WO2023015759A1 - 一种无pam限制的腺嘌呤碱基编辑器融合蛋白及应用 - Google Patents

一种无pam限制的腺嘌呤碱基编辑器融合蛋白及应用 Download PDF

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WO2023015759A1
WO2023015759A1 PCT/CN2021/131279 CN2021131279W WO2023015759A1 WO 2023015759 A1 WO2023015759 A1 WO 2023015759A1 CN 2021131279 W CN2021131279 W CN 2021131279W WO 2023015759 A1 WO2023015759 A1 WO 2023015759A1
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spry
seq
fusion protein
vector
fragment
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马旭
曹小芳
金孝华
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国家卫生健康委科学技术研究所
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Definitions

  • the disclosure belongs to the field of biomedicine, and relates to a PAM-free adenine base editor fusion protein and its application.
  • the CRISPR/Cas9 system was originally discovered in bacteria and archaea, and has been optimized and modified to form a powerful gene editing tool, which is widely used in the research of DNA knockout, knockin, and modification.
  • the CRISPR/Cas9 system is composed of Cas9 nuclease and sgRNA that recognizes the target sequence.
  • the sgRNA and the target sequence are complementary and paired to mediate the directional cutting of the genome by the Cas9 nuclease, resulting in double strand DNA breaks (double strand break, DSB).
  • the repair mechanism in the cell is homologous recombination (with a template) and non-homologous end joining (without a template) to achieve editing at the targeted site [1,2] .
  • ABE is formed by the fusion of adenine deaminase and nCas9. According to the data collected in the ClinVar database, 58% of the gene variations associated with human diseases are point mutations, and 47% of the disease-causing point mutations can be mediated by ABE. :T to G:C base conversion fixed [4] . A large number of studies have shown the application value of ABE in the field of disease repair.
  • the nonsense mutation of the disease-causing gene DMD can be repaired by viral delivery of ABE and corresponding sgRNA to the muscle of mice with Duchenne muscular dystrophy [5] ; In the liver of adult mice with hyperemia, the pathogenic splice site mutation was repaired, and the expression of FAH in hepatocytes was restored [6] .
  • the editing of ABE sites is limited by the editing window and PAM sequences.
  • the PAM sequence recognized by ABEmax is NGG. In order to further expand the editing range of base editors, ABEs that recognize different PAM sequences have emerged one after another.
  • the PAM sequence is xABE and ABE-NG [7] of NG, among which the most relaxed PAM restriction is ABEmax-SpRY published in March 2020, and its PAM sequence is NRN (R stands for A, G) and NYN (Y stands for C, T) [8] .
  • ABEmax-SpRY can target all sequences of the genome, but the editing efficiency of ABEmax-SpRY is low, and it does not solve the off-target problem of ABE at the transcriptome level, which limits the application of this base editor and needs to be improved and optimization.
  • the present disclosure provides an isolated mutant polypeptide, which sequentially includes an N-terminal fragment of SpRY(D10A), a TadA8e fragment and a C-terminal fragment of SpRY(D10A) polypeptide from N-terminus to C-terminus .
  • the amino acid sequence of the N-terminal fragment of the SpRY (D10A) protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% of the amino acid sequence shown in SEQ ID NO: 1 Or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity, or the amino acid sequence of the TadA8e fragment is identical to The amino acid sequence as shown in SEQ ID NO: 3 has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% % or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity, or the amino acid sequence of the C-terminal fragment of the SpRY (D10A) protein has at least the same amino acid sequence as shown in SEQ ID NO:5 90% or
  • the amino acid sequence of the N-terminal fragment of the SpRY (D10A) protein is shown in SEQ ID NO: 1
  • the amino acid sequence of the TadA8e fragment is shown in SEQ ID NO: 3
  • the SpRY (D10A) protein The amino acid sequence of the C-terminal fragment is shown in SEQ ID NO:5.
  • the nucleotide sequence encoding the N-terminal fragment of the SpRY (D10A) protein has at least 90% or at least 91% or at least 92% or at least 93% of the nucleotide sequence as shown in SEQ ID NO: 2 % or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity.
  • nucleotide sequence encoding the N-terminal fragment of the SpRY(D10A) protein is shown in SEQ ID NO:2.
  • the nucleotide sequence encoding the TadA8e fragment has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 90% or at least 93% or at least 94% of the nucleotide sequence as shown in SEQ ID NO:4 At least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, or 100% sequence identity.
  • the nucleotide sequence encoding the TadA8e fragment is shown in SEQ ID NO:4.
  • the nucleotide sequence encoding the C-terminal fragment of the SpRY (D10A) protein has at least 90% or at least 91% or at least 92% or at least 93% of the nucleotide sequence as shown in SEQ ID NO: 6 % or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity.
  • the nucleotide sequence encoding the C-terminal fragment of the SpRY(D10A) protein is shown in SEQ ID NO:6.
  • the mutant polypeptide is used for gene editing.
  • the editing window of the gene editing is about 3-10 bits.
  • the editing window of the gene editing is about 8-10 bits.
  • the mutant polypeptide comprises at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% of the sequence shown in SEQ ID NO: 13 or amino acid sequences with at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, or 100% sequence identity.
  • the mutant polypeptide comprises the sequence shown in SEQ ID NO: 13.
  • the present disclosure provides an isolated fusion protein comprising the mutant polypeptide.
  • the fusion protein containing the mutant polypeptide can target the whole genome, which broadens the editable range of the genome; it can cause base conversion from A:T to G:C more efficiently, with a large potential applications, including but not limited to the simulation or repair of pathogenic loci in genetic diseases.
  • the fusion protein containing the mutant polypeptide broadens the base editing window; and causes lower off-target at the transcriptome level, taking into account high efficiency and low off-target mutant form.
  • ABEmax-SpRY has no PAM restriction, effectively improves the targetable range of the genome, but its editing activity is not high.
  • the inventors use the adenine deaminase TadA8e in ABE8e to replace the adenine deaminase dimer in ABEmax-SpRY to construct 8e-SpRY.
  • 8e-SpRY can not only Causes base conversion more efficiently and also broadens the base editing window.
  • the inventors constructed four mutants based on 8e-SpRY, namely CE-8e-SpRY, V106W-SpRY, 8e-SpRY-HF and V106W-SpRY-HF.
  • CE-8e-SpRY is a mutant form with both high efficiency and low off-target.
  • the fusion protein further includes a connecting peptide, and the connecting peptide is located between the N-terminal fragment of the SpRY(D10A) protein and the TadA8e fragment, and/or between the TadA8e fragment and the C-terminal fragment of the SpRY(D10A) protein.
  • the connecting peptide sequence has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% of the amino acid sequence shown in SEQ ID NO:7 Or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, or 100% sequence identity.
  • the amino acid sequence of the connecting peptide is as shown in SEQ ID NO:7.
  • the nucleotide sequence encoding the connecting peptide has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 90% or at least 93% or at least 94% of the nucleotide sequence as shown in SEQ ID NO:8 At least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, or 100% sequence identity.
  • nucleotide sequence encoding the connecting peptide is shown in SEQ ID NO:8.
  • the fusion protein further includes a nuclear localization signal fragment.
  • the nuclear localization signal fragment is located at the N-terminal and/or C-terminal of the fusion protein.
  • the amino acid sequence of the nuclear localization signal fragment has at least 90% or at least 91% or at least 92% or at least 93% of the amino acid sequence shown in SEQ ID NO: 9 and/or SEQ ID NO: 11 Or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity.
  • amino acid sequence of the nuclear localization signal fragment is shown in SEQ ID NO:9 and/or SEQ ID NO:11.
  • the nucleotide sequence of the nuclear localization signal has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% of the nucleotide sequence as shown in SEQ ID NO: 10 or 12 % or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity.
  • nucleotide sequence of the nuclear localization signal is shown in SEQ ID NO: 10 or 12.
  • the nuclear localization signal segment includes about two copies.
  • the amino acid sequence of the fusion protein comprises at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% of the amino acid sequence shown in SEQ ID NO: 13 or Amino acid sequences having at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity.
  • the amino acid sequence of the fusion protein comprises the sequence shown in SEQ ID NO:13.
  • the fusion protein can achieve effective editing when the mutation site is located at positions 3-10 of the editing window.
  • the fusion protein can achieve effective editing when the mutation site is located at position 8-10 of the editing window.
  • the fusion protein can achieve effective editing when the mutation site is located at position 10 of the editing window.
  • the fusion protein is used for gene editing.
  • the editing window of the gene editing is about 3-10 bits.
  • the editing window of the gene editing is about 8-10 bits.
  • the present disclosure provides a polynucleotide encoding the mutant polypeptide or the fusion protein or its complementary sequence.
  • the polynucleotide is a nucleic acid construct.
  • the present disclosure provides a vector comprising the polynucleotide.
  • the vector is a recombinant expression vector.
  • the vector backbone is selected from pCMV or a derived plasmid thereof.
  • the pCMV-derived plasmid includes ABEmax-SpRY.
  • the vector comprises a plasmid or viral vector.
  • the vector is a plasmid or viral vector for expression in higher eukaryotic or prokaryotic cells.
  • the eukaryotic cells are selected from brain neuroma cells or embryonic kidney cells.
  • the human embryonic kidney cells comprise HEK293T cells.
  • the neuroma cells include N2a cells.
  • the present disclosure provides a method for producing the vector, adding a polynucleotide encoding the N-terminal fragment of the SpRY(D10A) protein, a polynucleotide encoding the TadA8e fragment, and an encoding SpRY(D10A) fragment to the backbone plasmid.
  • the polynucleotide of the C-terminal fragment of the protein thus obtaining the vector.
  • the vector comprises a plasmid or a viral vector.
  • the vector is a plasmid or viral vector for expression in higher eukaryotic or prokaryotic cells.
  • nucleotide sequence encoding the N-terminal fragment of the SpRY(D10A) protein is shown in SEQ ID NO:2.
  • the nucleotide sequence encoding the TadA8e fragment is shown in SEQ ID NO:4.
  • the nucleotide sequence encoding the C-terminal fragment of the SpRY(D10A) protein is shown in SEQ ID NO:6.
  • the backbone plasmid comprises pCMV or its derivative plasmid ABEmax-SpRY.
  • the eukaryotic cells are selected from brain neuroma cells or embryonic kidney cells.
  • the human embryonic kidney cells comprise HEK293T cells.
  • the neuroma cells include N2a cells.
  • the method includes removing the TadA fragment from the derived plasmid ABEmax-SpRY, and replacing the amino acids at positions 1048 to 1063 in SpRY (D10A) with TadA8e to construct the recombinant expression vector.
  • the vector is a CE-8e-SpRY plasmid.
  • the present disclosure provides an sgRNA.
  • the sequence of the sgRNA includes the sequence shown in SEQ ID NO: 18-65.
  • the present disclosure provides an expression system comprising the expression vector or the exogenous polynucleotide integrated in its genome.
  • the expression system expresses the fusion protein or the exogenous sequence integrated in its genome expresses the fusion protein or the expression system expresses the polynucleotide containing the polynucleotide or the exogenous sequence integrated in its genome source of polynucleotides as described above.
  • the expression system further comprises RNA.
  • the RNA is a guide RNA.
  • the RNA is sgRNA.
  • the sequence of the sgRNA comprises at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least the sequence shown in SEQ ID NO: 18-65 Sequences with 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity.
  • the sequence of the sgRNA includes the sequence shown in SEQ ID NO: 18-65.
  • the present disclosure provides a host cell comprising the polynucleotide or the vector or the expression system.
  • the present disclosure provides a composition comprising an effective amount of the mutant polypeptide, the fusion protein, the polynucleotide, the vector or the host cell at least one.
  • the composition is a kit.
  • the composition further comprises RNA.
  • the RNA is a guide RNA.
  • the RNA is sgRNA.
  • the sequence of the sgRNA comprises at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least the sequence shown in SEQ ID NO: 18-65 Sequences with 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity.
  • the sequence of the sgRNA includes the sequence shown in SEQ ID NO: 18-65.
  • the present disclosure provides any one of the mutant polypeptides or the fusion protein or the polynucleotide or the vector or the expression system or the host cell in Application in the preparation of medicines for treating genetic diseases.
  • the present disclosure provides any one of the mutant polypeptides or the fusion protein or the polynucleotide or the vector or the expression system or the host cell in Application in the preparation of gene editing reagents.
  • the editing window of the gene editing is about 3-10 bits.
  • the editing window of the gene editing is about 8-10 bits.
  • the present disclosure provides a base editing system, comprising any of the mutant polypeptides or the fusion protein or the polynucleotide or the vector or the expression system or The host cell.
  • the base editing system further comprises RNA.
  • the RNA is a guide RNA.
  • the RNA is sgRNA.
  • the present disclosure provides a method for gene editing, using the base editing system to perform gene editing.
  • the editing window of the gene editing is about 3-10 bits.
  • the editing window of the gene editing is about 8-10 bits.
  • the present disclosure provides a method for recombinantly producing any of the mutant polypeptides or the fusion protein, comprising the step of: introducing the vector into a host cell to produce a transfected or infected The transfected or infected host cells are cultured in vitro, the cell culture is recovered and the mutant polypeptide or fusion protein produced is optionally purified.
  • the present disclosure provides a method for preparing the mutant polypeptide or the fusion protein, comprising: (1) adding a polynucleotide encoding the N-terminal fragment of the SpRY (D10A) protein to the backbone plasmid , a polynucleotide encoding a TadA8e fragment and a polynucleotide encoding a SpRY (D10A) protein C-terminal fragment, thereby obtaining a recombinant expression vector; (2) transfecting the recombinant expression vector to a host cell to express the mutant Polypeptide or said fusion protein.
  • nucleotide sequence encoding the N-terminal fragment of the SpRY(D10A) protein is shown in SEQ ID NO:2.
  • the nucleotide sequence encoding the TadA8e fragment is shown in SEQ ID NO:4.
  • the nucleotide sequence encoding the C-terminal fragment of the SpRY(D10A) protein is shown in SEQ ID NO:6.
  • the backbone plasmid comprises pCMV or its derivative plasmid ABEmax-SpRY.
  • the method includes removing the TadA dimer from the derived plasmid ABEmax-SpRY, and replacing the amino acids at positions 1048 to 1063 in SpRY (D10A) with TadA8e to construct the recombinant expression vector.
  • the vector is a plasmid or viral vector.
  • the vector is a plasmid or viral vector for expression in higher eukaryotic or prokaryotic cells.
  • the eukaryotic cells are selected from brain neuroma cells or embryonic kidney cells.
  • the human embryonic kidney cells comprise HEK293T cells.
  • the neuroma cells include N2a cells.
  • the present disclosure provides a method for producing the vector, comprising the steps of: introducing the vector into a suitable cell line, culturing the cell line under suitable conditions so that the target vector can be produced, The resulting plasmid is recovered from the culture of the cell line and optionally purified.
  • the present disclosure provides a method for treating a genetic disease, comprising the following steps: administering to a subject a certain amount of the mutant polypeptide effective for the genetic disease, the fusion protein, At least one of said polynucleotides, or any combination thereof.
  • the genetic disease comprises phenylketonuria.
  • the aforementioned proteins are isolated polypeptides.
  • the aforementioned polypeptides are isolated polypeptides.
  • the aforementioned nucleic acid is an isolated polynucleic acid.
  • Figure 1 is a schematic diagram of ABEmax-SpRY and 8e-SpRY and their mutants.
  • Figure 2- Figure 7 shows the editing efficiency of ABEmax-SpRY and 8e-SpRY when the PAM is NNN.
  • Figure 8 shows the statistical results of the multi-point editing efficiency of ABEmax-SpRY and 8e-SpRY.
  • Figure 9 shows the editing windows of ABEmax-SpRY and 8e-SpRY.
  • Figure 10- Figure 15 shows the editing efficiency of 8e-SpRY and its mutants when the PAM is NNN.
  • Figure 16 is the statistical result of multi-point editing efficiency of 8e-SpRY and its mutants.
  • Figure 17 shows the statistical results of multi-point editing efficiency of 8e-SpRY and its mutants when PAM is NAN, NGN, NCN and NTN.
  • Figure 18 shows the editing windows of 8e-SpRY and its mutants.
  • Figure 19 shows the DNA targeting editing efficiency of ABEmax-SpRY, 8e-SpRY and their mutants.
  • Figure 20 shows the amount of RNA off-target of ABEmax-SpRY, 8e-SpRY and their mutants.
  • Fig. 21 is a schematic diagram of RNA off-target of ABEmax-SpRY, 8e-SpRY and its mutant A-to-I.
  • Figure 22 is the sanger sequencing diagram of the genotype of the PKU 728 G>A cell model and the sanger sequencing diagram of the repair efficiency of 8 repair sgRNAs.
  • Figure 23 is a histogram of the repair efficiency of three sgRNAs with repair effects.
  • Figure 24 is a sanger sequencing map of the repair efficiency of the other three ABE mutants.
  • Phenylketonuria is a kind of inborn metabolic disease. This disease is caused by the defect of phenylalanine hydroxylase (PAH) in the liver due to the mutation of chromosomal gene, which leads to the metabolism of phenylalanine (PA). due to obstacles.
  • PAH phenylalanine hydroxylase
  • V106W mutation was performed on TadA8e in 8e-SpRY to obtain V106W-SpRY, wherein the nucleotide sequence of TadA8e V106W is shown in SEQ ID NO:15, and the nucleotide sequence of SpRY D10A is shown in SEQ ID NO:16.
  • V106W mutation was performed on TadA8e in 8e-SpRY-HF to obtain V106W-SpRY-HF.
  • Both ends of 8e-SpRY and its mutants carry a nuclear localization signal, and the nuclear localization signal is bpNLS (the nucleotide sequence of the nuclear localization signal is shown in SEQ ID NO: 10; the amino acid sequence is shown in SEQ ID NO: 9) or SV40NLS (the nucleotide sequence of the nuclear localization signal is shown in SEQ ID NO: 12; the amino acid sequence is shown in SEQ ID NO: 11).
  • 8e-SpRY and its mutants are shown in Figure 1.
  • the amino acid sequence of the ABEmax-SpRY (fusion protein) is shown in SEQ ID NO: 67; the order of its composition from the N-terminal to the C-terminal is bpNLS, TadA dimer, SpRY D10A and bpNLS.
  • the nuclear localization signal carried at both ends can also be SV40NLS.
  • the amino acid sequence of the 8e-SpRY (fusion protein) is shown in SEQ ID NO: 68; the order of its composition from the N-terminal to the C-terminal is bpNLS, TadA8e, SpRY D10A and bpNLS.
  • the nuclear localization signal carried at both ends can also be SV40NLS.
  • the amino acid sequence of the CE-8e-SpRY (fusion protein) is shown in SEQ ID NO: 13 (the nucleotide sequence of the CE-8e-SpRY fusion protein is shown in SEQ ID NO: 14), and it consists of: From the N-terminal to the C-terminal, it includes bpNLS, the N-terminal fragment of SpRY (D10A), the TadA8e fragment, and the C-terminal fragment and bpNLS of the SpRY (D10A) polypeptide, between the SpRY (D10A) N-terminal fragment and the TadA8e fragment, between the TadA8e fragment and the There is a connecting peptide between the C-terminal fragments of SpRY (D10A), the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 7 (the nucleotide sequence of the coding CE-8e-SpRY connecting peptide is shown in SEQ ID NO: 8 ).
  • V106W-SpRY fusion protein
  • SEQ ID NO: 69 The amino acid sequence of the V106W-SpRY (fusion protein) is shown in SEQ ID NO: 69; the order of its composition from the N-terminus to the C-terminus is bpNLS, TadA8eV106W, SpRY D10A, and bpNLS, and nuclear localization signals can also be carried at both ends for SV40NLS.
  • the amino acid sequence of the 8e-SpRY-HF (fusion protein) is shown in SEQ ID NO:70; the order of its composition from the N-terminal to the C-terminal is bpNLS, TadA8e, SpRY D10A-HF, and bpNLS, and both ends carry nuclear
  • the positioning signal can also be SV40NLS.
  • V106W-SpRY-HF fusion protein
  • SEQ ID NO: 71 The amino acid sequence of the V106W-SpRY-HF (fusion protein) is shown in SEQ ID NO: 71; the order of its composition from the N-terminal to the C-terminal is bpNLS, TadA8eV106W, SpRY D10A-HF, and bpNLS, and both ends carry nuclear
  • the positioning signal can also be SV40NLS.
  • ABEmax-SpRY, 8e-SpRY and their mutants were used to edit endogenous sites in 293T cells.
  • sgRNAs were designed according to the PAM characteristics of SpRY nuclease, covering 16 different PAM sequences.
  • the sgRNA sequence is shown in SEQ ID NO: 18-65.
  • ACCG is added to the 5' end of the sgRNA sequence as the upstream sequence.
  • the sgRNA AAAC is added to the 5' end of the reverse complementary sequence as the downstream sequence, and the upstream and downstream sequences are annealed after the oligo is synthesized (program: 95°C, 5min; 95°C-85°C at-2°C/s; 85°C-25°C at-0.1 °C/s; hold at 16°C) and then ligated with the pGL3-U6-sgRNA (Addgene #51133) vector digested with BsaI (NEB: R3733L).
  • the enzyme digestion system is: pGL3-U6-sgRNA 2 ⁇ g; CutSmart buffer (NEB: B7204S) 6 ⁇ L; BsaI 1 ⁇ L; ddH 2 O to 60 ⁇ L, digest overnight at 37°C.
  • the ligation system is: 3 ⁇ L of Solution I (Takara: 6022Q); 1 ⁇ L of the vector after enzyme digestion; 6 ⁇ L of the annealed product, after 30 min of ligation at 16°C, transformation, picking, and identification. Shake the positive clones to extract the plasmid (Axygene: AP-MN-P-250G) and measure the concentration for later use.
  • HEK293T cells purchased from ATCC were inoculated and cultured in DMEM medium (Gibco: C11995500BT) supplemented with 10% serum (Gibco: 10270-106), which contained 1% double antibody (v/v) (Gibco: 15140122). The day before transfection, a 24-well plate was spread so that the cell density at the time of transfection reached about 80%, and the medium was changed 2 hours before transfection.
  • the amount of plasmid transfected in each well was 600ng of base editor plasmid and 300ng of sgRNA plasmid (sequence of sgRNA1-48 is shown in SEQ ID NO:18 ⁇ SEQ ID NO:65), and the plasmid was diluted in 40 ⁇ L of DMEM, Dilute 3 ⁇ L of EZ Trans cell transfection reagent (Shanghai Liji Biology: AC04L092) in 40 ⁇ L of DMEM, and finally add the diluted EZ Transfection Reagent to the diluted plasmid, mix well and let stand at room temperature for 15 minutes.
  • GFP is on the pGL3-U6-sgRNA vector.
  • a lysate 50mM KCl, 1.5mM MgCl 2 , 10mM Tris pH 8.0, 0.5% Nonidet P-40, 0.5% Tween 20, 100 ⁇ g/ml protease K was added to GFP-positive cell lysate was used as a template to amplify the target sequence.
  • the amplification system was: 2 ⁇ buffer (Vazyme: P505) 25 ⁇ L; dNTP 1 ⁇ L; Forward Primer (10 ⁇ mol/L) 1 ⁇ L; Reverse Primer (10 pmol/L) 1 ⁇ L; Cell lysate 1 ⁇ L; DNA polymerase (Vazyme: P505) 0.5 ⁇ L; ddH2O make up to 50 ⁇ L.
  • the sequences of Forward Primer and Reverse Primer are shown in SEQ ID NO: 72 to SEQ ID NO: 167 (corresponding to sgRNA1-48 respectively).
  • the amplified PCR product is purified with a recovery kit (Axygen: AP-PCR-250G).
  • the specific steps are: add 3 times the volume of PCR-A to the amplified product, mix it and add it to the adsorption column, 12000r/min Centrifuge for 1 min; discard the waste liquid, add 700 ⁇ L W2 (need to add a specified volume of ethanol) to the adsorption column, and centrifuge at 12000 r/min for 1 min; discard the waste-liquid, add 400 ⁇ L W2 to the adsorption column (need to add a specified volume of ethanol), 12000 r/min Centrifuge at 12000r/min for 1min; discard the waste liquid, and centrifuge at 12000r/min for 2min; open the lid to dry ethanol, add 28 ⁇ L ddH 2 O, centrifuge at 12000r/min for 1min to elute, and send the purified PCR product to Sanger sequencing or deep detection for analysis and editing Effect.
  • the relevant results are shown in Figure 2-9.
  • the results show that at all detection sites, covering PAMs of NAN, NGN, NCN and NTN, the editing efficiency of 8e-SpRY is significantly higher than that of ABEmax-SpRY; the statistical results of multi-point editing efficiency in Figure 8 show that 8e-SpRY is significantly Improved A to G editing efficiency.
  • the results of the editing window in Figure 9 show that the base editing window of ABEmax-SpRY is 5-6 positions; the base editing window of 8e-SpRY is 3-10 positions, and the window is wider.
  • FIGs 10-15 show the comparison results of the editing efficiency of 8e-SpRY mutants under NRN (R represents A or G), NYN (Y represents C or T) PAM; CE-8e-SpRY that inserts 8e into the middle of SpRY can It maintains its A-to-G editing activity very well.
  • the introduction of V106W into Tad8e's V106W-SpRY also did not significantly damage the original editing activity, but the introduction of four mutations into SpRY's 8e-SpRY-HF and V106W-SpRY-HF Then the editing activity is significantly reduced.
  • the statistical results of multi-point editing efficiency in NAN, NGN, NCN and NTN in Figure 17 show that the editing efficiency of CE-8e-SpRY in NGN and NTN has improved, and the editing efficiency of V106W-SpRY in the four PAMs has increased decreased, but not statistically significant.
  • the results of the editing window in Figure 18 show that V106W-SpRY maintains the same editing window as 8e-SpRY, both at 3-10 positions, and the high-activity editing window (editing efficiency greater than 40%) is 3-9 positions; CE-8e-SpRY Maintain the same editing window of 3-10 positions, the high-activity editing window (editing efficiency greater than 40%) is 3-10 positions, and the editing efficiency at 8-10 positions is higher than that of 8e-SpRY.
  • RNA off-target situation of ABEmax-SpRY, 8e-SpRY and their mutants in 293T cells was compared.
  • the sgRNA sequence used for RNA off-target detection is 5'-CTGGAACACAAAGCATAGAC-'3 (SEQ ID NO:66), which was constructed according to the plasmid construction method described in 2.1.
  • RNA isolater Total RNA extraction Reagent Vazyme: R401-01-AA
  • 200 ⁇ L chloroform was added, vigorously mixed up and down, left at room temperature for 3min, and placed at 4°C Centrifuge at 12000r/min for 15min; take 500 ⁇ L of the upper aqueous phase, add 500 ⁇ L of isopropanol, mix up and down, and centrifuge at 12000r/min at 4°C for 15min; discard the supernatant, add 1mL of 75% ethanol, gently invert several times to wash Precipitate, centrifuge at 12000r/min at 4°C for 5min; discard the supernatant, open the lid and dry for 5-10min, after the ethanol is completely volatilized, add 15 ⁇ L RNase-Free water to dissolve the precipitate, take 1 ⁇ L to measure the concentration
  • Figure 19 shows the editing efficiency of the 8th A on the DNA targeting site, ABEmax-SpRY, 8e-SpRY and its mutants can all cause effective editing, and the DNA targeting caused by 8e-SpRY and its mutants
  • the editing efficiency is comparable, and the editing efficiency of ABEmax-SpRY is relatively low.
  • the RNA off-target results in Figure 20 and Figure 21 show that, compared to other mutants of ABEmax-SpRY and 8e-SpRY, CE-8e-SpRY effectively reduces off-target editing at the transcriptome level.
  • the CE-8e-SpRY base editor obtained by the inventors can target the whole gene, while significantly improving the A-to-G editing efficiency, and effectively reducing the A-to-G editing efficiency at the transcriptome level. Off-target editing has great application potential.
  • design mut-sgRNA (SEQ ID NO: 168), and construct it according to the plasmid construction method described in 2.1.
  • Cell culture was carried out as described in 2.2.
  • a 24-well plate was spread one day before transfection, so that the cell density at the time of transfection reached about 80%, and the medium was changed 2 hours before transfection.
  • the amount of plasmid transfected in each well is 600ng of base editor plasmid and 300ng of sgRNA plasmid. Dilute the plasmid in 40 ⁇ L of DMEM, and dilute 3 ⁇ L of EZ Trans cell transfection reagent (Shanghai Lee Kee Bio: AC04L092) in 40 ⁇ L of In DMEM, finally add the diluted EZ transfection reagent to the diluted plasmid, mix well and let stand at room temperature for 15 minutes.
  • One positive cell was sorted from the well, and placed in an incubator for 14 days to identify the monoclonal genotype of the cell.
  • the monoclonal cells in each well part of the cells were centrifuged and added to the lysate (50mM KCl, 1.5mM MgCl 2 , 10mM Tris pH 8.0, 0.5% Nonidet P-40, 0.5% Tween 20, 100 ⁇ g/ml protease K).
  • the lysate 50mM KCl, 1.5mM MgCl 2 , 10mM Tris pH 8.0, 0.5% Nonidet P-40, 0.5% Tween 20, 100 ⁇ g/ml protease K.
  • the amplification system is: 2 ⁇ buffer (Vazyme: P505) 25 ⁇ L; dNTP 1 ⁇ L; Forward Primer (10 ⁇ mol/L) 1 ⁇ L; Reverse Primer (10 pmol/L) 1 ⁇ L; cell lysate 1 ⁇ L; DNA polymerase (Vazyme: P505) 0.5 ⁇ L; ddH 2 O to make up to 50 ⁇ L.
  • the forward primer sequence is: 5'-gtccctgggcagttatgtgtac-3' (SEQ ID NO: 177), and the reverse primer sequence is 5'-caactggtagctggaggacag-3' (SEQ ID NO: 178).
  • the PAH 728 G>A pure and mutant cells were selected as the human PAH 728 G>A cell model.
  • CE-8e-SpRY has high editing efficiency at positions 3-10, and recognizes PAM as NNN. According to the editing window of CE-8e-SpRY and the characteristics of PAM, the inventors designed 8 Recs around the pathogenic mutations that need to be repaired.
  • -sgRNA SEQ ID NO:169- ⁇ SEQ ID NO:176
  • Transfection was performed according to the cell culture and transfection method described in 2.2.
  • the repair efficiency was detected according to the method for detecting editing efficiency described in 2.3.
  • the repair sgRNA of these three base editors is SEQ ID NO: 173, which was constructed according to the plasmid construction method described in 2.1, and constructed according to the method described in 2.2.
  • the cell culture and transfection methods described above were used for transfection, and the repair efficiency was detected according to the method for detecting editing efficiency described in 2.3.
  • the results are shown in Figure 24.
  • the three base editors have no obvious repair effect on the 728 G>A mutation site.

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Abstract

一种无PAM限制的腺嘌呤碱基编辑器融合蛋白及应用。提供了一种突变体多肽，所述多肽自N端至C端依次包括SpRY(D10A)的N端片段、TadA8e片段和SpRY(D10A)多肽的C端片段。含有所述突变体多肽的融合蛋白可以靶向全基因组，拓宽了基因组的可编辑范围；能够更高效率地引起A:T到G:C的碱基转换，具有很大的应用潜力，包括但不限于基因疾病致病性位点的模拟或修复；且造成转录组水平上的脱靶更低，兼顾效率高和脱靶低的突变体形式。

Description

一种无PAM限制的腺嘌呤碱基编辑器融合蛋白及应用 技术领域
本公开属于生物医药领域，涉及一种无PAM限制的腺嘌呤碱基编辑器融合蛋白及应用。
背景技术
CRISPR/Cas9系统最初发现于细菌和古细菌中，后经过优化和改造形成强大的基因编辑工具，被广泛应用于DNA的敲除、敲入、修饰等研究。CRISPR/Cas9系统由Cas9核酸酶和识别靶序列的sgRNA两部分组成，sgRNA与靶序列互补配对介导Cas9核酸酶对基因组的定向切割，造成双链DNA断裂(double strand break,DSB)后，利用细胞中的修复机制同源重组(有模板情况下)和非同源末端连接(无模板情况下)实现靶向位点的编辑 [1,2]。随后，David Liu等人构建RuvC结构域失活的nickase Cas9(nCas9)，并在此基础上开发出单碱基编辑系统即胞嘧啶碱基编辑器(cytosine base editor,CBE)和腺嘌呤碱基编辑器(adenine base editor,ABE)，两种碱基编辑器在不引起DNA双链断裂的情况下可分别实现C:G到T:A、A:T到G:C的碱基转换，极大提升了单碱基编辑的效率和安全性 [2,3]。
ABE由腺嘌呤脱氨酶和nCas9融合而成，根据ClinVar数据库收录的数据，与人类疾病相关的基因变异有58％是点突变，而其中47％的致病点突变可通过ABE介导的A:T到G:C的碱基转换得到修复 [4]。已有大量研究显示ABE在疾病修复领域的应用价值。例如，通过病毒递送ABE和相应的sgRNA至杜氏肌营养不良的小鼠肌肉中，可修复致病基因DMD的无义突变 [5]；通过脂质纳米颗粒递送mRNA形式的ABE至患酪氨酸血症的成年小鼠肝脏中，修复了致病性的剪切位点变异，恢复肝细胞中FAH的表达 [6]。但ABE对于位点的编辑受到编辑窗口和PAM序列的限制，应用最为广泛的ABEmax识别的PAM序列为NGG，为进一步拓展碱基编辑器的编辑范围，识别不同PAM序列的ABE相继出现，如识别PAM序列为NG的xABE和ABE-NG [7]，其中PAM限制最为宽松的是2020年3月发表的ABEmax-SpRY，其PAM序列为NRN(R代表A、G)和NYN(Y代表C、T) [8]。ABEmax-SpRY可以靶向基因组的所有序列，但是ABEmax-SpRY编辑效率较低，且未解决ABE存在的在转录组水平上的脱靶问题，限制了该碱基编辑器的应用，需要对其进行改进和优化。
发明内容
一些实施方案中，本公开了提供了一种分离的突变体多肽，所述多肽自N端至C端依次包括SpRY(D10A)的N端片段、TadA8e片段和SpRY(D10A)多肽的C端片段。
一些实施方案中，所述SpRY(D10A)蛋白N端片段的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性，或所述TadA8e片段的氨基酸序列与如SEQ ID NO:3所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性，或所述SpRY(D10A)蛋白C端片段的氨基酸序列与如SEQ ID NO:5所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，所述SpRY(D10A)蛋白N端片段的氨基酸序列如SEQ ID NO:1所示，所述TadA8e片段的氨基酸序列如SEQ ID NO:3所示，所述SpRY(D10A)蛋白C端片段的氨基酸序列如SEQ ID NO:5所示。
一些实施方案中，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列与如SEQ ID NO:2所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示。
一些实施方案中，编码所述TadA8e片段的核苷酸序列与如SEQ ID NO:4所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示。
一些实施方案中，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列与如SEQ ID NO:6所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示。
一些实施方案中，所述突变体多肽用于基因编辑。
一些实施方案中，所述基因编辑的编辑窗口约为3～10位。
一些实施方案中，所述基因编辑的编辑窗口约为8～10位。
一些实施方案中，所述突变体多肽包含与如SEQ ID NO:13所示的序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性的氨基酸序列。
一些实施方案中，所述突变体多肽包含如SEQ ID NO:13所示的序列。
一些实施方案中，本公开提供了一种分离的融合蛋白，包含所述的突变体多肽。
在一些实施方案中，含有所述突变体多肽的融合蛋白可以靶向全基因组，拓宽了基因组的可编辑范围；能够更高效率地引起A:T到G:C的碱基转换，具有很大的应用潜力，包括但不限于基因疾病致病性位点的模拟或修复。一些实施例中，含有所述突变体多肽的融合蛋白拓宽了碱基编辑窗口；且造成转录组水平上的脱靶更低，兼顾效率高和脱靶低的突变体形式。
一些实施方案中，相较于现有的腺嘌呤碱基编辑器突变体，ABEmax-SpRY无PAM限制，有效提高了基因组的可靶向范围，但是其编辑活性不高。
在一些实施方案中，发明人用ABE8e中的腺嘌呤脱氨酶TadA8e代替ABEmax-SpRY中的腺嘌呤脱氨酶二聚体，构建形成8e-SpRY，8e-SpRY相比于ABEmax-SpRY不仅能够更高效率地引起碱基转换，还拓宽了碱基编辑窗口。
在一些实施方案中，发明人又在8e-SpRY的基础上构建了4种突变体，分别为CE-8e-SpRY、V106W-SpRY、8e-SpRY-HF和V106W-SpRY-HF。综合评价编辑效率和脱靶后，CE-8e-SpRY是兼顾效率高和脱靶低的突变体形式。
一些实施方案中，所述融合蛋白还包括连接肽，连接肽位于SpRY(D10A)蛋白N端片段与TadA8e片段之间，和/或位于TadA8e片段与SpRY(D10A)蛋白C端片段之间。
一些实施方案中，所述连接肽序列与如SEQ ID NO:7所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，所述连接肽氨基酸序列如SEQ ID NO:7所示。
一些实施方案中，编码所述连接肽的核苷酸序列与如SEQ ID NO:8所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，编码所述连接肽的核苷酸序列如SEQ ID NO:8所示。
一些实施方案中，所述融合蛋白还包括核定位信号片段。
一些实施方案中，所述核定位信号片段位于所述融合蛋白的N端和/或C端。
一些实施方案中，所述核定位信号片段的氨基酸序列与如SEQ ID NO:9和/或SEQ ID NO:11所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，所述核定位信号片段的氨基酸序列如SEQ ID NO:9和/或SEQ ID NO:11所示。
一些实施方案中，所述核定位信号的核苷酸序列与如SEQ ID NO:10或12所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性。
一些实施方案中，所述核定位信号的核苷酸序列如SEQ ID NO:10或12所示。
一些实施方案中，所述核定位信号片段包括约两个拷贝。
一些实施方案中，所述融合蛋白的氨基酸序列包含与如SEQ ID NO:13所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性的氨基酸序列。
一些实施方案中，所述融合蛋白的氨基酸序列包含如SEQ ID NO:13所示的序列。
一些实施方案中，所述融合蛋白在突变位点位于编辑窗口第3-10位时可实现有效的编辑。
一些实施方案中，所述融合蛋白在突变位点位于编辑窗口第8-10位时可实现有效的编辑。
一些实施方案中，所述融合蛋白在突变位点位于编辑窗口第10位时可实现有效的编辑。
一些实施方案中，所述融合蛋白用于基因编辑。
一些实施方案中，所述基因编辑的编辑窗口约为3～10位。
一些实施方案中，所述基因编辑的编辑窗口约为8～10位。
一些实施方案中，本公开提供了一种编码所述的突变体多肽或所述的融合蛋白的多核苷酸或其互补序列。
一些实施方案中，所述多核苷酸为核酸构建体。
一些实施方案中，本公开提供了一种载体，所述载体包含所述的多核苷酸。
一些实施方案中，所述载体为重组表达载体。
一些实施方案中，所述载体骨架选自pCMV或其衍生质粒。
一些实施方案中，所述pCMV的衍生质粒包括ABEmax-SpRY。
一些实施方案中，所述载体包括质粒或病毒载体。
一些实施方案中，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体。
一些实施方案中，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞。
一些实施方案中，所述人胚胎肾细胞包括HEK293T细胞。
一些实施方案中，所述脑神经瘤细胞包括N2a细胞。
一些实施方案中，本公开提供了一种产所述载体的方法，在骨架质粒中加入编码SpRY(D10A)蛋白N端片段的多核苷酸、编码TadA8e片段的多核苷酸和编码SpRY(D10A)蛋白C端片段的多核苷酸，由此获得所述的载体。
一些实施方案中，所还载体包括质粒或病毒载体。
一些实施方案中，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体。
一些实施方案中，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示。
一些实施方案中，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示。
一些实施方案中，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示。
一些实施方案中，所述骨架质粒包括pCMV或其衍生质粒ABEmax-SpRY。
一些实施方案中，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞。
一些实施方案中，所述人胚胎肾细胞包括HEK293T细胞。
一些实施方案中，所述脑神经瘤细胞包括N2a细胞。
一些实施方案中，所述方法包括从所述衍生质粒ABEmax-SpRY中去除TadA片段，并用TadA8e替换SpRY(D10A)中1048位至1063位的氨基酸，构建得所述重组表达载体。
一些实施方案中，所述载体为CE-8e-SpRY质粒。
一些实施方案中，本公开提供了一种sgRNA。
一些实施方案中，所述sgRNA的序列包括如SEQ ID NO：18-65所示的序列。
一些实施方案中，本公开提供了一种表达系统，所述表达系统含有所述的表达载体或其基因组中整合有外源的所述的多核苷酸。
一些实施方案中，所述表达系统表达所述的融合蛋白或其基因组中整合的外源序列表达所述的融合蛋白或所述表达系统表达含有所述的多核苷酸或其基因组中整合有外源的如上所述的多核苷酸。
一些实施方案中，所述表达系统还含有RNA。
一些实施方案中，所述RNA是引导RNA。
一些实施方案中，所述RNA是sgRNA。
一些实施方案中，所述sgRNA的序列包括与如SEQ ID NO：18-65所示的序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性的序列。
一些实施方案中，所述sgRNA的序列包括如SEQ ID NO：18-65所示的序列。
一些实施方案中，本公开提供了一种宿主细胞，包含所述的多核苷酸或所述的载体或所述的表达系统。
一些实施方案中，本公开提供了一种组合物，包含有效量的所述的突变体多肽，所述的融合蛋白，所述的多核苷酸，所述的载体或所述的宿主细胞中的至少一种。
一些实施方案中，所述组合物为试剂盒。
一些实施方案中，所述组合物还含有RNA。
一些实施方案中，所述RNA是引导RNA。
一些实施方案中，所述RNA是sgRNA。
一些实施方案中，所述sgRNA的序列包括与如SEQ ID NO：18-65所示的序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性 的序列。
一些实施方案中，所述sgRNA的序列包括如SEQ ID NO：18-65所示的序列。
一些实施方案中，本公开提供了一种任一所述的突变体多肽或所述的融合蛋白或所述的多核苷酸或所述的载体或所述的表达系统或所述的宿主细胞在制备治疗基因疾病的药物中的应用。
一些实施方案中，本公开提供了一种任一所述的突变体多肽或所述的融合蛋白或所述的多核苷酸或所述的载体或所述的表达系统或所述的宿主细胞在制备基因编辑试剂中的应用。
一些实施方案中，所述基因编辑的编辑窗口约为3～10位。
一些实施方案中，所述基因编辑的编辑窗口约为8～10位。
一些实施方案中，本公开提供了一种碱基编辑系统，包含任一所述的突变体多肽或所述的融合蛋白或所述的多核苷酸或所述的载体或所述的表达系统或所述的宿主细胞。
一些实施方案中，所述碱基编辑系统还含有RNA。
一些实施方案中，所述RNA是引导RNA。
一些实施方案中，所述RNA是sgRNA。
一些实施方案中，本公开提供了一种基因编辑方法，通过所述的碱基编辑系统进行基因编辑。
一些实施方案中，所述基因编辑的编辑窗口约为3～10位。
一些实施方案中，所述基因编辑的编辑窗口约为8～10位。
一些实施方案中，本公开提供了一种方法，用于重组产生任一所述的突变体多肽或所述的融合蛋白，包括步骤：将所述的载体引入宿主细胞以产生转染的或感染的宿主细胞，体外培养所述转染的或感染的宿主细胞，回收细胞培养物并任选地纯化所产生的突变体多肽或融合蛋白。
一些实施方案中，本公开提供了一种所述的突变体多肽或所述的融合蛋白的制备方法，包括：(1)在骨架质粒中加入编码SpRY(D10A)蛋白N端片段的多核苷酸、编码TadA8e片段的多核苷酸和编码SpRY(D10A)蛋白C端片段的多核苷酸，由此获得重组表达载体；(2)转染所述重组表达载体至宿主细胞使其表达所述突变体多肽或所述融合蛋白。
一些实施方案中，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示。
一些实施方案中，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示。
一些实施方案中，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示。
一些实施方案中，所述骨架质粒包括pCMV或其衍生质粒ABEmax-SpRY。
一些实施方案中，所述方法包括从所述衍生质粒ABEmax-SpRY中去除TadA二聚体，并用TadA8e替换SpRY(D10A)中1048位至1063位的氨基酸，构建得所述重组表达载体。
一些实施方案中，所还载体质粒或病毒载体。
一些实施方案中，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体。
一些实施方案中，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞。
一些实施方案中，所述人胚胎肾细胞包括HEK293T细胞。
一些实施方案中，所述脑神经瘤细胞包括N2a细胞。
一些实施方案中，本公开提供了一种产生所述载体的方法，包括步骤：将所述载体引入至适宜的细胞系，在适宜条件下培养所述细胞系从而使所述目的载体能够产生，从所述细胞系的培养物回收所产生的质粒和任选地纯化所述质粒。
一些实施方案中，本公开提供了一种基因疾病的治疗方法，包括以下步骤：向受试者施予一定量的对所述基因疾病有效的所述的突变体多肽，所述的融合蛋白，所述的多核苷酸中的至少一种，或其任意组合。
一些实施方案中，所述基因疾病包括包括苯丙酮尿症。
在一些实施方案中，上述的蛋白为分离的多肽。
在一些实施方案中，上述的多肽为分离的多肽。
在一些实施方案中，上述的核酸为分离的多核酸。
附图说明
图1为ABEmax-SpRY和8e-SpRY及其突变体的示意图。
图2-图7为ABEmax-SpRY和8e-SpRY在PAM为NNN时的编辑效率。
图8为ABEmax-SpRY和8e-SpRY多点编辑效率的统计结果。
图9为ABEmax-SpRY和8e-SpRY的编辑窗口。
图10-图15为8e-SpRY及其突变体在PAM为NNN时的编辑效率。
图16为8e-SpRY及其突变体多点编辑效率的统计结果。
图17为8e-SpRY及其突变体在PAM为NAN、NGN、NCN和NTN时多点编辑效率的统计结果。
图18为8e-SpRY及其突变体的编辑窗口。
图19为ABEmax-SpRY、8e-SpRY及其突变体的DNA靶向编辑效率。
图20为ABEmax-SpRY、8e-SpRY及其突变体的RNA脱靶量。
图21为ABEmax-SpRY、8e-SpRY及其突变体A-to-I的RNA脱靶示意图。
图22为PKU 728 G>A细胞模型基因型的sanger测序图以及8种修复sgRNA修复效率的sanger测序图。
图23为3种有修复效果的sgRNA修复效率的柱状图。
图24为其他3种ABE突变体修复效率的sanger测序图。
具体实施方式
以下通过具体的实施例进一步说明本公开的技术方案，具体实施例不代表对本公开保护范围的限制。其他人根据本公开理念所做出的一些非本质的修改和调整仍属于本公开的保护范围。
苯丙酮尿症(Phenylketonuria，简称PKU)是先天代谢性疾病的一种，本症是由于染色体基因突变导致肝脏中苯丙氨酸羟化酶(PAH)缺陷从而引起苯丙氨酸(PA)代谢障碍所致。
实施例1碱基编辑器质粒的构建
首先构建8e-SpRY以及相应的突变体。参照ClonExpress MultiS One Step Cloning Kit(Vazyme,C113-01)说明书设计引物，扩增ABE8e(Addgene#138489)中的TadA8e片段，并用TadA8e替代ABEmax-SpRY(Addgene#140003)中的TadA二聚体，构建得到8e-SpRY质粒。
先将8e-SpRY中的TadA8e从原位置删除，再用TadA8e替代SpRY D10A中第1048位至1063位的氨基酸，构建得到CE-8e-SpRY质粒，从5’端到3’端的顺序依次为SpRY(D10A)N端、TadA8e和SpRY(D10A)C端，其中SpRY(D10A)N端的核苷酸序列如SEQ ID NO:2(氨基酸序列如SEQ ID NO:1所示)，TadA8e的核苷酸序列如SEQ ID NO:4(氨基酸序列如SEQ ID NO:3所示)，SpRY(D10A)C端的核苷酸序列如SEQ ID NO:6(氨基酸序列如SEQ ID NO:5所示)。
将8e-SpRY中的TadA8e进行V106W突变，得到V106W-SpRY，其中TadA8e V106W核苷酸序列如SEQ ID NO:15，SpRY D10A核苷酸序列如SEQ ID NO:16。
将8e-SpRY中的SpRY D10A进行N497A,R661A,Q695A，和Q926A突变，得到8e-SpRY-HF，其中SpRY D10A-HF核苷酸序列如SEQ ID NO:17。
将8e-SpRY-HF中的TadA8e进行V106W突变，得到V106W-SpRY-HF。
8e-SpRY及其突变体两端均携带核定位信号，核定位信号为bpNLS(核定位信号的核苷酸序列如SEQ ID NO:10所示；氨基酸序列如SEQ ID NO:9所示)或SV40NLS(核定位信号的核苷酸序列如SEQ ID NO:12所示；氨基酸序列如SEQ ID NO:11所示)。8e-SpRY及其突变体具体图示如图1所示。
(1)ABEmax-SpRY(融合蛋白)
所述ABEmax-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:67所示；其组成从N端到C端的顺序依次为bpNLS、TadA二聚体、SpRY D10A和bpNLS。一些实施例中，两端携带核定位信号也可为SV40NLS。
(2)8e-SpRY(融合蛋白)
所述8e-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:68所示；其组成从N端到C端的顺序依次为bpNLS、TadA8e、SpRY D10A和bpNLS。一些实施例中，两端携带核定位信号也可为SV40NLS。
(3)CE-8e-SpRY(融合蛋白)
所述CE-8e-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:13所示(CE-8e-SpRY融合蛋白的核苷酸序列如SEQ ID NO:14所示)，其组成为:自N端至C端依次包括bpNLS、SpRY(D10A)的N端片段、TadA8e片段和SpRY(D10A)多肽的C端片段和bpNLS，在SpRY(D10A)N端片段与TadA8e片段之间、TadA8e片段与SpRY(D10A)C端片段之间具有连接肽，所述连接肽的氨基酸序列如SEQ ID NO:7所示(编码CE-8e-SpRY连接肽的核苷酸序列如SEQ ID NO:8所示)。一些实施例中，两端携带核定位信号也可为SV40NLS。
(4)V106W-SpRY(融合蛋白)
所述V106W-SpRY(融合蛋白)的氨基酸序列如SEQ ID NO:69所示；其组成从N端到C端的顺序依次为bpNLS、TadA8eV106W、SpRY D10A、和bpNLS，两端携带核定位信号也可为SV40NLS。
(5)8e-SpRY-HF(融合蛋白)
所述8e-SpRY-HF(融合蛋白)的氨基酸序列如SEQ ID NO:70所示；其组成从N端到C端的顺序依次为bpNLS、TadA8e、SpRY D10A-HF、和bpNLS，两端携带核定位信号也可为SV40NLS。
(6)V106W-SpRY-HF
所述V106W-SpRY-HF(融合蛋白)的氨基酸序列如SEQ ID NO:71所示；其组成从N端到C端的顺序依次为bpNLS、TadA8eV106W、SpRY D10A-HF、和bpNLS，两端携带核定位信号也可为SV40NLS。
实施例2
在本实施例中，利用ABEmax-SpRY、8e-SpRY及其突变体在293T细胞中进行内源位点的编辑。
2.1sgRNA质粒的构建
参考人基因组序列，根据SpRY核酸酶的PAM特征设计48条sgRNA，涵盖16种不同的PAM序列，sgRNA序列如SEQ ID NO:18-65所示，sgRNA序列5’端加入ACCG为上游序列，sgRNA反向互补序列的5’端加入AAAC为下游序列，合成oligo后上下游序列退火(程序为：95℃，5min；95℃-85℃at-2℃/s；85℃-25℃at-0.1℃/s；hold at 16℃)后与经过BsaI(NEB：R3733L)酶切后的pGL3-U6-sgRNA(Addgene#51133)载体连接。酶切体系为：pGL3-U6-sgRNA 2μg；CutSmart buffer(NEB：B7204S)6μL；BsaI 1μL；ddH 2O补齐到60μL，37℃酶切过夜。连接体系为：Solution I(Takara：6022Q)3μL；酶切后载体1μL；退火产物6μL，16℃连接30min后转化、挑菌、鉴定。对阳性克隆菌摇菌提取质粒(Axygene：AP-MN-P-250G)、测定浓度后备用。
2.2细胞培养与转染
HEK293T细胞(购自ATCC)接种培养于添加10％血清(Gibco:10270-106)的DMEM培养基中(Gibco：C11995500BT)，其中含1％双抗(v/v)(Gibco：15140122)。转染前一天铺24孔板，使转染时的细胞密度达到80％左右，转染前2h换液。每孔转染的质粒量分别为碱基编辑器质粒600ng，sgRNA质粒(sgRNA1-48的序列如SEQ ID NO:18～SEQ ID NO:65所示)300ng，将质粒稀释于40μL的DMEM中，将3μL的EZ Trans细胞转染试剂(上海李记生物：AC04L092)稀释于40μL的DMEM中，最后将稀释好的EZ转染试剂加入到稀释好的质粒中，混匀后室温静置15min。将混有质粒和EZ的DMEM加入24孔板中，6h后用含有10％血清的完全培养基换液，转染48h后显微镜观察绿色荧光蛋白(Green fluorescent protein,GFP)的表达，流式细胞分选仪分选GFP阳性细胞。
其中GFP是pGL3-U6-sgRNA载体上的。
2.3检测编辑效率
将分选得到的GFP阳性细胞离心去上清后，加入裂解液(50mM KCl,1.5mM MgCl 2,10mM Tris pH 8.0,0.5％Nonidet P-40,0.5％Tween 20,100μg/ml protease K)，以GFP阳性细胞裂解液为模板，扩增靶向序列，扩增体系为：2×buffer(Vazyme：P505)25μL；dNTP 1μL；Forward Primer(10μmol/L)1μL；Reverse Primer(10pmol/L)1μL；细胞裂解产物1μL；DNA聚合酶(Vazyme：P505)0.5μL；ddH2O补齐到50μL。Forward Primer和Reverse Primer序列如SEQ ID NO:72～SEQ ID NO:167所示(分别对应sgRNA1-48)。
扩增出来的PCR产物用回收试剂盒进行纯化(Axygen：AP-PCR-250G)，具体步骤为：扩增产物中加入3倍体积的PCR-A，混匀后加入吸附柱中，12000r/min离心1min；弃废液，吸附柱中加入700μL W2(需加入指定体积的乙醇)，12000r/min离心1min；弃废-液，吸附柱中加入400μL W2(需加入指定体积的乙醇)，12000r/min离心1min；弃废液，12000r/min离心2min；开盖晾干乙醇后，加入28μL ddH 2O，12000r/min离心1min进行洗脱，纯化后的PCR产物送Sanger测序或深度检测，分析编辑效果。
相关结果如图2-9所示。结果显示在所有检测位点上，涵盖NAN、NGN、NCN和NTN的PAM，8e-SpRY的编辑效率均明显高于ABEmax-SpRY；图8的多点编辑效率的统计结果显示，8e-SpRY显著改善了A到G的编辑效率。图9的编辑窗口结果显示，ABEmax-SpRY碱基编辑窗口为5-6位；8e-SpRY碱基编辑窗口为3-10位，窗口更宽。
图10-15显示在NRN(R代表A或G)、NYN(Y代表C或T)PAM下，8e-SpRY突变体编辑效率的对比结果；将8e插入到SpRY中间的CE-8e-SpRY可以很好的维持其A-to-G的编辑活性，将V106W引入Tad8e的V106W-SpRY同样未明显损伤原来的编辑活性，但是将4种突变引入SpRY的8e-SpRY-HF和V106W-SpRY-HF则使编辑活性明显下降。
图16的多点编辑效率的统计结果显示，8e-SpRY-HF和V106W-SpRY-HF显著降低了活性，CE-8e-SpRY编辑效率提高但无显著性差异，V106W-SpRY编辑效率降低同样无显著性差异。
图17在NAN、NGN、NCN和NTN的多点编辑效率的统计结果显示，CE-8e-SpRY在NGN和NTN的编辑效率有所提高，V106W-SpRY在4种PAM下的编辑效率均有所下降，但均无统计学意义。图18的编辑窗口结果显示，V106W-SpRY维持与8e-SpRY相同的编辑窗口，均为3-10位，高活编辑窗口(编辑效率大于40％)为3-9位；CE-8e-SpRY维持相同的编辑窗口即3-10位，高活编辑窗口(编辑效率大于40％)为3-10位，且在8-10位的编辑效率高于8e-SpRY。
表1.实施例2中转染细胞所用的质粒组合(1)
Figure PCTCN2021131279-appb-000001
Figure PCTCN2021131279-appb-000002
Figure PCTCN2021131279-appb-000003
表2.实施例2中转染细胞所用的质粒组合(2)
Figure PCTCN2021131279-appb-000004
Figure PCTCN2021131279-appb-000005
实施例3
在本实施例中，对比ABEmax-SpRY、8e-SpRY及其突变体在293T细胞中的RNA脱靶情况。
3.1 sgRNA的构建
用于RNA脱靶检测的sgRNA序列为5’-CTGGAACACAAAGCATAGAC-‘3(SEQ ID NO:66)，按照2.1所述的质粒构建方法进行构建。
3.2细胞培养与转染
细胞培养按照2.2所述进行，转染前一天用293T细胞铺6cm Dish，使转染时的细胞密度达到80％左右。每皿转染的质粒量为碱基编辑器质粒4μg，sgRNA质粒2μg，将质粒稀释于250μL的DMEM中，将18μL的EZ Trans细胞转染试剂(上海李记生物：AC04L092)稀释于250μL的DMEM中，最后将稀释好的EZ转染试剂加入到稀释好的质粒中，混匀后室温静置15min。将混有质粒和EZ的DMEM加入6cm Dish中，6h后用含有10％血清的完全培养基换液(DMEM+10％FBS)，转染48h后显微镜观察GFP(GFP是pGL3-U6-sgRNA载体上的)表达，流式细胞分选仪分选GFP阳性细胞。取少数阳性细胞按照2.3所述检测编辑效率，其余阳性细胞提取RNA后送RNA-Seq。
3.3 RNA提取
GFP阳性细胞3000r/min离心10min后弃上清，加入1mL RNA isolater Total RNA extraction Reagent(Vazyme：R401-01-AA)充分裂解细胞；加入200μL氯仿，上下剧烈混匀，室温放置3min后，4℃12000r/min离心15min；取上层水相500μL，加入500μL的异丙醇，上下颠倒混匀，4℃12000r/min离心15min；吸弃上清，加入1mL 75％的乙醇，轻轻颠倒几次清洗沉淀，4℃12000r/min离心5min；吸弃上清，开盖干燥5-10min，待乙醇完全挥发后，加入15μL RNase-Free水溶解沉淀，取1μL测浓度。取1μg RNA送RNA-Seq。
相关结果如19-21所示。图19显示在DNA靶向位点上的第8位A的编辑效率，ABEmax-SpRY、8e-SpRY及其突变体均能引起有效的编辑，其中8e-SpRY及其突变体引起的DNA靶向编辑效率相当，ABEmax-SpRY的编辑效率相对较低。图20和图21的RNA脱靶结果显示，相对于ABEmax-SpRY和8e-SpRY的其他突变体，CE-8e-SpRY有效降低了在转录组水平的脱靶编辑。
综合编辑效率检测和脱靶检测结果，发明人获得的CE-8e-SpRY碱基编辑器可以靶向全基因，同时显著提高了A-to-G的编辑效率，并且有效降低了在转录组水平上的脱靶编辑，具有很大的应用潜力。
表3.实施例3中转染细胞所用的质粒组合
Figure PCTCN2021131279-appb-000006
实施例4 CE-8e-SpRY在修复疾病致病位点中的应用
4.1构建人PAH 728 G>A细胞模型
4.1.1突变mut-sgRNA构建
参考人基因组序列，设计mut-sgRNA(SEQ ID NO:168)，按照2.1所述的质粒构建方法进行构建。
4.1.2细胞培养与转染
细胞培养按照2.2所述进行，转染前一天铺24孔板，使转染时的细胞密度达到80％左右，转染前2h换液。每孔转染的质粒量分别为碱基编辑器质粒600ng，sgRNA质粒300ng，将质粒稀释于40μL的DMEM中，将3μL的EZ Trans细胞转染试剂(上海李记生物：AC04L092)稀释于40μL的DMEM中，最后将稀释好的EZ转染试剂加入到稀释好的质粒中，混匀后室温静置15min。将混有 质粒和EZ的DMEM加入24孔板中，6h后用含有10％血清的完全培养基换液，转染48h后流式细胞分选仪分选GFP阳性细胞到96孔板中，每孔分选1个阳性细胞，放置于培养箱培养14天后鉴定细胞单克隆基因型。
4.1.3单克隆细胞基因型鉴定
每孔的单克隆细胞取部分细胞离心后加入裂解液(50mM KCl,1.5mM MgCl 2,10mM Tris pH 8.0,0.5％Nonidet P-40,0.5％Tween 20,100μg/ml protease K)，以细胞裂解液为模板，扩增靶向序列，扩增体系为：2×buffer(Vazyme：P505)25μL；dNTP 1μL；Forward Primer(10μmol/L)1μL；Reverse Primer(10pmol/L)1μL；细胞裂解产物1μL；DNA聚合酶(Vazyme：P505)0.5μL；ddH 2O补齐到50μL。Forward Primer序列为：5’-gtccctgggcagttatgtgtac-3’(SEQ ID NO:177)，Reverse Primer序列为5’-caactggtagctggaggacag-3’(SEQ ID NO:178)。扩增产物送Sanger测序后挑选PAH 728 G>A纯和突变的细胞即为人PAH 728 G>A细胞模型。
4.2修复PAH 728 G>A突变
CE-8e-SpRY在3-10位有较高的编辑效率，且识别PAM为NNN，根据CE-8e-SpRY的编辑窗口和PAM特点，发明人围绕需要修复的致病突变设计了8条Rec-sgRNA(SEQ ID NO:169-～SEQ ID NO:176)，并按照2.1所述的质粒构建方法进行构建。按照2.2所述的细胞培养与转染方法进行转染。按照2.3所述的检测编辑效率的方法进行修复效率的检测。
结果如图22和图23所示：Mut-sgRNA成功造成728G>A的纯和突变；8条Rec-sgRNA中，Rec-sgRNA1(即图22和图23中的sg1)对728 G>A的修复效率最高，Rec-sgRNA 2(即图22和图23中的sg2)和Rec-sgRNA 3(即图22和图23中的sg3)具有微弱的修复效果。
根据x-ABEmax，ABEmax-NG和ABEmax-SpRY的PAM特点和编辑窗口，这3种碱基编辑器的修复sgRNA为SEQ ID NO:173，按照2.1所述的质粒构建方法进行构建，按照2.2所述的细胞培养与转染方法进行转染，按照2.3所述的检测编辑效率的方法进行修复效率的检测。结果如图24所示，3种碱基编辑器对728 G>A的突变位点均无明显修复效果。
本实施例说明由于CE-8e-SpRY识别的PAM为NNN，围绕需要修复的位点有多种sgRNA的选择，可经过sgRNA的筛选选择最符合修复要求的sgRNA，有效提高了可修复位点的范围和修复效果的灵活性。另外，现有的3种碱基编辑器在各自的编辑窗口内均无法修复728 G>A的突变位点，发明人提供的CE-8e-SpRY在突变位点位于编辑窗口第10位时可实现有效的编辑，拓展了现有碱基编辑工具的可编辑范围，显示出独特的编辑特性。
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Claims (16)

  1. 一种分离的突变体多肽，其特征在于，所述多肽自N端至C端依次包括SpRY(D10A)的N端片段、TadA8e片段和SpRY(D10A)多肽的C端片段。
  2. 如权利要求1所述的突变体多肽，其特征在于，所述SpRY(D10A)蛋白N端片段的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性，或所述TadA8e片段的氨基酸序列与如SEQ ID NO:3所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性，或所述SpRY(D10A)蛋白C端片段的氨基酸序列与如SEQ ID NO:5所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列与如SEQ ID NO:2所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，编码所述TadA8e片段的核苷酸序列与如SEQ ID NO:4所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列与如SEQ ID NO:6所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，所述突变体多肽用于基因编辑；
    优选地，所述基因编辑的编辑窗口约为3～10位；
    优选地，所述基因编辑的编辑窗口约为8～10位；
    优选地，所述突变体多肽包含与如SEQ ID NO:13所示的序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性的氨基酸序列。
  3. 一种分离的融合蛋白，其特征在于，包含权利要求1-2任一所述的突变体多肽；
    优选地，所述融合蛋白还包括连接肽，连接肽位于SpRY(D10A)蛋白N端片段与TadA8e片段之间，和/或位于TadA8e片段与SpRY(D10A)蛋白C端片段之间；
    优选地，所述连接肽序列与如SEQ ID NO:7所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，编码所述连接肽的核苷酸序列与如SEQ ID NO:8所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，所述融合蛋白还包括核定位信号片段；
    优选地，所述核定位信号片段位于所述融合蛋白的N端和/或C端；
    优选地，所述核定位信号片段的氨基酸序列与如SEQ ID NO:9和/或SEQ ID NO:11所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，所述核定位信号的核苷酸序列与如SEQ ID NO:10或12所示的核苷酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性；
    优选地，所述核定位信号片段包括约两个拷贝；优选地，所述融合蛋白包含与如SEQ ID NO:13所示的氨基酸序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性的氨基酸序列；
    优选地，所述融合蛋白用于基因编辑；
    优选地，所述基因编辑的编辑窗口约为3～10位；
    优选地，所述基因编辑的编辑窗口约为8～10位；
    优选地，所述融合蛋白可以靶向全基因组，能够更高效率地引起A:T到G:C的碱基转换；
    优选地，所述融合蛋白在突变位点位于编辑窗口第3-10位时可实现有效的编辑；
    优选地，所述融合蛋白在突变位点位于编辑窗口第8-10位时可实现有效的编辑；
    优选地，所述融合蛋白在突变位点位于编辑窗口第10位时可实现有效的编辑。
  4. 编码权利要求1-2任一所述的突变体多肽或权利要求3所述的融合蛋白的多核苷酸，或其互补序列；
    优选地，所述多核苷酸为核酸构建体。
  5. 一种载体，其特征在于，所述载体包含权利要求4所述的多核苷酸；
    优选地，所述载体为重组表达载体；
    优选地，所述载体骨架选自pCMV或其衍生质粒；
    优选地，所述pCMV的衍生质粒包括ABEmax-SpRY；
    优选地，所述载体包括质粒或病毒载体；
    优选地，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体；
    优选地，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞；
    优选地，所述人胚胎肾细胞包括HEK293T细胞；
    优选地，所述脑神经瘤细胞包括N2a细胞。
  6. 一种产权利要求5所述载体的方法，其特征在于，在骨架质粒中加入编码SpRY(D10A)蛋白N端片段的多核苷酸、编码TadA8e片段的多核苷酸和编码SpRY(D10A)蛋白C端片段的多核苷酸，由此获得所述的载体；
    优选地，所还载体包括质粒或病毒载体；
    优选地，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体；
    优选地，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示；
    优选地，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示；
    优选地，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示；
    优选地，所述骨架质粒包括pCMV或其衍生质粒ABEmax-SpRY；
    优选地，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞；
    优选地，所述人胚胎肾细胞包括HEK293T细胞；
    优选地，所述脑神经瘤细胞包括N2a细胞；
    优选地，所述方法包括从所述衍生质粒ABEmax-SpRY中去除TadA片段，并用TadA8e替换SpRY(D10A)中1048位至1063位的氨基酸，构建得所述重组表达载体；
    优选地，所述载体为CE-8e-SpRY质粒。
  7. 一种表达系统，其特征在于，所述表达系统表达如权利要求3所述的融合蛋白或其基因组中整合的外源序列表达如权利要求3所述的融合蛋白或所述表达系统表达含有如权利要求4所述的多核苷酸或其基因组中整合有外源的如权利要求4所述的多核苷酸；
    优选地，所述表达系统还含有RNA；
    优选地，所述RNA是引导RNA；
    优选地，所述RNA是sgRNA；
    优选地，所述sgRNA的序列包括与如SEQ ID NO：18-65所示的序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性的序列。
  8. 宿主细胞，其特征在于，包含权利要求4所述的多核苷酸或权利要求5所述的载体或权利要求7所述的表达系统。
  9. 一种组合物，其特征在于，包含有效量的权利要求1-2任一所述的突变体多肽，权利要求3所述的融合蛋白，权利要求4所述的多核苷酸，权利要求5所述的载体或权利要求8所述的宿主细胞中的至少一种；
    优选地，所述组合物为试剂盒；
    优选地，所述组合物还含有RNA；
    优选地，所述RNA是引导RNA；
    优选地，所述RNA是sgRNA；
    优选地，所述sgRNA的序列包括与如SEQ ID NO：18-65所示的序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性 的序列。
  10. 权利要求1-2任一所述的突变体多肽或权利要求3所述的融合蛋白或权利要求4所述的多核苷酸或权利要求5所述的载体或权利要求7所述的表达系统或权利要求8所述的宿主细胞在制备治疗基因疾病的药物中的应用；
    优选地，所述基因疾病包括苯丙酮尿症。
  11. 权利要求1-2任一所述的突变体多肽或权利要求3所述的融合蛋白或权利要求4所述的多核苷酸或权利要求5所述的载体或权利要求7所述的表达系统或权利要求8所述的宿主细胞在制备基因编辑试剂中的应用；
    优选地，所述基因编辑的编辑窗口约为3～10位；
    优选地，所述基因编辑的编辑窗口约为8～10位。
  12. 一种碱基编辑系统，其特征在于，包含如权利要求1-2任一所述的突变体多肽或权利要求3所述的融合蛋白或权利要求4所述的多核苷酸或权利要求5所述的载体或权利要求7所述的表达系统或权利要求8所述的宿主细胞；
    优选地，所述碱基编辑系统还含有RNA；
    优选地，所述RNA是引导RNA；
    优选地，所述RNA是sgRNA；
    优选地，所述sgRNA的序列包括与如SEQ ID NO：18-65所示的序列具有至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％或至少99.5％或至少99.8％或至少99.9％、或100％的序列同一性的序列。
  13. 一种基因编辑方法，其特征在于，通过权利要求12所述的碱基编辑系统进行基因编辑；
    优选地，所述基因编辑的编辑窗口约为3～10位；
    优选地，所述基因编辑的编辑窗口约为8～10位。
  14. 一种方法，其特征在于，用于重组产生权利要求1-2任一所述的突变体多肽或权利要求3所述的融合蛋白，其特征在于，包括步骤：将权利要求5所述的载体引入宿主细胞以产生转染的或感染的宿主细胞，体外培养所述转染的或感染的宿主细胞，回收细胞培养物并任选地纯化所产生的突变体多肽或融合蛋白。
  15. 一种权利要求1-2任一所述的突变体多肽或权利要求3所述的融合蛋白的制备方法，其特征在于，包括：
    (1)在骨架质粒中加入编码SpRY(D10A)蛋白N端片段的多核苷酸、编码TadA8e片段的多核苷酸和编码SpRY(D10A)蛋白C端片段的多核苷酸，由此获得重组表达载体；
    (2)转染所述重组表达载体至宿主细胞使其表达所述突变体多肽或所述融合蛋白；
    优选地，编码所述SpRY(D10A)蛋白N端片段的核苷酸序列如SEQ ID NO:2所示；
    优选地，编码所述TadA8e片段的核苷酸序列如SEQ ID NO:4所示；
    优选地，编码所述SpRY(D10A)蛋白C端片段的核苷酸序列如SEQ ID NO:6所示；
    优选地，所述骨架质粒包括pCMV或其衍生质粒ABEmax-SpRY；
    优选地，所述方法包括从所述衍生质粒ABEmax-SpRY中去除TadA二聚体，并用TadA8e替换SpRY(D10A)中1048位至1063位的氨基酸，构建得所述重组表达载体；
    优选地，所还载体质粒或病毒载体；
    优选地，所述载体是用于在高等真核细胞或原核细胞中表达的质粒或病毒载体；
    优选地，所述真核细胞选自脑神经瘤细胞或胚胎肾细胞；
    优选地，所述人胚胎肾细胞包括HEK293T细胞；
    优选地，所述脑神经瘤细胞包括N2a细胞。
  16. 一种基因疾病的治疗方法，其特征在于，包括以下步骤：向受试者施予一定量的对所述基因疾病有效的权利要求1-2任一所述的突变体多肽，权利要求3所述的融合蛋白，权利要求4所述的多核苷酸中的至少一种，或其任意组合；
    优选地，所述基因疾病为苯丙酮尿症。
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