, A48M, or A48H, and further contains mutations selected from: (1) N46C; (2) V82T, I76Y, Y147R and / or Q154R; (3) R26G, V28A, Y73P and / or H96N; (4) (a) N46C, and (b) V82T, I76Y, Y147R and / or Q154R; (5) (a) N46C, and (b) R26G, V28A, Y73P and / or H96N; (6) (a) V82T, I76Y, Y147R and / or Q154R, and (b) R26G, V28A, Y73P and / or H96N, and (c) the TadA mutant does not contain a mutation at the N46 position.

[0083] In some embodiments, the functional fragment possesses the adenosine deaminase activity and cytidine deaminase activity of the base deaminase from which it is derived.

[0084] In some embodiments, the TadA mutant comprises a mutation selected from the group consisting of TadA containing the sequence shown in SEQ ID NO: 1, compared to TadA containing the sequence shown in SEQ ID NO: 1: (i) A48G and N46C; (ii) A48G, N46C, V82T, I76Y, Y147R and Q154R; (iii) A48G, N46C, R26G, V28A, Y73P and H96N; (iv) A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N, and the TadA mutant does not contain a mutation at position N46; (v) A48M and N46C; (vi) A48M, N46C, V82T, I76Y, Y147R and Q154R; (vii) A48M, N46C, R26G, V28A, Y73P and H96N; (viii) A48M, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N, and the TadA mutant does not contain a mutation at position N46; (ix) A48H and N46C; (x) A48H, N46C, V82T, I76Y, Y147R and Q154R; (xi) A48H, N46C, R26G, V28A, Y73P and H96N; (xii) A48H, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N, and the TadA mutant does not contain a mutation at position N46.

[0085] In some embodiments, the TadA mutant has mutations A48G and N46C compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0086] In some embodiments, the TadA mutant has mutations A48G, N46C, V82T, I76Y, Y147R, and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0087] In some embodiments, compared to TadA containing the sequence shown in SEQ ID NO: 1, the TadA mutant has the mutations A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P, and H96N, and the TadA mutant does not contain a mutation at position N46.

[0088] In some embodiments, the guide RNA molecule binding domain comprises an amino acid sequence as shown in SEQ ID NO: 10; and / or, the backbone sequence of the guide RNA molecule comprises a nucleotide sequence as shown in SEQ ID NO: 13.

[0089] In some embodiments, the base deaminase comprises an amino acid sequence as shown in SEQ ID NO: 8 or 9.

[0090] In some embodiments, the polypeptide construct is a fusion protein containing an amino acid sequence as shown in SEQ ID NO: 19 or 20.

[0091] In a second aspect, this application provides a composite comprising: (a) A guide RNA molecule, said guide RNA molecule as defined in the first aspect; as well as, (b) A polypeptide construct, said polypeptide construct as defined in the first aspect.

[0092] In a third aspect, this application provides a carrier system comprising one or more carriers, wherein the one or more carriers comprise: (1) A first nucleotide sequence encoding a guide RNA molecule as defined in the first aspect; optionally, the first nucleotide sequence is operatively linked to a first regulatory element; and, (2) A second nucleotide sequence encoding a polypeptide construct as defined in the first aspect; optionally, the second nucleotide sequence is operatively linked to a second regulatory element; The first nucleotide sequence and the second nucleotide sequence may be present on the same or different vectors.

[0093] In some implementations, the first regulating element is a promoter, such as an inducible promoter.

[0094] In some implementations, the second regulating element is an promoter, such as an inductive promoter.

[0095] In a fourth aspect, this application provides an engineered RNA molecule comprising the sequence of a guide RNA molecule as defined in the first aspect.

[0096] On the other hand, this application also provides nucleic acid molecules, vectors, or host cells encoding said engineered RNA molecules. In some embodiments, the cells are not plant cells. In some embodiments, the cells are microbial cells.

[0097] In a fifth aspect, this application provides a kit comprising the composition as described in the first aspect, the complex as described in the second aspect, the vector system as described in the third aspect, the engineered RNA molecule as described in the fourth aspect, or a nucleic acid molecule or vector or host cell encoding the engineered RNA molecule.

[0098] In some embodiments, the kit further includes instructions for using the composition, complex, vector system, engineered RNA molecule, nucleic acid molecule encoding the engineered RNA molecule, or vector or host cell.

[0099] In a sixth aspect, this application provides a delivery composition comprising a delivery vector and one or more selected from the following: the composition as described in the first aspect, the complex as described in the second aspect, the vector system as described in the third aspect, the engineered RNA molecule as described in the fourth aspect, or a nucleic acid molecule or vector encoding the engineered RNA molecule.

[0100] In some implementations, the delivery carrier is a particle.

[0101] In some embodiments, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0102] In another aspect, this application provides a cell comprising: a composition as described in the first aspect, a complex as described in the second aspect, a carrier system as described in the third aspect, an engineered RNA molecule as described in the fourth aspect, a nucleic acid molecule or carrier encoding the engineered RNA molecule, or a delivery composition as described in the sixth aspect. In some embodiments, the cell is not a plant cell. In some embodiments, the cell is a microbial cell.

[0103] In a seventh aspect, this application provides a method for extracellular editing of target RNA, comprising contacting the target RNA with one or more of the composition of the first aspect, the complex of the second aspect, the engineered RNA molecule of the fourth aspect, and the delivery composition of the sixth aspect under conditions suitable for target RNA editing, thereby inducing deamination of the bases to be edited in the target RNA.

[0104] In an eighth aspect, this application provides a method for editing target RNA in cells, comprising delivering the composition of the first aspect, the complex of the second aspect, the vector system of the third aspect, the engineered RNA molecule of the fourth aspect, the nucleic acid molecule or vector encoding the engineered RNA molecule, or the delivery composition of the sixth aspect into a cell containing the target RNA, thereby inducing deamination of the base to be edited at the target site.

[0105] In a ninth aspect, this application provides a cell or its progeny obtained by the method of the eighth aspect, wherein the cell contains modifications not present in its wild type. In some embodiments, the cell is not a plant cell. In some embodiments, the cell is a microbial cell.

[0106] In a tenth aspect, this application provides a composition of the first aspect, a complex of the second aspect, a vector system of the third aspect, an engineered RNA molecule of the fourth aspect, a nucleic acid molecule or vector encoding said engineered RNA molecule, a kit of the fifth aspect, or a delivery composition of the sixth aspect, for use in RNA editing, or for use in the preparation of formulations for RNA editing.

[0107] In some implementations, the RNA editing is base editing performed on RNA.

[0108] II. Base editing system based on novel TadA mutants.

[0109] II-I. A novel base editing system based on TadA mutants—TadA mutants for adenine base editing body.

[0110] In an eleventh aspect, this application provides a TadA mutant or a functional fragment thereof, wherein, compared to TadA comprising the sequence shown in SEQ ID NO: 1, the TadA mutant comprises a mutation selected from the following: (1) V28R, (2) V30A, (3) I76Y, (4) V82T, (5) F84H, F84V, F84N, F84S, F84I, F84T, F84C or F84A, (6) R98E, (7) N108H, (8) R111C or R111L, (9) Y147R, (10) Q154R, (11) Any combination of (1) to (10).

[0111] In some embodiments, the functional fragment possesses the adenosine deaminase activity from which it is derived from the TadA mutant.

[0112] In some embodiments, the TadA mutant comprises a mutation selected from the group consisting of TadA containing the sequence shown in SEQ ID NO: 1, compared to TadA containing the sequence shown in SEQ ID NO: 1: (i) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and / or, (e) Q154R; (ii) (a) F84H, F84V or F84I, and (b) V82T; (iii) (a) F84H, F84V or F84I, (b) V82T, and (c) Y147R; (iv) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and (e) Q154R; (v) F84I, V82T, Y147R, I76Y, and / or, Q154R; (vi) F84I and V82T; (vii) F84I, V82T and Y147R; (viii) F84I, V82T, Y147R, I76Y and Q154R.

[0113] In some embodiments, the TadA mutant has mutations F84I and V82T compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0114] In some embodiments, the TadA mutant has mutations F84I, V82T, and Y147R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0115] In some embodiments, the TadA mutant has mutations F84I, V82T, Y147R, I76Y and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0116] In some embodiments, the TadA mutant comprises an amino acid sequence as shown in SEQ ID NO: 4 or 5.

[0117] In some embodiments, the TadA mutant has adenosine deaminase activity. In some embodiments, the TadA mutant does not have cytidine deaminase activity, or has significantly low cytidine deaminase activity (e.g., having up to 30%, up to 20%, up to 10%, up to 5%, up to 2%, or up to 1% of the cytidine deaminase activity of the deaminase shown in SEQ ID NO: 6).

[0118] In some implementations, the TadA mutant is used for adenine base editing.

[0119] II-II. A novel base editing system based on TadA mutants – TadA mutations for cytosine base editing body.

[0120] In a twelfth aspect, this application provides a TadA mutant or a functional fragment thereof, wherein, compared to TadA comprising the sequence shown in SEQ ID NO: 1, the TadA mutant comprises mutations: (i) N46P or N46V, and (ii) A48G, and further comprises mutations selected from: V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P, H96N and any combination thereof.

[0121] In some embodiments, the functional fragment possesses the cytidine deaminase activity from which it is derived from the TadA mutant.

[0122] In some embodiments, the TadA mutant comprises a mutation selected from the group consisting of TadA containing the sequence shown in SEQ ID NO: 1, compared to TadA containing the sequence shown in SEQ ID NO: 1: (i) (a) N46P and A48G, and (b) V82T, I76Y, Y147R and / or Q154R; (ii) (a) N46P and A48G, (b) V82T, I76Y, Y147R and / or Q154R, and (c) R26G, V28A, Y73P and / or H96N; (iii) N46P, A48G and V82T; (iv) N46P, A48G and I76Y; (v) N46P, A48G and Y147R; (vi) N46P, A48G and Q154R; (vii) N46P, A48G, V82T, I76Y, Y147R and Q154R; (viii) N46P, A48G, V82T, I76Y, Y147R, Q154R and R26G; (ix) N46P, A48G, V82T, I76Y, Y147R, Q154R and V28A; (x) N46P, A48G, V82T, I76Y, Y147R, Q154R and Y73P; (xi) N46P, A48G, V82T, I76Y, Y147R, Q154R and H96N; (xii) N46P, A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N; (xiii) (a) N46V and A48G, and (b) V82T, I76Y, Y147R and / or Q154R; (xiv) (a) N46V and A48G, (b) V82T, I76Y, Y147R and / or Q154R, and (c) R26G, V28A, Y73P and / or H96N; (xv) N46V, A48G and V82T; (xvi) N46V, A48G and I76Y; (xvii) N46V, A48G and Y147R; (xviii) N46V, A48G and Q154R; (xix) N46V, A48G, V82T, I76Y, Y147R and Q154R; (xx) N46V, A48G, V82T, I76Y, Y147R, Q154R and R26G; (xxi) N46V, A48G, V82T, I76Y, Y147R, Q154R and V28A; (xxii) N46V, A48G, V82T, I76Y, Y147R, Q154R and Y73P; (xxiii) N46V, A48G, V82T, I76Y, Y147R, Q154R and H96N; (xxiv) N46V, A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N.

[0123] In some embodiments, the TadA mutant has mutations N46P, A48G, and V82T compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0124] In some embodiments, the TadA mutant has mutations N46P, A48G, and I76Y compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0125] In some embodiments, the TadA mutant has mutations N46P, A48G, and Y147R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0126] In some embodiments, the TadA mutant has mutations N46P, A48G, and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0127] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0128] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and R26G compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0129] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and V28A compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0130] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and Y73P compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0131] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, and H96N compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0132] In some embodiments, the TadA mutant has mutations N46P, A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P, and H96N compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0133] In some embodiments, the TadA mutant comprises an amino acid sequence as shown in SEQ ID NO: 6 or 7.

[0134] In some embodiments, the TadA mutant has cytidine deaminase activity. In some embodiments, the TadA mutant does not have adenosine deaminase activity, or has significantly low adenosine deaminase activity (e.g., having up to 30%, up to 20%, up to 10%, up to 5%, up to 2%, or up to 1% of the adenosine deaminase activity of the deaminase shown in SEQ ID NO: 4).

[0135] In some implementations, the TadA mutant is used for cytosine base editing.

[0136] II-III. Novel TadA mutant-based base editing system for adenine / cytosine dual-base editing The TadA mutant.

[0137] In a thirteenth aspect, this application provides a TadA mutant or a functional fragment thereof, wherein, compared to TadA comprising the sequence shown in SEQ ID NO: 1, the TadA mutant comprises the mutations A48G, A48M, or A48H, and further comprises mutations selected from: (1) N46C; (2) V82T, I76Y, Y147R and / or Q154R; (3) R26G, V28A, Y73P and / or H96N; (4) (a) N46C, and (b) V82T, I76Y, Y147R and / or Q154R; (5) (a) N46C, and (b) R26G, V28A, Y73P and / or H96N; (6) (a) V82T, I76Y, Y147R and / or Q154R, and (b) R26G, V28A, Y73P and / or H96N, and (c) the TadA mutant does not contain a mutation at the N46 position.

[0138] In some embodiments, the functional fragment possesses adenosine deaminase activity and cytidine deaminase activity from which it is derived from the TadA mutant.

[0139] In some embodiments, the TadA mutant comprises a mutation selected from the group consisting of TadA containing the sequence shown in SEQ ID NO: 1, compared to TadA containing the sequence shown in SEQ ID NO: 1: (i) A48G and N46C; (ii) A48G, N46C, V82T, I76Y, Y147R and Q154R; (iii) A48G, N46C, R26G, V28A, Y73P and H96N; (iv) A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N, and the TadA mutant does not contain a mutation at position N46; (v) A48M and N46C; (vi) A48M, N46C, V82T, I76Y, Y147R and Q154R; (vii) A48M, N46C, R26G, V28A, Y73P and H96N; (viii) A48M, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N, and the TadA mutant does not contain a mutation at position N46; (ix) A48H and N46C; (x) A48H, N46C, V82T, I76Y, Y147R and Q154R; (xi) A48H, N46C, R26G, V28A, Y73P and H96N; (xi)A48H, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P and H96N, and the TadA mutant does not contain a mutation at position N46.

[0140] In some embodiments, the TadA mutant has mutations A48G and N46C compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0141] In some embodiments, the TadA mutant has mutations A48G, N46C, V82T, I76Y, Y147R, and Q154R compared to TadA containing the sequence shown in SEQ ID NO: 1.

[0142] In some embodiments, compared to TadA containing the sequence shown in SEQ ID NO: 1, the TadA mutant has the mutations A48G, V82T, I76Y, Y147R, Q154R, R26G, V28A, Y73P, and H96N, and the TadA mutant does not contain a mutation at position N46.

[0143] In some embodiments, the TadA mutant contains an amino acid sequence as shown in SEQ ID NO: 8 or 9.

[0144] In some implementations, the TadA mutant is used for adenine / cytosine dual-base editing.

[0145] In a fourteenth aspect, this application provides a polypeptide construct comprising a base deaminase and a guide RNA molecule binding domain linked to the base deaminase, the guide RNA molecule binding domain being capable of binding to the backbone sequence of the guide RNA molecule. The base deaminase is selected from any of the eleventh to thirteenth aspects of the TadA mutant or its functional fragment.

[0146] In some embodiments, the guide RNA molecule binding domain is selected from: Cas effector proteins of the CRISPR system lacking nuclease activity, MS2 bacteriophage coat protein (MCP), and bacteriophage Lambda N protein (λ N-peptide).

[0147] In some embodiments, the binding domain of the guide RNA molecule is selected from CRISPR-Cas13 effector proteins lacking nuclease activity. In some embodiments, the backbone sequence of the guide RNA molecule is selected from the backbone sequence of crRNAs that the CRISPR-Cas13 effector protein can recognize and bind to.

[0148] In some embodiments, the binding domain of the guide RNA molecule is selected from the MS2 phage coat protein (MCP). In some embodiments, the backbone sequence of the guide RNA molecule is selected from an MS2 aptamer that the MS2 phage coat protein (MCP) can recognize and bind to.

[0149] In some embodiments, the binding domain of the guide RNA molecule is selected from the phage Lambda N protein. In some embodiments, the backbone sequence of the guide RNA molecule is selected from the boxB sequence that the phage Lambda N protein can recognize and bind to.

[0150] In some embodiments, the guide RNA molecule binding domain is a PspCas13b lacking nuclease activity. In some embodiments, the guide RNA molecule binding domain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0151] In some embodiments, in the polypeptide construct, the guide RNA molecule binding domain is linked to the N-terminus and / or C-terminus of the base deaminase.

[0152] In some embodiments, the peptide construct further comprises a nuclear localization sequence (NLS) and / or a nuclear export sequence (NES). In some embodiments, the peptide construct further comprises one or more NLS and / or one or more NES. In some embodiments, the NLS comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 22-23. In some embodiments, the NES comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0153] In some embodiments, the polypeptide construct is a fusion protein, for example, a fusion protein comprising the base deaminase and the guide RNA molecule binding domain.

[0154] In some embodiments, the polypeptide construct is a fusion protein, and adjacent domains of the fusion protein are independently linked by or without a linker (e.g., a peptide linker, such as a peptide linker containing one or more glycines and / or one or more serines). In some embodiments, the linker comprises the amino acid sequence GS or the amino acid sequence shown in SEQ ID NO: 21.

[0155] In some embodiments, the guide RNA molecule binding domain comprises an amino acid sequence as shown in SEQ ID NO: 10; and / or, the backbone sequence of the guide RNA molecule comprises a nucleotide sequence as shown in SEQ ID NO: 13.

[0156] In some embodiments, the base to be edited is adenine, and the base deaminase is selected from the TadA mutant of aspect eleven or a functional fragment thereof. In some embodiments, the base deaminase is selected from the TadA mutant comprising the amino acid sequence shown in SEQ ID NO: 4 or 5 or a functional fragment thereof. In some embodiments, the polypeptide construct is a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 15 or 16.

[0157] In some embodiments, the base to be edited is cytosine, and the base deaminase is selected from the TadA mutant of aspect 12 or a functional fragment thereof. In some embodiments, the base deaminase is selected from the TadA mutant comprising the amino acid sequence shown in SEQ ID NO: 6 or 7 or a functional fragment thereof. In some embodiments, the polypeptide construct is a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 17 or 18.

[0158] In some embodiments, the base to be edited is adenine, cytosine, or a combination thereof, and the base deaminase is selected from the TadA mutant of aspect thirteen or a functional fragment thereof. In some embodiments, the base deaminase is selected from the TadA mutant comprising the amino acid sequence shown in SEQ ID NO: 8 or 9 or a functional fragment thereof. In some embodiments, the polypeptide construct is a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 19 or 20.

[0159] In the fifteenth aspect, this application provides a nucleic acid molecule that encodes a TadA mutant or a functional fragment thereof from any of the eleventh to thirteenth aspects, or a polypeptide construct from the fourteenth aspect.

[0160] In a sixteenth aspect, this application provides a vector comprising the nucleic acid molecule described in aspect fifteen. In some embodiments, the vector is a cloning vector or an expression vector.

[0161] In its seventeenth aspect, this application provides a host cell comprising the nucleic acid molecule of aspect fifteen or the vector of aspect sixteen. In some embodiments, the cell is not a plant cell. In some embodiments, the cell is a microbial cell.

[0162] In the eighteenth aspect, this application provides a method for preparing a TadA mutant or a functional fragment thereof from any of the eleventh to thirteenth aspects or a polypeptide construct from the fourteenth aspect, comprising culturing a host cell of the seventeenth aspect under conditions that allow protein expression, and recovering the TadA mutant or a functional fragment thereof or the polypeptide construct from the cultured host cell culture.

[0163] In a nineteenth aspect, this application provides a composition comprising: (a) A first component, selected from: (i) a guide RNA molecule comprising a backbone sequence and a guide sequence, the guide sequence being capable of hybridizing with a target RNA sequence comprising bases to be edited; (ii) a nucleotide sequence encoding the guide RNA molecule; (iii) any combination of (i) and (ii); as well as, (b) A second component selected from: (i) a polypeptide construct comprising a base deaminase and a guide RNA molecule binding domain linked to the base deaminase, the guide RNA molecule binding domain being capable of binding to the backbone sequence of the guide RNA molecule; (ii) a nucleotide sequence encoding the polypeptide construct; (iii) any combination of (i) and (ii); The base deaminase is selected from any of the eleventh to thirteenth aspects of the TadA mutant or its functional fragment.

[0164] In an embodiment where the first component is a nucleotide sequence encoding the guide RNA molecule and the second component is a nucleotide sequence encoding the polypeptide construct, the nucleotide sequences of the first component and the second component may exist in different nucleic acid molecules or vectors, or they may exist in the same nucleic acid molecule or vector.

[0165] In some embodiments, the guide sequence of the guide RNA molecule is designed to cause the target RNA sequence to form a single-stranded RNA loop region containing the bases to be edited after hybridization with the target RNA sequence.

[0166] In some embodiments, the target RNA sequence comprises: a target circularizable region containing the bases to be edited, a first region located at the 5' end of the target circularizable region, and a second region located at the 3' end of the target circularizable region; The guide sequence of the guide RNA molecule is designed to include a first region, a second region, and an optional third region located between the first and second regions, the second region being upstream of the first region; and the first region is complementary to a first region of the target RNA sequence, the second region is complementary to a second region of the target RNA sequence, and the third region is absent or cannot be complementary to a target circularizable region of the target RNA sequence. Therefore, after the guide RNA molecule hybridizes with the target RNA sequence, the target loopable region located between region I and region II of the target RNA sequence exists as a single strand, thereby forming a single-stranded RNA loop region containing the bases to be edited.

[0167] In some implementations, the third region of the guide RNA molecule is absent.

[0168] In some embodiments, the third region of the guide RNA molecule has fewer nucleotide residues than the target circularizable region of the target RNA sequence.

[0169] In some embodiments, the first and second regions of the guide RNA molecule have the same or different numbers of nucleotide residues.

[0170] In some embodiments, the guide RNA molecule binding domain is selected from: Cas effector proteins of the CRISPR system lacking nuclease activity, MS2 bacteriophage coat protein (MCP), and bacteriophage Lambda N protein (λ N-peptide).

[0171] Based on the disclosure of this application, those skilled in the art will readily understand that the design of the backbone sequence of the guide RNA molecule in the composition of this application corresponds to the selection of the guide RNA molecule binding domain of the polypeptide construct. That is, the backbone sequence of the guide RNA molecule is designed to be recognized and bound by the guide RNA molecule binding domain contained in the polypeptide construct.

[0172] In some embodiments, the backbone sequence of the guide RNA molecule is selected from the backbone sequence of a crRNA (or crRNA / tracrRNA complex or construct) that the Cas effector protein can recognize and bind to.

[0173] In some embodiments, the binding domain of the guide RNA molecule is selected from CRISPR-Cas13 effector proteins lacking nuclease activity. In some embodiments, the backbone sequence of the guide RNA molecule is selected from the backbone sequence of crRNAs that the CRISPR-Cas13 effector protein can recognize and bind to.

[0174] In some embodiments, the binding domain of the guide RNA molecule is selected from the MS2 phage coat protein (MCP). In some embodiments, the backbone sequence of the guide RNA molecule is selected from an MS2 aptamer that the MS2 phage coat protein (MCP) can recognize and bind to.

[0175] In some embodiments, the binding domain of the guide RNA molecule is selected from the phage Lambda N protein. In some embodiments, the backbone sequence of the guide RNA molecule is selected from a boxB sequence that the phage Lambda N protein can recognize and bind to. In some embodiments, the guide RNA molecule binding domain is a PspCas13b lacking nuclease activity. In some embodiments, the guide RNA molecule binding domain comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the backbone sequence of the guide RNA molecule comprises the amino acid sequence shown in SEQ ID NO: 13.

[0176] In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule contains one or more bases to be edited. In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases to be edited.

[0177] In some embodiments, the base to be edited is selected from adenine, cytosine, and any combination thereof.

[0178] In some embodiments, the first region of the guide RNA molecule consists of 3-50 (e.g., 5-50, 5-40, 5-30, 5-25, 5-20, 5-18, 5-15, 10-50, 10-40, 10-30, 10-25, 10-20, 10-18, 12-50, 12-40, 12-30, 12-25, 12-20, 12-18) nucleotide residues.

[0179] In some embodiments, the second region of the guide RNA molecule consists of 3-50 (e.g., 5-50, 5-40, 5-30, 5-25, 5-20, 5-18, 5-15, 10-50, 10-40, 10-30, 10-25, 10-20, 10-18, 12-50, 12-40, 12-30, 12-25, 12-20, 12-18) nucleotide residues.

[0180] In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of at least 3, at least 4, at least 5, or at least 6 nucleotide residues. In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of 3-30 (e.g., 6-30, 6-25, 6-20, 6-19, 6-18, or 6-17) nucleotide residues. In some embodiments, the single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotide residues.

[0181] In some embodiments, the base to be edited in the target RNA sequence is separated from the first region by at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotide residues, and / or is separated from the second region by at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotide residues.

[0182] In some embodiments, in the polypeptide construct, the guide RNA molecule binding domain is linked to the N-terminus and / or C-terminus of the base deaminase.

[0183] In some embodiments, the polypeptide construct further comprises a nuclear localization sequence (NLS) and / or a nuclear export sequence (NES). In some embodiments, the NLS comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 22-23. In some embodiments, the NES comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0184] In some embodiments, the polypeptide construct is a fusion protein, for example, a fusion protein comprising the base deaminase and the guide RNA molecule binding domain.

[0185] In some embodiments, the polypeptide construct is a fusion protein, and adjacent domains of the fusion protein are independently linked by or without a linker (e.g., a peptide linker, such as a peptide linker containing one or more glycines and / or one or more serines). In some embodiments, the linker comprises the amino acid sequence GS or the amino acid sequence shown in SEQ ID NO: 21.

[0186] In some embodiments, the guide RNA molecule binding domain comprises an amino acid sequence as shown in SEQ ID NO: 10; and / or, the backbone sequence of the guide RNA molecule comprises a nucleotide sequence as shown in SEQ ID NO: 13.

[0187] In some embodiments, the base to be edited is adenine, and the base deaminase is selected from the TadA mutant of aspect eleven or a functional fragment thereof. In some embodiments, the base deaminase is selected from the TadA mutant comprising the amino acid sequence shown in SEQ ID NO: 4 or 5 or a functional fragment thereof. In some embodiments, the polypeptide construct is a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 15 or 16.

[0188] In some embodiments, the base to be edited is cytosine, and the base deaminase is selected from the TadA mutant of aspect 12 or a functional fragment thereof. In some embodiments, the base deaminase is selected from the TadA mutant comprising the amino acid sequence shown in SEQ ID NO: 6 or 7 or a functional fragment thereof. In some embodiments, the polypeptide construct is a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 17 or 18.

[0189] In some embodiments, the base to be edited is adenine, cytosine, or a combination thereof, and the base deaminase is selected from the TadA mutant of aspect thirteen or a functional fragment thereof. In some embodiments, the base deaminase is selected from the TadA mutant comprising the amino acid sequence shown in SEQ ID NO: 8 or 9 or a functional fragment thereof. In some embodiments, the polypeptide construct is a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 19 or 20.

[0190] In a twentieth aspect, this application provides a complex comprising: (a) A guide RNA molecule, said guide RNA molecule as defined in aspect nineteen; as well as, (b) A polypeptide construct, said polypeptide construct as defined in aspect nineteen.

[0191] In a twentieth aspect, this application provides a carrier system comprising one or more carriers, said one or more carriers comprising: (1) A first nucleotide sequence encoding a guide RNA molecule as defined in the nineteenth aspect; optionally, the first nucleotide sequence is operatively linked to a first regulatory element; and, (2) A second nucleotide sequence encoding a polypeptide construct as defined in the nineteenth aspect; optionally, the second nucleotide sequence is operatively linked to a second regulatory element; The first nucleotide sequence and the second nucleotide sequence may be present on the same or different vectors.

[0192] In some implementations, the first regulating element is a promoter, such as an inducible promoter.

[0193] In some implementations, the second regulating element is an promoter, such as an inductive promoter.

[0194] In a twentieth aspect, this application provides a kit comprising a TadA mutant or a functional fragment thereof as described in any one of aspects eleven to thirteen, a polypeptide construct as described in aspect fourteen, a nucleic acid molecule as described in aspect fifteen, a vector as described in aspect sixteen, a host cell as described in aspect seventeen, a composition as described in aspect nineteen, a complex as described in aspect twenty, or a vector system as described in aspect twenty-one.

[0195] In a twentieth aspect, this application provides a delivery composition comprising a delivery vector and one or more of the following: a TadA mutant or a functional fragment thereof as described in any one of aspects eleven to thirteen, a polypeptide construct as described in aspect fourteen, a nucleic acid molecule as described in aspect fifteen, a vector as described in aspect sixteen, a composition as described in aspect nineteen, a complex as described in aspect twentieth, and a vector system as described in aspect twenty-one.

[0196] In some implementations, the delivery carrier is a particle.

[0197] In some embodiments, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0198] In another aspect, this application provides a cell comprising: a TadA mutant or a functional fragment thereof as described in any one of aspects eleven through thirteen, a polypeptide construct as described in aspect fourteen, a nucleic acid molecule as described in aspect fifteen, a vector as described in aspect sixteen, a composition as described in aspect nineteen, a complex as described in aspect twentieth, a vector system as described in aspect twenty-one, and a delivery composition as described in aspect twenty-three. In some embodiments, the cell is not a plant cell. In some embodiments, the cell is a microbial cell.

[0199] In its twenty-fourth aspect, this application provides a method for extracellular editing of target RNA, comprising, under conditions suitable for target RNA editing, contacting the target RNA with one or more of the following: a TadA mutant or a functional fragment thereof as described in any one of aspects eleven to thirteen, a polypeptide construct as described in aspect fourteen, a nucleic acid molecule as described in aspect fifteen, a vector as described in aspect sixteen, a composition as described in aspect nineteen, a complex as described in aspect twentieth, and a delivery composition as described in aspect twenty-third, thereby inducing deamination of the bases to be edited in the target RNA.

[0200] In a twentieth aspect, this application provides a method for editing target RNA in cells, comprising delivering a TadA mutant or a functional fragment thereof as described in any one of aspects eleven to thirteen, a polypeptide construct as described in aspect fourteen, a nucleic acid molecule as described in aspect fifteen, a vector as described in aspect sixteen, a composition as described in aspect nineteen, a complex as described in aspect twentieth, a vector system as described in aspect twenty-one, or a delivery composition as described in aspect twenty-three into a cell containing the target RNA, thereby inducing deamination of the base to be edited at the target site.

[0201] In a twenty-sixth aspect, this application provides a cell or its progeny obtained by the method of the twenty-fifth aspect, wherein said cell contains modifications not present in its wild type. In some embodiments, said cell is not a plant cell. In some embodiments, said cell is a microbial cell.

[0202] In its twenty-seventh aspect, this application provides the use of the TadA mutant or a functional fragment thereof as described in any one of aspects eleven to thirteen, the polypeptide construct as described in aspect fourteen, the nucleic acid molecule as described in aspect fifteen, the vector as described in aspect sixteen, the host cell as described in aspect seventeen, the composition as described in aspect nineteen, the complex as described in aspect twenty, the vector system as described in aspect twenty-one, the kit as described in aspect twenty-two, or the delivery composition as described in aspect twenty-three, for RNA editing, or for use in the preparation of formulations for RNA editing.

[0203] In some implementations, the RNA editing is base editing performed on RNA.

[0204] On the other hand, this application provides the following implementation scheme: Implementation Scheme 1. A TadA mutant or a functional fragment thereof, wherein, compared to TadA comprising the sequence shown in SEQ ID NO: 1, the TadA mutant comprises a mutation selected from: (1) V28R, (2) V30A, (3) I76Y, (4) V82T, (5) F84H, F84V, F84N, F84S, F84I, F84T, F84C or F84A, (6) R98E, (7) N108H, (8) R111C or R111L, (9) Y147R, (10) Q154R, (11) Any combination of (1) to (10).

[0205] Implementation Scheme 2. The TadA mutant or a functional fragment thereof as described in Implementation Scheme 1, wherein, compared to TadA comprising the sequence shown in SEQ ID NO: 1, the TadA mutant comprises a mutation selected from: (i) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and / or, (e) Q154R; (ii) (a) F84H, F84V or F84I, and (b) V82T; (iii) (a) F84H, F84V or F84I, (b) V82T, and (c) Y147R; (iv) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and (e) Q154R; (v) F84I, V82T, Y147R, I76Y, and / or, Q154R; (vi) F84I and V82T; (vii) F84I, V82T and Y147R; (viii) F84I, V82T, Y147R, I76Y and Q154R.

[0206] Implementation Scheme 3. The TadA mutant or a functional fragment thereof as described in Implementation Scheme 1, wherein, compared to TadA comprising the sequence shown in SEQ ID NO: 1, the TadA mutant comprises a mutation selected from: (i) F84I and V82T; (ii) F84I, V82T and Y147R; (iii) F84I, V82T, Y147R, I76Y and Q154R.

[0207] Implementation Scheme 4. The TadA mutant or a functional fragment thereof as described in Implementation Scheme 1, wherein the TadA mutant comprises an amino acid sequence as shown in SEQ ID NO: 4 or 5.

[0208] Implementation Scheme 5. A polypeptide construct comprising a base deaminase and a guide RNA molecule binding domain linked to said base deaminase; The base deaminase is selected from the TadA mutant or its functional fragment as described in any one of embodiments 1-4.

[0209] Implementation Scheme 6. The polypeptide construct described in Implementation Scheme 5, wherein the guide RNA molecule binding domain is selected from: the Cas effector protein of the CRISPR system lacking nuclease activity, MS2 bacteriophage coat protein (MCP), and bacteriophage Lambda N protein (λN-peptide).

[0210] Implementation Scheme 7. The polypeptide construct described in Implementation Scheme 5, having one or more of the following characteristics: (1) In the polypeptide construct, the guide RNA molecule binding domain is linked to the N-terminus and / or C-terminus of the base deaminase; (2) The polypeptide construct further comprises a nuclear localization sequence (NLS) and / or a nuclear export sequence (NES). (3) The binding domain of the guide RNA molecule contains an amino acid sequence as shown in SEQ ID NO: 10; and / or, the backbone sequence of the guide RNA molecule contains a nucleotide sequence as shown in SEQ ID NO: 13; (4) The polypeptide construct is a fusion protein; (5) The polypeptide construct is a fusion protein containing an amino acid sequence as shown in SEQ ID NO: 15 or 16.

[0211] Implementation Scheme 8. A nucleic acid molecule encoding a TadA mutant or a functional fragment thereof as described in any one of Implementation Schemes 1-4, or a polypeptide construct as described in any one of Implementation Schemes 5-7.

[0212] Implementation Scheme 9. A carrier comprising the nucleic acid molecules described in Implementation Scheme 8.

[0213] Implementation Scheme 10. A host cell comprising the nucleic acid molecule described in Implementation Scheme 8 or the vector described in Implementation Scheme 9.

[0214] Implementation Scheme 11. A composition comprising: (a) A first component, selected from: (i) a guide RNA molecule comprising a backbone sequence and a guide sequence, the guide sequence being capable of hybridizing with a target RNA sequence comprising bases to be edited; (ii) a nucleotide sequence encoding the guide RNA molecule; (iii) any combination of (i) and (ii); as well as, (b) A second component selected from: (i) a polypeptide construct comprising a base deaminase and a guide RNA molecule binding domain linked to the base deaminase, the guide RNA molecule binding domain being capable of binding to the backbone sequence of the guide RNA molecule; (ii) a nucleotide sequence encoding the polypeptide construct; (iii) any combination of (i) and (ii); The base deaminase is selected from the TadA mutant or its functional fragment as described in any one of embodiments 1-4.

[0215] Implementation Scheme 12. The composition of Implementation Scheme 11, wherein the guide sequence of the guide RNA molecule is designed to cause the target RNA sequence to form a single-stranded RNA loop region containing the bases to be edited after hybridization with the target RNA sequence.

[0216] Implementation Scheme 13. The composition of Implementation Scheme 12, wherein the target RNA sequence comprises: a target circularizable region containing the base to be edited, a first region located at the 5' end of the target circularizable region, and a second region located at the 3' end of the target circularizable region; The guide sequence of the guide RNA molecule is designed to include a first region, a second region, and an optional third region located between the first and second regions, the second region being upstream of the first region; and the first region is complementary to a first region of the target RNA sequence, the second region is complementary to a second region of the target RNA sequence, and the third region is absent or cannot be complementary to a target circularizable region of the target RNA sequence.

[0217] Implementation Scheme 14. The composition of Implementation Scheme 13, having one or more of the following characteristics: (1) The third region of the guide RNA molecule does not exist; (2) The number of nucleotide residues in the third region of the guide RNA molecule is less than the number of nucleotide residues in the target circular region of the target RNA sequence; (3) The first region and the second region of the guide RNA molecule have the same or different numbers of nucleotide residues; (4) The single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule contains one or more bases to be edited; (5) The single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of at least 3, at least 4, at least 5, or at least 6 nucleotide residues; (6) The polypeptide construct is as defined in any one of embodiments 5-7.

[0218] Implementation Scheme 15. A complex comprising: (a) A guide RNA molecule, said guide RNA molecule as defined in any one of embodiments 11-14; as well as, (b) A polypeptide construct, said polypeptide construct as defined in any one of embodiments 5-7.

[0219] Implementation Scheme 16. A carrier system comprising one or more carriers, said one or more carriers comprising: (1) A first nucleotide sequence encoding a guide RNA molecule, said guide RNA molecule as defined in any one of embodiments 11-14; and, (2) A second nucleotide sequence encoding a polypeptide construct as defined in any one of embodiments 5-7; The first nucleotide sequence and the second nucleotide sequence may be present on the same or different vectors.

[0220] Implementation Scheme 17. A kit comprising the TadA mutant or a functional fragment thereof as described in any one of Implementation Schemes 1-4, the polypeptide construct as described in any one of Implementation Schemes 5-7, the nucleic acid molecule as described in Implementation Scheme 8, the vector as described in Implementation Scheme 9, the composition as described in any one of Implementation Schemes 11-14, the complex as described in Implementation Scheme 15, or the vector system as described in Implementation Scheme 16.

[0221] Implementation Scheme 18. A delivery composition comprising a delivery vector and one or more of the following: a TadA mutant or a functional fragment thereof as described in any one of Implementation Schemes 1-4, a polypeptide construct as described in any one of Implementation Schemes 5-7, a nucleic acid molecule as described in Implementation Scheme 8, a vector as described in Implementation Scheme 9, a composition as described in any one of Implementation Schemes 11-14, a complex as described in Implementation Scheme 15, and a vector system as described in Implementation Scheme 16.

[0222] Implementation Scheme 19. A cell comprising: the TadA mutant or a functional fragment thereof as described in any one of Implementation Schemes 1-4, the polypeptide construct as described in any one of Implementation Schemes 5-7, the nucleic acid molecule as described in Implementation Scheme 8, the vector as described in Implementation Scheme 9, the composition as described in any one of Implementation Schemes 11-14, the complex as described in Implementation Scheme 15, the vector system as described in Implementation Scheme 16, or the delivery composition as described in Implementation Scheme 18.

[0223] Implementation Scheme 20. A method for extracellular editing of target RNA, comprising, under conditions suitable for target RNA editing, contacting the target RNA with one or more of the following: a TadA mutant or a functional fragment thereof as described in any one of Implementation Schemes 1-4, a polypeptide construct as described in any one of Implementation Schemes 5-7, a nucleic acid molecule as described in Implementation Scheme 8, a vector as described in Implementation Scheme 9, a composition as described in any one of Implementation Schemes 11-14, a complex as described in Implementation Scheme 15, a vector system as described in Implementation Scheme 16, and a delivery composition as described in Implementation Scheme 18, thereby inducing deamination of the bases to be edited in the target RNA.

[0224] Implementation Scheme 21. A method for editing target RNA in cells, comprising delivering a TadA mutant or a functional fragment thereof as described in any one of Implementation Schemes 1-4, a polypeptide construct as described in any one of Implementation Schemes 5-7, a nucleic acid molecule as described in Implementation Scheme 8, a vector as described in Implementation Scheme 9, a composition as described in any one of Implementation Schemes 11-14, a complex as described in Implementation Scheme 15, a vector system as described in Implementation Scheme 16, and / or a delivery composition as described in Implementation Scheme 18 into a cell containing the target RNA, thereby inducing deamination of the base to be edited at the target site.

[0225] Implementation Scheme 22. A cell or its progeny obtained by the method of Implementation Scheme 21, wherein the cell contains modifications not present in its wild type.

[0226] Implementation Scheme 23. The TadA mutant or its functional fragment as described in any one of Implementation Schemes 1-4, the polypeptide construct as described in any one of Implementation Schemes 5-7, the nucleic acid molecule as described in Implementation Scheme 8, the vector as described in Implementation Scheme 9, the composition as described in any one of Implementation Schemes 11-14, the complex as described in Implementation Scheme 15, the vector system as described in Implementation Scheme 16, the kit as described in Implementation Scheme 17, or the delivery composition as described in Implementation Scheme 18, for use in RNA editing, or for use in the preparation of a formulation for RNA editing.

[0227] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0228] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0229] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0230] As used herein, the term “base editing” has the meaning commonly understood by those skilled in the art as referring to genome editing techniques involving the conversion of a specific nucleic acid base into another nucleic acid base at a target nucleic acid (target nucleic acid) site (e.g., included in RNA).

[0231] As used herein, the term "deaminase" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine (or adenine) deaminase that catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, an adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in ribonucleic acid (RNA) to inosine. In some embodiments, the deaminase is a cytidine (or cytosine) deaminase that catalyzes the hydrolytic deamination of cytosine or cytosine. In some embodiments, a cytidine deaminase catalyzes the hydrolytic deamination of cytosine or cytosine in ribonucleic acid (RNA) to uridine. In some embodiments, the deaminase possesses both adenosine deaminase and cytidine deaminase activities, catalyzing the hydrolytic deamination of both adenine or adenosine and cytosine or cytosine. In some implementations, the deaminase is a single-stranded RNA deaminase, or a modified, evolved, or otherwise altered deaminase that can utilize single-stranded RNA as a substrate for deamination.

[0232] As used herein, the term "TadA," tRNA adenosine deaminase A, refers to a deaminase capable of catalyzing the deamination of adenine (A) into hypoxanthine (I) using RNA as a substrate. Specific amino acid sequences of various naturally occurring or artificially modified TadA disclosed in the prior art (e.g., the amino acid sequences shown in any one of SEQ ID NO: 1-3) are available from public databases (e.g., the GenBank database).

[0233] Based on the disclosure of this application, it will be readily understood by those skilled in the art that this application provides a series of mutations for modifying the deamination activity of TadA. These mutations are applicable to existing TadA (including not only naturally occurring TadA derived directly from organisms but also artificially modified / screened non-natural TadA variants) to impart altered base deamination activity (e.g., enhanced or reduced adenosine deamination activity, and / or enhanced or reduced cytidine deamination activity). Therefore, as used in this application, the term "TadA mutant" includes not only TadA mutants obtained by implementing the mutations of this application on naturally occurring TadA derived directly from organisms but also TadA mutants obtained by implementing the mutations of this application on artificially modified / screened non-natural TadA (i.e., modified / screened non-natural TadA derived indirectly from organisms).

[0234] In this document, the term "peptide construct" is used in its broadest sense. Generally, "peptide construct" is used to refer to a construct comprising one or more peptide or protein components, wherein the one or more peptide or protein components may each have different origins or different biological activities or functions, and are linked by covalent and / or non-covalent means (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalently linked by hydrogen bonds). The peptide constructs of the present invention are not limited in the number of their molecular chains (e.g., peptide chains). For example, the peptide constructs of the present invention may contain only one molecular chain (e.g., a peptide chain), or two or more molecular chains (e.g., peptide chains) linked covalently and / or non-covalently (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalently linked by hydrogen bonds). Similarly, those skilled in the art will readily understand that, in embodiments comprising multiple polypeptide or protein components, the multiple polypeptide or protein components contained in the polypeptide construct of the present invention may be all or partly located in the same molecular chain (e.g., peptide chain), or may each be located in different molecular chains (e.g., peptide chains). In some preferred embodiments, the polypeptide construct of this application is a fusion protein (e.g., a fusion protein comprising a base deaminase and a guide RNA molecule binding domain).

[0235] As used herein, the term “CRISPR system” has the meaning commonly understood by those skilled in the art, and typically includes transcripts or other elements relating to the expression of CRISPR-associated (“Cas”) genes, or transcripts or other elements capable of directing the activity of said Cas genes. Such transcripts or other elements may include sequences encoding Cas effector proteins and guide RNA containing CRISPR RNA (crRNA), or other sequences or transcripts derived from CRISPR loci.

[0236] As used herein, the term "guide RNA" has the meaning commonly understood by those skilled in the art. Generally, a guide RNA may comprise a repetitive sequence (also called a backbone sequence, which typically forms a stem-loop structure that allows for specific recognition and binding by a particular effector protein) and a guide sequence, or may consist essentially of a repetitive sequence and a guide sequence. In some cases, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize with said target sequence and guide the effector protein and guide RNA complex to specifically bind to said target sequence. In some embodiments, when optimal alignment occurs, the complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of those skilled in the art. For example, publicly available and commercially available alignment algorithms and programs exist, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan.

[0237] In some cases, the guide sequence has a length of at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides. In some cases, the guide sequence has a length of no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15, 10, or fewer nucleotides. In some embodiments, the guide sequence has a length of 10-35 nucleotides.

[0238] In some cases, the repeating sequence has a length of at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, or at least 70 nucleotides. In some cases, the repeating sequence has a length of no more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15, 10, or fewer nucleotides. In some embodiments, the repeating sequence has a length of 55-70 nucleotides. In some embodiments, the repeating sequence has a length of 15-50 nucleotides.

[0239] In this invention, the term "target sequence" can refer to any endogenous or exogenous polynucleotide for a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. In some cases, the target sequence or target polynucleotide may include multiple disease-associated polynucleotides and polynucleotides associated with signal transduction biochemical pathways.

[0240] As used herein, the terms “non-naturally occurring” or “engineered” are used interchangeably and indicate artificial involvement. When these terms are used to describe nucleic acid molecules or peptides or compositions or complexes, they indicate that the nucleic acid molecule or peptide or composition or complex is at least substantially free from at least one other component bound to it, either in nature or as found in nature, or that the nucleic acid molecule or peptide contains at least one component or portion formed with artificial involvement (e.g., artificial design or modification).

[0241] As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences), for which detailed descriptions can be found in Goeddel, *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, California (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct expression of that nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). In some cases, regulatory elements can also be directed to express in a time-dependent manner (such as in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" covers enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0242] As used herein, the term "promoter" has the meaning known to those skilled in the art, referring to a non-coding nucleotide sequence located upstream of a gene that initiates the expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to that promoter.

[0243] As used herein, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that allows the expression of that nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).

[0244] As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of these nucleotide residues. Hydrogen bonding can occur via Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific mechanism. The complex can consist of two strands forming a duplex, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. Hybridization can constitute a step in a broader process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is called the "complement" of that given sequence.

[0245] As used herein, the term “upstream” describes the relative position of two nucleic acid sequences (or two nucleic acid molecules) and has a meaning commonly understood by those skilled in the art. For example, stating that “one nucleic acid sequence is upstream of another nucleic acid sequence” means that, when aligned in a 5’ to 3’ direction, the former is located further forward (i.e., closer to the 5’ end) than the latter. As used herein, the term “downstream” has the opposite meaning to “upstream”.

[0246] As described herein, the terms “complementary” or “complementarity” are used when referring to sequences of nucleotides linked by the base pairing rule. For example, sequence 5'-AGT-3' is complementary to sequence 3'-TCA-5'. Complementarity can be “partial,” where only some bases in the nucleic acid match according to the base pairing rule. Alternatively, there can be “complete” or “full” complementarity between nucleic acids.

[0247] As used herein, the term "mutant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide containing an amino acid sequence altered by the introduction of amino acid residue substitutions, deletions, or additions. In some cases, the term "mutant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, mutants have similar, identical, or improved functions to the polypeptide or peptide from which they are derived.

[0248] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0249] In this invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0250] Those skilled in the art know that during mRNA translation, the first amino acid in the resulting polypeptide chain is often the amino acid encoded by the start codon (e.g., methionine (M)) due to the effect of the start codon. Therefore, the polypeptides or proteins involved in this invention (e.g., the base deaminase of this invention, TadA of this invention, TadA mutant of this invention, polypeptide constructs of this invention) encompass not only amino acid sequences that do not contain an amino acid encoded by a start codon (e.g., methionine) at their N-terminus, but also amino acid sequences that contain an amino acid encoded by a start codon (e.g., methionine) at their N-terminus.

[0251] Unless otherwise specified herein or clearly contradicted by the context, “A, B and / or C” or similar expressions should be understood as “A, B, C or any combination thereof”, for example, it can be understood as any one of A, B, C, A and B, A and C, B and C, A and B and C.

[0252] Beneficial effects of the invention

[0253] The RNA base editing tool provided by this invention has one or more of the following beneficial effects: (1) Existing RNA editing tools (including DECOR) are all designed for editing a single site and have no controllable editing window. This invention utilizes single-stranded deaminases (e.g., TadA) and special LF-gRNA to develop, for the first time, an RNA editing tool with a controllable editing window; (2) This invention provides a novel TadA mutant by rationally designing and mutating TadA8e, which significantly reduces off-target effects of bystander editing and whole transcriptome, thereby achieving A-to-I base editing on RNA with fewer off-target effects and higher precision (i.e., AIM-A and AIM-Amax systems). (3) This invention provides a novel TadA mutant by rationally designing and mutating TadA8e, which realizes efficient and residual A-to-I activity C-to-U base editing on RNA (i.e. AIM-C and AIM-Cmax system). (4) Currently, there is a lack of bifunctional base editors for RNA in this field. This invention provides a novel TadA mutant by rationally designing and mutating TadA8e, thereby realizing simultaneous A-to-I and C-to-U base editing on a single RNA molecule (i.e., AIM-A&C and AIM-A&Cmax systems). (5) The AIM-A, AIM-C and AIM-A&C systems provided in this application can all achieve controllable number of RNA base editing within a certain range.

[0254] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0255] Figure 1 AIM design principle diagram. A. Main components of AIM: single-stranded deaminase TadA; dead Psp Cas13b; and specially designed LF-gRNA that can generate loops at the target site. B. Schematic diagram of AIM achieving a controllable window. By designing different LF-gRNAs to generate loops of different sizes, editing windows of different widths can be achieved.

[0256] Figure 2 Programmable A-to-I editing was achieved using the single-stranded deaminase TadA. A. Schematic diagram of TadA and its substrate tRNA. B. Schematic diagram of the AIM tool. C. Schematic diagram of the Reporter-UAG structure, and explanation of the positions of crRNA (-38) and crRNA (+40) targeting Reporter-UAG. D. Editing efficiency of different TadA variants on Reporter-UAG.

[0257] Figure 3 Circular guide RNA (LF-gRNA) was used to construct the editing window. A. LF-gRNA-10 nt induces a 10-nucleotide single-stranded RNA loop on the target mRNA. B. EGFP expression signals of conventional crRNA and LF-gRNA-10 nt. C. A-to-I editing efficiency of combinations of LF-gRNA and conventional crRNA with TadA7.10 and TadA8e.

[0258] Figure 4 Parameter exploration of circularizable guide RNAs (LF-gRNAs). A. Editing efficiency of various LF-gRNAs with different arm length combinations. B. Editing efficiency of LF-gRNAs with asymmetric arms. C. Designing a series of LF-gRNAs with the target base A located at positions 1 through 10 of the RNA loop and evaluating A-to-I editing efficiency.

[0259] Figure 5The width of the editing window can be controlled by changing the loop size. A. “Reporter Vector-4A” and its design with different LF-gRNAs. B. TadA7.10 can achieve different sized editing windows in different LF-gRNA designs. The larger the loop induced by the LF-gRNA at the target site, the wider the editing window. C. TadA8e can achieve different sized editing windows in different LF-gRNA designs. The larger the loop induced by the LF-gRNA at the target site, the wider the editing window.

[0260] Figure 6 A novel TadA mutant enables precise and efficient A-to-I editing. A. Analysis of A-to-I editing events and proportions caused by TadA8e within the 100 bp region of the reporter transcript (reporter vector-TAG). Red indicates the editing efficiency of loop A caused by LF-gRNA, blue indicates the editing efficiency of out-of-loop A caused by LF-gRNA, and blue represents off-target editing. B. Structure of ABE8e in single-stranded DNA-binding state, aligned with the co-crystal structure of Staphylococcus aureus TadA-tRNA. Key amino acid sites are divided into 4 groups according to different functional regions. C. Analysis of A-to-I editing events and proportions caused by TadA8e (F84I) within the 100 bp region of the reporter transcript (reporter vector-TAG). D. Editing efficiency of TadA8e (F84I) and TadA8e (F84I-V82T) on the reporter vector-TAG. E. Comparison of mutation sites among different TadA variants. F, G. Analysis of A-to-I editing events and proportions caused by AIM-A (F) and AIM-Amax (G) within the 100 bp region of the reporter transcript (reporter vector-TAG).

[0261] Figure 7 AIM-A enables controlled editing of quantity and location within a specific region. A. Representative sequences of novel reporter genes containing isolated and continuous adenosines are presented. The corresponding LF-gRNAs are shown, with loop sizes ranging from 4 to 14 nucleotides. B. A-to-I editing efficiency mediated by AIM-A under LF-gRNA designs with different loop sizes. C. Representative sequences of novel reporter genes containing isolated and continuous adenosines are presented. LF-gRNA designs at different loop positions are shown. D. A-to-I editing efficiency mediated by AIM-A under LF-gRNA designs at different loop positions is demonstrated.

[0262] Figure 8A novel TadA mutant achieves C-to-U editing. A. Structural representation of the Reporter-CAC. B. Analysis of A-to-I and C-to-U editing events and proportions caused by TadA8e within the 100 bp region of the reporter transcript (reporter vector-TAG). C. Structure of ABE8e in single-stranded DNA-binding state, aligned with the co-crystal structure of Staphylococcus aureus TadA-tRNA. Key amino acids that may affect C-to-U editing efficiency are marked in dashed circles. D. A-to-I and C-to-U editing efficiencies of the N46 and A48 combined mutants. E. A-to-I and C-to-U editing efficiencies of the activating mutant based on N46P-A48G.

[0263] Figure 9 A novel TadA mutant enables precise and efficient C-to-U editing. A. Analysis of A-to-I and C-to-U editing events and proportions caused by AIM-C within the 100 bp region of the reporter transcript (reporter vector-TAG). B. Sequence alignment of the TadA variant used for DNACBE with TadA8e and AIM-C. C. A-to-I and C-to-U editing efficiency of the AIM-C-based activating mutant. D. Analysis of A-to-I and C-to-U editing events and proportions caused by AIM-Cmax within the 100 bp region of the reporter transcript (reporter vector-TAG).

[0264] Figure 10 The novel TadA mutant enables precise and efficient C-to-U editing. The left side shows a set of Reporter sequences; the same LF-gRNA can generate loops ranging from 5 to 17 nt in size. The right side shows the editing efficiency of AIM-C under different loop sizes.

[0265] Figure 11 A novel TadA mutant achieves simultaneous A-to-I and C-to-U editing. A. Statistical analysis of A-to-I and C-to-U editing efficiencies in mutants after saturation mutation at the N46 site of TadA8e (A48G). B. Statistical analysis of A-to-I and C-to-U editing efficiencies in activated mutants based on TadA8e (A48 series mutations-N46C). HE represents four mutations: R26G / V28A / Y73P / H96N. C. The proportion of AIM-A&C simultaneously producing A-to-I and C-to-U editing in the same transcript. D. Further activation of AIM-A&C yields AIM-A&Cmax. E. A single Reporter sequence, using the same LF-gRNA, can generate loops ranging from 5-17 nt in size. F. Editing efficiency of AIM-&C under different loop sizes. Sequence information

[0266] The descriptions of the sequences involved in this application are provided in the table below.

[0267] Table 1: Sequence Information

[0268] Note: Amino acid residues that differ from TadA8e in each TadA mutant are indicated by underscores. Detailed Implementation

[0269] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention.

[0270] Experimental methods

[0271] (1) Construction of TadA.

[0272] For the TadA expression plasmid, based on information provided by Addgene, the coding sequences of inactivated PspCas13b (amino acid sequence as shown in SEQ ID NO: 10) and TadA (TadA8e and the amino acid sequences of various TadA mutants based thereon are shown in Table 1) were synthesized and ligated into the pcDNA3.1 backbone using the Gibson cloning method (transcription was driven by the CMV promoter and terminated by the BGHpoly-A signal). The fragments were ligated in the middle using synthesized linker sequences containing NLS / NES (the amino acid sequences of exemplary fusion proteins are shown in SEQ ID NOs: 14-20). Sequence PCR amplification was performed using the high-fidelity enzyme 2×PhantaMax MasterMix (Vazyme). The DNA fragments obtained by PCR amplification were purified and recovered using the TIANprep Midi purification kit (Tiangen). The concentrations of plasmids, purified DNA fragments, and extracted RNA were measured using a Nanodrop spectrophotometer.

[0273] For the crRNA expression plasmid, the PspCas13b crRNA backbone plasmid was constructed based on information provided by Addgene (number 103854). The crRNA spacer sequence was synthesized as a fragment and inserted into the PspCas13b crRNA backbone (SEQ ID NO: 13) using the Goldengate cloning method (transcription was driven by the human U6 promoter and terminated by a poly-U signal). Goldengate assembly was performed using the Type IIS restriction enzyme Esp3I (Thermo Fisher Scientific) and the T4 DNA ligase (New England Biolabs).

[0274] All assembled plasmid vectors were transformed into Trans1-T1 phage-resistant chemically competitive bacteria and cultured on Luria-Bertani (LB) agar containing penicillin. The plasmid sequences were validated using Sanger sequencing, and the validated plasmid-containing bacteria were cultured on LB agar containing penicillin. Endotoxin-free plasmid DNA was extracted using the TIANprep Midi Plasmid Kit for subsequent cell transfection.

[0275] (2) Cell culture and liposome transfection.

[0276] HEK293T cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. At cell passage, cells were washed with PBS, digested with 0.25% trypsin, and incubated at 37°C for 2 minutes. The trypsin was then neutralized with FBS-containing medium. After centrifugation at 600 rpm for 3 minutes, cells were counted and plated. The cell line was negative for mycoplasma contamination.

[0277] HEK293T cells were used at a concentration of 1.75 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 24-well plates and transfected when the cell density reached approximately 70% after 16-18 hours of incubation. Transfection was performed using Lipofectamine LTX and Plus reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0278] To assess the RNA editing efficiency on the exogenous reporter gene, HEK293T cells were transfected with 500 ng of the AIM expression vector (containing the coding sequence of the dCas13b-TadA fusion protein), 500 ng of the LF-gRNA plasmid, and 125 ng of the exogenous reporter gene plasmid. After 72 hours, RNA was extracted from the cells, and the editing efficiency was assessed using Sanger sequencing or high-throughput sequencing.

[0279] (3) RNA extraction and reverse transcription.

[0280] 72 hours after cell transfection, the cell culture medium was aspirated from the wells of the culture dish. 500 μL of Trizol total RNA extraction reagent (Thermo Fisher Scientific) was added to each well, and the mixture was incubated at room temperature for 10 minutes, followed by the addition of 100 µL of chloroform. The mixture was vortexed and allowed to separate into layers at room temperature. This process was repeated three times, followed by centrifugation at 12,000 rpm at 4°C for 15 minutes. The supernatant was transferred to a new tube, and an equal volume of isopropanol was added. After thorough mixing, the tube was incubated at -20°C for at least 1 hour, followed by centrifugation at 14,500 rpm at 4°C for 30 minutes. After centrifugation, a flocculent gel-like precipitate (RNA precipitate) was observed on the tube wall and bottom. The RNA precipitate was washed twice with 1 ml of 75% ethanol, and the supernatant was removed. After air-drying the precipitate for 5–10 minutes, the RNA precipitate was resuspended in RNase-free water, and its concentration was determined. Using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme) and following the manufacturer's instructions, cDNA was generated from extracted RNA using random primers and oligo-dT primers for downstream experiments.

[0281] (4) Construction of amplicon libraries for next-generation sequencing.

[0282] Amplicon libraries were obtained through two rounds of PCR amplification. The first round of PCR (total reaction volume 10 µl) was performed using Q5 Hot Start High-Fidelity 2X Master Mix (NEB, #M0543) with 0.5 µM of gene-specific primers carrying Illumina adapter sequences. After 10 cycles of PCR amplification, the PCR products were purified using 1 volume of AMPure XP magnetic beads (BECKMAN, #A63882). The second round of PCR (30 µl) was then performed using unique Illumina barcode primers (0.5 µM each) containing the adapter sequences. After 15 cycles of the second round of PCR amplification, the products were purified using 0.8 volume of XP magnetic beads. Fragment size and distribution of the libraries were assessed using an Agilent 4150 TapeStation system (Agilent, G2992AA). Qualified libraries were sequenced at 150 bp paired ends using the Illumina NovaSeq platform.

[0283] Experimental results

[0284] I. Programmable A-to-I editing using the single-stranded deaminase TadA.

[0285] Inspired by CRISPR-based DNA base editors, which utilize single-stranded DNA deaminases to introduce point mutations in specific single-stranded DNA regions, the inventors of this application sought a single-stranded RNA deaminase to achieve RNA editing. Currently, the APOBEC family and TadA are known to possess single-stranded RNA deamination activity. However, while APOBEC1 and APOBEC3A have been used for RNA editing, they exhibit significant editing activity against single-stranded DNA and even detectable activity against double-stranded DNA, resulting in off-target editing at the genome level. Bacterial TadA catalyzes the editing of tRNA... Arg2 Deamination reaction on the stem-ring structure of the anticodon ( Figure 2 A); while the evolved Escherichia coli TadA (EcTadA) can act on single-stranded DNA and has low activity on single-stranded RNA, but no activity on double-stranded DNA.

[0286] The inventors of this application attempt to utilize TadA as an effector protein for a programmable RNA editor. To perform programmable RNA base editing using TadA, the inventors of this application combine a single TadA with a Cas13b (dCas13b) that targets RNA but lacks nuclease activity. Figure 2 B) were fused together. To assess the efficiency of RNA A-to-I editing, a reporter vector containing UAG was constructed ( Figure 2 C), which contains a UAG premature stop codon located within the protein linker sequence connecting two fluorescent proteins (mCherry and EGFP). The adenine-specific deamination reaction within the UAG stop codon reactivates EGFP expression, thus allowing for the quantification of A-to-I editing efficiency. Following standard Cas13b crRNA design principles, two crRNAs were generated, located upstream (-38) or downstream (+40) of the adenine in the stop codon, respectively. Figure 2 C). The programmable RNA A-to-I editing capabilities of wild-type EcTadA (SEQ ID NO: 3), TadA7.10 (SEQ ID NO: 2), and TadA8e (SEQ ID NO: 1) were then evaluated. The results showed that TadA7.10 and TadA8e exhibited A-to-I conversion rates of 20%–48% at the target sites, while WT-TadA and completely inactivated TadA (E59A) showed only background-level A-to-I editing. Figure 2 D). Therefore, it is concluded that TadA7.10 and TadA8e, rather than WT-TadA, can be used for programmable RNA A-to-I editing.

[0287] II. Use circularizable guide RNA (LF-gRNA) to construct editing windows.

[0288] Building upon the discovery that TadA7.10 and TadA8e can be used for programmable RNA A-to-I editing, the inventors of this application further sought to construct an editing window for TadA. The inventors designed a crRNA to form a single-stranded RNA loop surrounded by double-stranded RNA within the target transcript. This application names this non-standard crRNA design as a circularizable guide RNA (LF-gRNA). Figure 1 A and Figure 2 B). Furthermore, it was confirmed that the induced single-stranded RNA loop can serve as a highly efficient substrate for TadA, while adjacent nucleotides located within the double-stranded RNA region are expected to be shielded and protected from deamination.

[0289] First, the editing efficiency mediated by LF-gRNA was evaluated. LF-gRNA-10 nt was designed to induce a 10-nucleotide single-stranded RNA loop on the target mRNA, with the intended target A located at position 6. This single-stranded RNA loop was surrounded by symmetrical complementary double-stranded RNA arms (15 nucleotides on the left + 15 nucleotides on the right). Figure 3 A). Compared to conventional crRNA, LF-gRNA-10nt showed an average 2.3-fold increase in EGFP expression signal ( Figure 3 B). Next-generation sequencing (NGS) analysis confirmed that the combination of LF-gRNA with TadA7.10 and TadA8e achieved A-to-I editing efficiencies of up to 62% and 73%, respectively. Figure 3 C). Further testing of various LF-gRNAs with different arm length combinations revealed that complementary arms of symmetrical RNAs ranging from 24 bp to 36 bp achieved highly efficient editing (>30%). Figure 4 A). Furthermore, LF-gRNAs with asymmetric arms also showed good editing efficiency (20%-65%). Figure 4 B).

[0290] Next, we investigated how the position of base A within a 10-nucleotide single-stranded RNA loop affects editing efficiency. A series of LF-gRNAs were designed to target base A at positions 1 through 10 of the RNA loop. Figure 4C). Data shows that TadA7.10 exhibits efficient A-to-I conversion (>10%) at positions 4-8 of the 10-nucleotide ring, with the major window (A-to-I editing rate >30%) located at positions 5-8. TadA-8e shows a wider window, covering positions 3-9, with the major window located at positions 4-9 ( Figure 4 C).

[0291] III. Control the width of the editing window by changing the ring size.

[0292] Further attempts were made to control the width of the editing window by manipulating the size of the RNA loop induced by LF-gRNA. To facilitate observation of the editing window, a "reporter vector-4A" containing a 4-linked adenine (A) targeting site was constructed. Figure 5 A). Various LF-gRNAs were designed for the reporter vector-4A, with induced loop sizes ranging from 4 nucleotides (LF-gRNA-4 nt) to 14 nucleotides (LF-gRNA-14 nt). The editing efficiency of A within the ssRNA loop and adjacent dsRNA arms was detected using NGS.

[0293] The results showed that all LF-gRNAs capable of inducing RNA loops of at least 6 nucleotides in size exhibited significant A-to-I editing, with TadA at 7.10 (the most efficient A at 5.6%-23.3%). Figure 5 B) and TadA8e (the most efficient A at 11.9%-33.8%), Figure 5 C). LF-gRNA-4 nt showed low A-to-I editing efficiency (TadA7.10: 0.6%-2.9%; TadA8e: 1.2%-6.1%), revealing a potential lower limit for RNA loop size. The editing window expands with increasing loop size. Specifically, LF-gRNA-6 nt forms a 1-2 nucleotide narrow window, with the most efficient editing site located at position 5 of the loop. LF-gRNA-8 nt expands the window size to 3-4 nucleotides, with the most efficient editing site also located at position 5. Furthermore, LF-gRNA-14 nt can edit multiple A's within the loop, forming editing windows of 7 and 10 nucleotides wide in TadA7.10 and TadA8e, respectively. Figure 5 B and Figure 5 C). Therefore, this application successfully controls the width of the editing window by manipulating the size of the ssRNA loop induced by LF-gRNA.

[0294] IV. Identify novel TadA mutants for precise and efficient A-to-I editing.

[0295] To investigate the editing specificity of TadA7.10 and TadA8e under LF-gRNA design, we analyzed A-to-I editing events located within the 100 bp region of the reporter transcript. We found numerous A-to-I off-target sites, located outside the double-stranded RNA arm region, and even within the double-stranded RNA arm region itself. Figure 6 A). Therefore, there is an urgent need to identify novel TadA mutants to reduce off-target reactions.

[0296] The inventors of this application attempted to guide the design of mutants based on the TadA structure of the RNA base editor. Since there is no structural information to elucidate how the evolved TadA recognizes and catalyzes circular RNA substrates, the inventors used the structure of ABE8e in its single-stranded DNA-binding state, aligned with the structure of Staphylococcus aureus TadA-tRNA, to guide the redesign of TadA8e, aiming to reduce off-target RNA editing. To assess the precision of the mutants, the efficiency of targeting the target was divided by the average editing efficiency within a 100 bp range around the target on the transcript, with the precision of TadA8e normalized to 1. The inventors designed four strategies to influence the substrate binding and catalytic activity of TadA (…). Figure 6 B).

[0297] (1) First, we focused on residues that interact with RNA substrates (R74, R98, R129, R64, V88, H96, V120). These residues were replaced with representative amino acids with different properties to weaken non-specific RNA binding affinity. Among them, R98A retained high target editing activity (>50%) and the precision was improved compared with the original TadA8e (Table 2).

[0298] (2) Next, the residues (R111, L145, Y149, Q154, V155) at the TadA dimer interface were examined. These residues were replaced with representative amino acids with different properties to weaken TadA's ability to form dimers. Studies have shown that the formation of TadA dimers can promote TadA's deamination activity. Among them, mutations at the R111 site can significantly reduce bystander editing activity, although targeted editing activity is weakened (Table 1).

[0299] (3) Subsequently, saturation mutations were performed on N108 and V106, residues that appeared in the early stages of directed evolution of TadA7.10 (for A-to-I editing on single-stranded DNA). The inventors of this application found that most N108 mutations significantly reduced A-to-I editing activity, while most V106 variants showed little change (Table 1). This indicates that the N108D mutation not only initiated single-stranded DNA editing activity during directed evolution but also had a significant impact on ssRNA activity.

[0300] (4) Subsequently, residues in the adenine-binding pocket (V28, V30, N46, F84) were tested, as these residues may affect the catalytic activity of evolved TadA. As expected, several mutations at the edge of the active pocket (V28D; V30W, Q, R and all N46 variants) significantly reduced A-to-I editing activity. Interestingly, four mutations in F84 showed significantly reduced bystander editing but retained some targeting efficiency, resulting in high specificity values ​​(Table 2). Inspired by these observations, saturation mutations were performed on F84, and F84I, F84V, and F84H were found to balance efficiency and specificity well (Table 2). Further analysis of A-to-I editing events in all amplicon sequencing (amplicon sequencing was performed in regions approximately 100 bp upstream and downstream of the target site) again demonstrated the high specificity of these F84 variants. Figure 6 C). In summary, this application identified F84 as the key residue determining the specificity of TadA RNA editing by screening approximately 20 residue sites in TadA for mutations.

[0301] Table 2. Evaluation of the targeting efficiency and accuracy of TadA mutants

[0302] To further enhance its activity, a mutant V82T was superimposed on TadA8e(F84I), showing a ~1.5-fold increase in activity. Figure 6 D). Furthermore, we analyzed the sequence of TadA-8.20, a highly active TadA mutant ( Figure 6E), and introduced I76Y, Y147R, and Q154R into TadA8e (F84I-V82T). Results showed that the triple mutant F84I-V82T-Y147R and the quintuplet mutant F84I-V82T-I76Y-Y147R-Q154R achieved targeted editing efficiencies of 62% and 76%, respectively. Further analysis of A-to-I editing events in all amplicon sequencing (amplicon sequencing was performed on regions approximately 100 bp upstream and downstream of the target site) further demonstrated the high specificity of these F84 variants. Figure 6 F and G). Therefore, we refer to the novel TadA8e mutants linked to dCas13b and carrying triple mutations (F84I-V82T-Y147R) and quintuple mutations (F84I-V82T-I76Y-Y147R-Q154R) as AIM-A and AIM-Amax, respectively.

[0303] To further evaluate whether AIM-A possesses controllable editing capabilities within a certain range, a novel reporter gene containing isolated and continuous adenosine was constructed. Figure 7 A). Various LF-gRNAs were designed to target this reporter gene, inducing loops ranging in size from 4 nucleotides (LF-gRNA-4 nt) to 14 nucleotides (LF-gRNA-14 nt). Results showed that LF-gRNAs inducing loops of at least 6 nucleotides could all induce significant A-to-I editing via AIM-A (the most efficient adenosine editing rate was 11.4%-28.0%). Figure 7 B). LF-gRNA-4 nt resulted in a lower A-to-I editing rate, revealing the lower limit of the loop size. The number of editable bases increased with loop enlargement: LF-gRNA-6 nt mediated the editing of two adenosines, with the most efficient site located at position 4 of the loop; while LF-gRNA-14 nt edited 6 adenosines within the loop, covering a window up to 10 nucleotides wide. Figure 7 B). All LF-gRNAs showed low levels of background editing within the double-stranded RNA arm regions, indicating that the bases in these regions were effectively protected.

[0304] To demonstrate selective editing of adenosine in reporter genes, various LF-gRNAs with a fixed 7-nt loop were designed, allowing the loop to gradually shift one nucleotide towards the 5' end. Figure 7 C). The results showed that, based on different designs of LF-gRNA, it was possible to selectively edit a single isolated adenosine, or preferentially edit two or more consecutive adenosines. Figure 7 D). Therefore, it can be concluded that AIM can edit a specified number and position of bases.

[0305] V. Identify novel TadA mutants for precise and efficient C-to-U editing.

[0306] Furthermore, the inventors of this application have engineered the TadA protein to achieve precise and efficient C-to-U editing. First, a Reporter-CAC quantification model was constructed to assess the level of C-to-U editing (…). Figure 8 A). However, the results showed that TadA8e had very weak C-to-U activity on RNA (~2%). Figure 8 B).

[0307] Subsequently, the inventors of this application sought novel TadA variants to achieve efficient C-to-U RNA editing. They initially hypothesized that a smaller cytosine loop (compared to a larger adenine loop) should be located more deeply within the active pocket to achieve efficient deamination; by analyzing the structures of the binding substrates (EcTadA-ssDNA and SaTadA-tRNA), three loop regions that might promote deeper cytosine repositioning were identified. Figure 8 C). Mutations were performed on E27, V28, P29, V30 (in ring I), N46, A48 (in ring II), and K110, R111 (in ring III), and the editing efficiency of the target C-to-U and adjacent A-to-I variants was examined. Screening over 130 variants revealed that A48F and A48G exhibited higher C-to-U activity than TadA-8e (4.6% and 2.6%, respectively, Table 3). However, these variants still possessed significant A-to-I activity (approximately 40%).

[0308] To obtain a pure C-to-U editor, A-to-I activity must be eliminated. During the screening process, the inventors noted that N46 is crucial for the A-to-I RNA activity of TadA8e, as 18 out of 20 N46 mutations lost activity (<1%, Table 3). Notably, four N46 variants lacked A-to-I activity but still exhibited weak and detectable C-to-U activity (>0.2%); among them, N46P performed best, showing almost no A-to-I activity (0.04%). Therefore, combining A48F or A48G with N46P to achieve efficient and pure C-to-U RNA editing is very attractive. N46P-A48F and N46P-A48G showed 0.68% and 7.4% C-to-U activity, respectively, with no detectable A-to-I editing (…). Figure 8D). To enhance C-to-U activity, the compatibility of the hyperactive mutation of AIM-A with the N46P-A48G double mutation was examined. It was found that adding V82T, I76Y, Y147R, or Q154R mutations individually improved efficiency by approximately 2-fold; adding all four mutations together to N46P-A48G achieved approximately 38.1% C-to-U editing, equivalent to approximately a 5-fold increase in C-to-U efficiency. Figure 8 E). This application names this novel variant (N46P-A48G-V82T-I76Y-Y147R-Q154R) as AIM-C. Further analysis of A-to-I and C-to-U editing events in all amplicon sequencing (amplicon sequencing was performed on a region approximately 100 bp upstream and downstream of the target site) demonstrated that AIM-C lacks A-to-I activity and exhibits highly accurate C-to-U editing (…). Figure 9 A).

[0309] To further enhance AIM-C activity, sequence alignment was performed on the TadA variant used for DNA CBE, and several regions with different amino acid residues compared to TadA8e were identified, particularly in the R26-V28 region and the TadA dimerization interface (H96 and Y73). Figure 9 B). Through single and combined mutations, we identified four mutations (R26G, V28A, Y73P, and H96N), which individually showed an improvement of 1.2 to 1.4 times (B). Figure 9 C). Combining the four mutations improved efficiency by 2.1 times compared to the original AIM-C, achieving 66% editing efficiency in Reporter-CAC. Figure 9 C). This application names this new variant (AIM-C with the addition of R26G / V28A / Y73P / H96N mutations) as AIM-Cmax. Further analysis of A-to-I and C-to-U editing events in all amplicon sequencing (amplicon sequencing of approximately 100 bp upstream and downstream of the target site) demonstrated that AIM-Cmax lacks A-to-I activity and exhibits highly accurate C-to-U editing (…). Figure 9 D).

[0310] Table 3. Statistics on A-to-I and C-to-U efficiencies of novel TadA mutants

[0311] Furthermore, the ability of AIM-C to achieve controllable C-to-U editing within a certain range was further tested. The results showed that C-to-U editing was achievable with ring sizes ranging from 7 nt to 17 nt (the most efficient cytidine editing rate was 7.5%–23.5%); while a 5 nt ring exhibited lower activity (0.5%). Consistent with AIM-A, the number of editable bases increased with ring expansion: a 7 nt ring mediated a single cytidine edit at position 5, while extending the ring size to 17 nt enabled editing between positions 4 and 13, forming an editing window up to 10 nt wide. Figure 10 Overall, these results demonstrate that AIM-C enables controlled C-to-U editing within user-defined areas.

[0312] VI. Identify novel TadA mutants to achieve simultaneous A-to-I and C-to-U editing on a single transcript Simultaneous editing of A and C bases provides new opportunities for RNA information manipulation. For example, compared to single-function editing, it allows for an additional 30 codon changes, resulting in 10 amino acid substitutions. Therefore, a further objective of this application is to develop a bifunctional AIM system capable of simultaneously achieving A-to-I and C-to-U alterations.

[0313] During the screening of AIM-C, the inventors of this application discovered that TadA8e (A48G) exhibited strong A-to-I activity and relatively weak but clearly defined C-to-U activity (Table 3), providing a good starting point for the development of a bifunctional base editor. Since N46 is particularly important for A-to-I activity, a saturation mutation was performed on the N46 site of TadA8e (A48G) to balance the activities of A and C. N46C-A48G was identified, exhibiting moderate but most balanced A&C activity (A-to-I: 6.8%; C-to-U: 3.4%). Figure 11 A). To improve editing efficiency, a highly active mutation was integrated into N46C-A48G, resulting in a six-mutant (N46C-A48G-V82T-I76Y-Y147R-Q154R), with an editing rate of approximately 60% A-to-I and approximately 40% C-to-U. Figure 11 B). These six mutants fused with dCas13b were named the AIM-A&C system. The frequency of simultaneous A&C editing was further investigated. The results showed that AIM-A&C exhibited high levels of simultaneous A&C editing within a single RNA molecule, accounting for 40.2% of all transcripts (B). Figure 11 C). Therefore, AIM-A&C is highly efficient in simultaneous A&C editing.

[0314] Given that C-to-U editing efficiency in the AIM-A&C system is typically lower than A-to-I efficiency, the inventors of this application attempted to further improve C-to-U activity. By integrating four mutations (R26G, V28A, Y73P, and H96N) from the AIM-Cmax system into AIM-A&C, the C-to-U efficiency was improved by 1.4 times (from 40% to 56%). Figure 11 D), but A-to-I activity decreased significantly (from 63% to 9%). As mentioned above, N46 is crucial for A-to-I activity (Table 3). Therefore, the N46 mutation in the AIM-A&C(G)-R26G / V28A / Y73P / H96N mutant was restored to its original Asn residue. Interestingly, we observed that the A-to-I and C-to-U editing efficiencies reached equilibrium at this point (approximately 68.7% and 71.5%, respectively), representing increases of 1.1-fold and 1.7-fold compared to the original AIM-A&C. Figure 11 D). This application names this optimized system AIM-A&Cmax. This application further confirms that AIM-A&C can also achieve controlled editing. Using a 7 nt ring, AIM-A&C can precisely edit A5 and C6. Further expanding the ring size to 17 nt, it edits four A's and four C's spanning 11 nt. Figure 11 E, Figure 11 F).

[0315] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

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Claims

1. A TadA mutant or a functional fragment thereof, characterized in that, Compared to TadA containing the sequence shown in SEQ ID NO: 1, the TadA mutant contains mutations selected from the following: (1) V28R, (2) V30A, (3) I76Y, (4) V82T, (5) F84H, F84V, F84N, F84S, F84I, F84T, F84C or F84A, (6) R98E, (7) N108H, (8) R111C or R111L, (9) Y147R, (10) Q154R, (11) Any combination of (1) to (10).

2. The TadA mutant or its functional fragment as described in claim 1, characterized in that, Compared to TadA containing the sequence shown in SEQ ID NO: 1, the TadA mutant contains mutations selected from the following: (i) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and / or, (e) Q154R; (ii) (a) F84H, F84V or F84I, and (b) V82T; (iii) (a) F84H, F84V or F84I, (b) V82T, and (c) Y147R; (iv) (a) F84H, F84V or F84I, (b) V82T, (c) Y147R, (d) I76Y, and (e) Q154R; (v) F84I, V82T, Y147R, I76Y, and / or, Q154R; (vi) F84I and V82T; (vii) F84I, V82T and Y147R; (viii) F84I, V82T, Y147R, I76Y and Q154R.

3. The TadA mutant or its functional fragment as described in claim 1, characterized in that, Compared to TadA containing the sequence shown in SEQ ID NO:1, the TadA mutant contains mutations selected from: (i) F84I and V82T; (ii) F84I, V82T and Y147R; (iii) F84I, V82T, Y147R, I76Y and Q154R.

4. The TadA mutant or its functional fragment as described in claim 1, characterized in that, The TadA mutant contains an amino acid sequence as shown in SEQ ID NO: 4 or 5.

5. A polypeptide construct, characterized in that, The polypeptide construct includes a base deaminase and a guide RNA molecule binding domain linked to the base deaminase. The base deaminase is selected from the TadA mutant or its functional fragment as described in any one of claims 1-4.

6. The polypeptide construct of claim 5, characterized in that, The guide RNA molecule binding domain is selected from: Cas effector protein of the CRISPR system lacking nuclease activity, MS2 bacteriophagecoat protein (MCP), and bacteriophage Lambda N protein (λ N-peptide).

7. The polypeptide construct of claim 5, characterized in that, The polypeptide construct possesses one or more of the following characteristics: (1) In the polypeptide construct, the guide RNA molecule binding domain is linked to the N-terminus and / or C-terminus of the base deaminase; (2) The polypeptide construct further comprises a nuclear localization sequence (NLS) and / or a nuclear export sequence (NES). (3) The binding domain of the guide RNA molecule contains an amino acid sequence as shown in SEQ ID NO: 10; and / or, the backbone sequence of the guide RNA molecule contains a nucleotide sequence as shown in SEQ ID NO: 13; (4) The polypeptide construct is a fusion protein; (5) The polypeptide construct is a fusion protein containing an amino acid sequence as shown in SEQ ID NO: 15 or 16.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the TadA mutant or its functional fragment as described in any one of claims 1-4, or the polypeptide construct as described in any one of claims 5-7.

9. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 8.

10. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 8 or the vector of claim 9.

11. A composition, characterized in that, The composition comprises: (a) A first component, selected from: (i) a guide RNA molecule comprising a backbone sequence and a guide sequence, the guide sequence being capable of hybridizing with a target RNA sequence comprising bases to be edited; (ii) a nucleotide sequence encoding the guide RNA molecule; (iii) any combination of (i) and (ii); as well as, (b) A second component selected from: (i) a polypeptide construct comprising a base deaminase and a guide RNA molecule binding domain linked to the base deaminase, the guide RNA molecule binding domain being capable of binding to the backbone sequence of the guide RNA molecule; (ii) a nucleotide sequence encoding the polypeptide construct; (iii) any combination of (i) and (ii); The base deaminase is selected from the TadA mutant or its functional fragment as described in any one of claims 1-4.

12. The composition of claim 11, characterized in that, The guide sequence of the guide RNA molecule is designed to cause the target RNA sequence to form a single-stranded RNA loop region containing the bases to be edited after hybridization with the target RNA sequence.

13. The composition of claim 12, characterized in that, The target RNA sequence comprises: a target circular region containing the bases to be edited, a first region located at the 5' end of the target circular region, and a second region located at the 3' end of the target circular region; The guide sequence of the guide RNA molecule is designed to include a first region, a second region, and an optional third region located between the first and second regions, the second region being upstream of the first region; and the first region is complementary to a first region of the target RNA sequence, the second region is complementary to a second region of the target RNA sequence, and the third region is absent or cannot be complementary to a target circularizable region of the target RNA sequence.

14. The composition of claim 13, characterized in that, The composition has one or more of the following characteristics: (1) The third region of the guide RNA molecule does not exist; (2) The number of nucleotide residues in the third region of the guide RNA molecule is less than the number of nucleotide residues in the target circular region of the target RNA sequence; (3) The first region and the second region of the guide RNA molecule have the same or different numbers of nucleotide residues; (4) The single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule contains one or more bases to be edited; (5) The single-stranded RNA loop region formed after the target RNA sequence hybridizes with the guide RNA molecule consists of at least 3, at least 4, at least 5, or at least 6 nucleotide residues; (6) The polypeptide construct is defined as in any one of claims 5-7.

15. A complex, characterized in that, The complex comprises: (a) A guide RNA molecule, as defined in any one of claims 11-14; as well as, (b) A polypeptide construct as defined in any one of claims 5-7.

16. A carrier system comprising one or more carriers, said one or more carriers comprising: (1) A first nucleotide sequence encoding a guide RNA molecule, said guide RNA molecule as defined in any one of claims 11-14; and, (2) A second nucleotide sequence encoding a polypeptide construct as defined in any one of claims 5-7; in, The first nucleotide sequence and the second nucleotide sequence may be present on the same or different vectors.

17. A reagent kit, characterized in that, The kit comprises the TadA mutant or its functional fragment as described in any one of claims 1-4, the polypeptide construct as described in any one of claims 5-7, the nucleic acid molecule as described in claim 8, the vector as described in claim 9, the composition as described in any one of claims 11-14, the complex as described in claim 15, or the vector system as described in claim 16.

18. A delivery composition, characterized in that, The composition comprises a delivery vector and one or more of the following: a TadA mutant or a functional fragment thereof as described in any one of claims 1-4, a polypeptide construct as described in any one of claims 5-7, a nucleic acid molecule as described in claim 8, a vector as described in claim 9, a composition as described in any one of claims 11-14, a complex as described in claim 15, and a vector system as described in claim 16.

19. A cell characterized by, The cell comprises: the TadA mutant or a functional fragment thereof as described in any one of claims 1-4, the polypeptide construct as described in any one of claims 5-7, the nucleic acid molecule as described in claim 8, the vector as described in claim 9, the composition as described in any one of claims 11-14, the complex as described in claim 15, the vector system as described in claim 16, or the delivery composition as described in claim 18.

20. A method for editing target RNA outside of cells, characterized in that, The method includes, under conditions suitable for editing the target RNA, contacting the target RNA with one or more of the following: the TadA mutant or its functional fragment as described in any one of claims 1-4, the polypeptide construct as described in any one of claims 5-7, the nucleic acid molecule as described in claim 8, the vector as described in claim 9, the composition as described in any one of claims 11-14, the complex as described in claim 15, the vector system as described in claim 16, and the delivery composition as described in claim 18, thereby inducing deamination of the bases to be edited in the target RNA.

21. A method for editing target RNA within cells, characterized in that, The method comprises delivering the TadA mutant or its functional fragment as described in any one of claims 1-4, the polypeptide construct as described in any one of claims 5-7, the nucleic acid molecule as described in claim 8, the vector as described in claim 9, the composition as described in any one of claims 11-14, the complex as described in claim 15, the vector system as described in claim 16, and / or the delivery composition as described in claim 18 into a cell containing the target RNA, thereby inducing deamination of the base to be edited at the target site.

22. A cell characterized in that, The cells are cells or their progeny obtained by the method of claim 21, wherein the cells contain modifications not present in their wild type.

23. The TadA mutant or its functional fragment according to any one of claims 1-4, the polypeptide construct according to any one of claims 5-7, the nucleic acid molecule according to claim 8, the vector according to claim 9, the composition according to any one of claims 11-14, the complex according to claim 15, the vector system according to claim 16, the kit according to claim 17, or the delivery composition according to claim 18, for use in RNA editing, or for use in the preparation of a formulation for RNA editing.

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