CN114560946A - Product, method and application of adenine single base editing without PAM limitation - Google Patents

Abstract

The application discloses an adenine single base editing product without PAM limitation, a method and application. The product comprises a fusion protein which comprises the following two parts: the adenine nucleoside deaminase comprises an amino acid sequence shown as SEQ ID No.1 and an endonuclease comprises an amino acid sequence shown as SEQ ID No. 2. The product can realize the replacement and edition of introducing A > G without being limited by PAM in the gene of cells, especially hematopoietic stem/progenitor cells, and can realize the purpose of long-acting and thorough disease cure by the reinfusion of the edited autologous hematopoietic stem cells of patients. The product is applied to repairing beta-thalassemia (thalassemia) -related mutation IVS2-654C > T, has high editing efficiency, can obviously and effectively improve the expression level of beta-globin genes, and has application potential in clinical treatment of beta-anemia.

Description

Product, method and application of adenine single base editing without PAM limitation

Technical Field

The invention relates to the technical field of genetic engineering and protein modification, in particular to an adenine single base editing product without PAM limitation, a method and application.

Background

The CRISPR/Cas9 gene editing system can cut a specific part of DNA to generate DNA double-strand break (DSB), and two repair mechanisms of non-homologous end connection and homologous recombination repair exist in a cell, so that genome editing is realized. But the primary generation CRISPR technology involves DNA double strand breaks, which pose potential risks. The development of a single-base gene editing system enables precise modification of a specific gene site without causing DNA double strand breaks. The base editor in the single-base gene editing system mainly comprises two parts: cas protein and DNA modifying enzymes. To date, two types of base editors have been developed: a Cytosine Base Editor (CBE) to effect conversion of C > T (from C to T); adenine Base Editor (ABE), effecting a > G (from a to G) conversion.

Since the invention was originally invented in 2017, ABE was limited in editing efficiency and editing scope during application. First, the existing seventh generation ABE, ABE7.10max, has a very low editing efficiency in human hematopoietic stem/progenitor cells or even approaches to 0, which limits the application of ABE in hematopoietic stem/progenitor cells. Secondly, because the editing range of the ABE is still influenced by the Cas protein part, if the existing nuclease SpCas9 is limited by NGG PAM, the application range of the adenine base editor in the whole genome is greatly limited.

Researchers have made many breakthrough studies this year. In addition, in the 3 rd month 2020, David.R.liu research group obtains the eighth generation deaminase Tad8e with greatly improved deamination efficiency and Cas compatibility of other species through phage assisted evolution, in addition, in the 4 th month 2020, scientists of Beam Therapeutics also carry out experiments of amino acid saturation mutation of each existing mutation site through another strategy based on mutation carried by ABE7.10 to obtain the eighth generation deaminase ABE8s with obviously improved deamination efficiency in hematopoietic stem/progenitor cells, and in the same year, in the 3 rd month 2020, the KlCas 9-SPRY mutant of SpCas9 with almost no PAM limitation is obtained through structure-based modification by the Benjin P.einstein research group.

Beta-thalassemia is a common hereditary disease with abnormal hemoglobin in adults caused by beta-globin gene defect, and about 3000 thousands of people are born by the 'thalassemia' gene carriers in China, wherein about 30 thousands of people are born by the severe and intermediate 'thalassemia'. The IVS2-654C > T genotype is a common genotype in 'poor land' in China, and the pathogenesis is that the 654 th base in the 2 nd intron of the HBB gene generates C > T (from C to T) mutation, so that an abnormal splice site is generated, 73nt of exon is additionally added in the beta-globin mRNA, and translation is terminated in advance. Although the corresponding mutation on the complementary strand of the mutation site is (G > A) which can be repaired by ABE theoretically, the region on the complementary strand containing the mutation site (G > A) does not have PAM which is suitable for ABE7.10 to play a role, and the ABE7.10 has limitation on the editing efficiency of hematopoietic stem/progenitor cells, so that the effective repair of the pathogenic mutation cannot be realized by a single-base editing mode.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an adenine single base editing product without PAM limitation, a method and application.

Aiming at the two bottlenecks of limiting the application of the ABE to the human hematopoietic stem/progenitor cells, the ABE8e and the Cas9-SPRY are combined for the first time on the basis of the prior art, so that on one hand, the editing range of the ABE is expanded, a disease model can be simulated or pathogenic mutation can be repaired by manufacturing mutation of A > G in a genome region without proper sgRNA originally, and on the other hand, a new way is provided for treating diseases related to the human hematopoietic stem/progenitor cells, particularly diseases caused by PAM-free point mutation.

The invention can realize the replacement and edition of introduced A > G without being limited by PAM in the gene of the cell, especially the hematopoietic stem/progenitor cell, and can realize the purpose of long-acting and thorough disease cure by the reinfusion of the edited patient autologous hematopoietic stem/progenitor cell.

In one aspect, the present invention provides a fusion protein for editing adenine single base without PAM restriction, comprising the following two parts: adenine nucleoside deaminase and endonuclease,

the amino acid sequence of the adenosine deaminase comprises a sequence shown in SEQ ID No.1 (ABE8e) or a sequence which has homology of more than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with the sequence shown in SEQ ID No.1, preferably, the sequence shown in SEQ ID No.1,

the amino acid sequence of the endonuclease comprises a sequence shown in SEQ ID No.2 (Cas9-SPRY) or a sequence which has homology of more than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with the sequence shown in SEQ ID No.2, preferably the sequence shown in SEQ ID No. 2.

In the above fusion protein, the order of joining the two parts of the fusion protein may be: the adenosine deaminase is positioned at the N end or the C end of the endonuclease, and preferably, the adenosine deaminase is positioned at the N end of the endonuclease.

The fusion protein provided by the invention is a novel protein, can solve the problems that the application of an adenine base editor in hematopoietic stem/progenitor cells has low editing efficiency and/or the editing range is limited by PAM,

specifically, as in the specific embodiment of the present invention, the efficiency of ABE7.10 in adenine base editing in hematopoietic stem/progenitor cells is 0, and the fusion protein provided by the present invention (named ABE-SPRY in the examples) can be used to introduce a > G substitution at a normal site with sgRNA of NGG PAM, such as sgRNA-sg 1620 of ngl PAM, which is the enhancer region of BCL11A gene (PAM is GGG), and also can introduce a > G substitution at a normal site with sgRNA of non-NGG PAM, such as sgRNA-sgnat-2 of non-NGG PAM in the intergenic region (PAM is GGCT). The application of ABE-SPRY has greater significance in introducing A > G (T > C corresponding to the mutation introduced into the complementary strand) mutation with disease prevention or protection or disease alleviation effect in a wide range and high efficiency of a genome, or repairing pathogenic G > A mutation (C > T corresponding to the pathogenic mutation in the complementary strand). For example, IVS2-654C > T (i.e., mutation on the complementary strand to G > A) mutations that result in aberrant splicing of introns by repair of HBB (β -globin gene) while sgRNAs suitable for ABE introduction of A > G substitutions to repair the mutated site are all non-NGG PAMs.

Based on the above contribution of the present invention, the present invention also protects the following biomaterials:

in one aspect, the invention provides a nucleic acid molecule capable of encoding a fusion protein as defined in any one of the above, said nucleic acid molecule comprising a DNA molecule and/or an RNA molecule comprising a eukaryotic mRNA and/or a viral RNA,

preferably, the DNA coding sequence of the adenosine deaminase in the fusion protein is shown as SEQ ID No.3, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the sequence shown as SEQ ID No.3,

preferably, the DNA coding sequence of the endonuclease in the fusion protein is shown in SEQ ID No.4, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the sequence shown in SEQ ID No. 4.

In another aspect, the invention also provides a recombinant vector comprising the nucleic acid molecule, the recombinant vector comprising a viral vector, and/or a non-viral vector; the viral vector comprises an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector, and/or an oncolytic viral vector, and the non-viral vector comprises a cationic high molecular polymer, a liposome, and/or a plasmid vector.

In another aspect, the present invention also provides a recombinant cell or a recombinant bacterium, wherein the recombinant cell or the recombinant bacterium comprises the fusion protein, the nucleic acid molecule, or the recombinant vector;

preferably, the recombinant cell is a hematopoietic stem/progenitor cell or an erythroid progenitor cell, more preferably, the hematopoietic stem/progenitor cell is CD34⁺The hematopoietic stem/progenitor cells of (a),

preferably, the recombinant cell is of mammalian origin, more preferably of human origin.

In another aspect, the present invention also provides a single-base gene editing system, the system comprising any one of the above fusion proteins, and/or the nucleic acid molecule, and/or the recombinant vector, and/or the recombinant cell or recombinant bacterium, and/or a sgRNA, the sgRNA guiding the fusion protein to perform single-base gene editing on a gene target site in a cell;

preferably, the cells are hematopoietic stem/progenitor cells or erythroid progenitor cells;

more preferably, the gene target site is IVS2-654C > T mutation site of HBB gene, the target sequence of sgRNA includes at least one of SEQ ID No.7-11, more preferably, at least one of SEQ ID No.8, 9, 10,

and/or the gene target site is the IVS 1-110G > A mutation site of HBB gene, the target sequence of sgRNA includes at least one of SEQ ID No.26, 27, 28, 29, 30, 31 and 32,

and/or the gene target site is HbE G > A mutation site of HBB gene (G > A mutation of 79 th nucleotide of exon region of HBB gene, namely codon GAG mutation encoding 26 th amino acid is AAG), and the target sequence of the sgRNA comprises at least one of SEQ ID No.33, 34, 35, 36, 37, 38 and 39.

On the other hand, the invention also protects the application of the fusion protein, the nucleic acid molecule, the recombinant vector, the recombinant cell or recombinant bacterium and the single-base gene editing system in preparing a gene editing product, a disease treatment and/or prevention product, an animal model or a new plant variety;

preferably, the cell to which the gene editing product is directed is a hematopoietic stem/progenitor cell or an erythroid progenitor cell, more preferably, the hematopoietic stem/progenitor cell is CD34⁺The hematopoietic stem/progenitor cells of (a),

preferably, the target site edited by the gene editing product is a genome site without PAM nearby,

the cell is of mammalian, more preferably, human,

the disease comprises beta-thalassemia, more preferably, the beta-thalassemia is caused by a mutation in Hb E G > a comprising IVS2-654C > T, IVS 1-110G > a, and/or HBB gene.

In another aspect, the invention also provides a method for repairing abnormal splicing of intron caused by mutation of HBB (beta-globin gene) IVS2-654C > T in cells, wherein the mutation of IVS2-654C > T introduces an additional splice donor site to cause abnormal splicing of intron,

the method comprises the following steps: introducing any of the fusion proteins and sgrnas thereof into a cell, and performing a > G and/or T > C editing on the mutation site of IVS2-654C > T in the HBB to restore normal splicing;

preferably, the target sequence of the sgRNA includes 15-25bp sequence, more preferably, 17-22bp, more preferably, 20bp, more preferably, the mutation site of the IVS2-654C > T is located at position 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, preferably, 3, 4, 5, 6, or 7, more preferably, 4, 5, or 6, more preferably, the target sequence of the sgRNA includes at least one of SEQ ID nos. 7-11, more preferably, at least one of SEQ ID nos. 8, 9, 10;

preferably, the sequence of the additional splice donor site is AAGGTAATA.

In another aspect, the invention also provides a repair method for aberrant splicing of introns due to HBB (beta-globin gene) IVS 1-110G > A mutation in cells, said IVS 1-110G > A mutation introducing an additional splice donor site resulting in aberrant splicing of introns,

the method comprises the following steps: introducing into a cell a fusion protein of any of the above and a sgRNA thereof that directs the fusion protein to perform A > G and/or T > C editing of the mutation site of IVS 1-110G > A in the HBB to restore normal splicing,

preferably, the target sequence of the sgRNA includes 15-25bp sequence including the mutation site of IVS 1-110G > a, more preferably, 17-22bp, more preferably, 20bp, more preferably, the mutation site of IVS 1-110G > a is located at position 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, more preferably, 3, 4, 5, 6, 7, 8, 9, or more preferably, the target sequence of the sgRNA includes at least one of SEQ ID nos. 26, 27, 28, 29, 30, 31, and 32.

In another aspect, the present invention provides a method for repairing a defective synthesis of globin caused by mutation of Hb E G > A of HBB gene in a cell,

the method comprises the following steps: introducing the fusion protein of claim 1 or 2 and sgRNAs thereof into a cell, the sgRNAs directing the fusion protein to perform A > G and/or T > C editing on the Hb E G > A mutation site of the HBB gene to restore normal globin synthesis,

preferably, the target sequence of the sgRNA includes 15 to 25bp of sequence including the mutation site of Hb E G > a of the HBB gene, more preferably, 17 to 22bp, more preferably, 20bp, more preferably, the mutation site of Hb E G > a of the HBB gene is located at position 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, more preferably, 3, 4, 5, 6, 7, 8, or 9, more preferably, the target sequence of the sgRNA includes at least one of SEQ ID nos. 33, 34, 35, 36, 37, 38, and 39.

In the above three repair methods, preferably, the cells are hematopoietic stem/progenitor cells or erythroid progenitor cells, and more preferably, the hematopoietic stem/progenitor cells are CD34⁺The hematopoietic stem/progenitor cells of (a);

preferably, the cell is of mammalian origin, more preferably, human origin;

preferably, the sgRNA comprises a chemical modification of a base; in a preferred embodiment, the sgRNA comprises a chemical modification of any one or any few of the 1 st to n th bases at the 5 'end, and/or a chemical modification of any one or any few of the 1 st to n th bases at the 3' end; and n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, the sgRNA comprises a chemical modification of one, two, three, four or five bases at the 5 'end and/or a chemical modification of one, two, three, four or five bases at the 3' end. For example, the sgRNA is chemically modified at the 1 st, 2 nd, 3 rd, 4 th, 5 th, or 1 to 2 nd, 1 to 3 rd, 1 to 4 th, and 1 to 5 th bases at the 5' end of the sgRNA; and/or chemically modifying the 1 st, 2 nd, 3 rd, 4 th, 5 th or 1-2 th, 1-3 rd, 1-4 th, or 1-5 th bases at the 3' end of the sgRNA; in a preferred embodiment, the chemical modification is one or any of methylation modification, fluorination modification or thio modification;

preferably, a complex comprising the fusion protein and the sgRNA is introduced into the cell by means of electrotransformation;

further, the molar ratio of the fusion protein to the sgRNA is 1 (1-3), preferably 1:2 or 1: 3;

further, the fusion protein and the sgRNA form a complex by incubation; preferably, the temperature of the incubation is 20-50 ℃, preferably, 25-37 ℃; preferably, the incubation time is 2-30 minutes, preferably, 5-20 minutes, more preferably, 10 minutes;

further, the ratio of the amount of the complex comprising the fusion protein and the sgRNA to the amount of the cells is 20-100 μ g of the complex: (1X 10)²-1×10⁶Individual) cells, preferably, 30 μ g of complex: (1X 10)³-1×10⁵One) cell;

further, the cells after electroporation were cultured in CD34⁺Extracting the genome DNA of the cell obtained in the step for genotype identification in an EDM-1 differentiation system for 7 days, and determining the mutation efficiency; after the mutation is determined, EDM-2 stage differentiation is carried out for 4 days, and EMD-3 stage differentiation is carried out for 7 days. After each stage of differentiation is finished, RNA is extracted, reverse transcription is carried out to detect abnormal splicing of the intron by cDNA gel electrophoresis, and whether the CDS region of the restored hematopoietic stem/progenitor cell can recover the normal coding function is verified.

The invention has the following beneficial effects:

the invention provides a novel ABE-SPRY protein without PAM restriction, the site-specific repair can be realized by designing sgRNA near a mutation site, the restriction problem that suitable PAM is lacked near the site where mutation needs to be introduced is solved, and the ABE-SPRY protein can be used for the related repair of hematopoietic stem/progenitor cells or erythroid progenitor cells and the treatment/prevention of related diseases.

The invention designs sgRNA near the pathogenic site of the hematopoietic stem/progenitor cells of a beta-thalassemia IVS2-654C > T patient, introduces sgRNA and ABE-SPRY protein which can specifically repair the mutant site into the defective hematopoietic stem/progenitor cells, and enables the IVS2-654C > T mutant site of HBB gene to realize partial or complete repair of T > C (complementary strand is A > G) through specific deamination editing. The intron of the HBB gene in the recombinant hematopoietic stem/progenitor cells obtained by the invention can be normally spliced, the CDS region can restore the normal coding function, and the aim of curing beta-thalassemia can be achieved only by transplanting the autologous hematopoietic stem/progenitor cells repaired after gene editing back into the body clinically. The invention has good application prospect in the aspect of treatment and prevention of beta-thalassemia.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 shows the results of Sanger sequencing for determining the editing efficiency of different RNP electrotransfers into healthy human CD34+ hematopoietic stem/progenitor cells 96h, wherein sgNACT-2(ABE7.10) represents the RNP formed by ABE7.10 protein and sgNACT-2, sg1620(ABE7.10) represents the RNP formed by ABE7.10 protein and sg1620, sgNACT-2(ABE-SPRY) represents the RNP formed by ABE-SPRY protein and sgNACT-2, sg1620(ABE-SPRY) represents the RNP formed by ABE-SPRY protein and sg1620, and CK represents the unedited control.

FIG. 2 is the statistics of the editing efficiency (%) from A to G of deep sequencing for detecting different RNP electrotransforms into healthy human CD34+ hematopoietic stem/progenitor cells 96 h.

Figure 3 is a Sanger sequencing graph that detects the editing efficiency of different sgrnas on CD34+ hematopoietic stem/progenitor cells of patients with β -thalassemia who have IVS2-654C > T mutations (i.e., mutations on the complementary strand to G > a), with P representing an unedited control.

Fig. 4 is a statistical result of allele ratio (%) at the mutation site of CD34+ hematopoietic stem/progenitor cells from β -thalassemia patients who detected different sgrnas for IVS2-654C > T mutations (i.e., mutations on the complementary strand to G > a) by deep sequencing assay.

FIG. 5 shows the detection of repair of abnormal splicing by different sgRNAs due to IVS2-654C > T mutation in patient CD34+ hematopoietic stem/progenitor cells by PCR amplification of exons of HBB, wherein the upper slower migrating band A (468bp) is the abnormally spliced amplification product and the lower faster migrating band N (395bp) is the normally spliced amplification product.

Fig. 6 shows qPCR detection of changes in the ratio of mRNA expression of two globin proteins by different sgrnas on IVS2-654C > T mutations in CD34+ hematopoietic stem/progenitor cells of patients.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings, and the present invention is not limited to the following embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited. Such as those described in Sambrook et al, molecular cloning, A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

The invention utilizes CRISPR-Cas9 gene editing technology and adenine base editor ABE technology to extend and invent an adenine base editing system ABE-SPRY without PAM restriction. The editing system can be applied to the whole genome site targeted by sgRNA of 15-25bp of any or several NGG PAM or non-NGG PAM in the human hematopoietic stem/progenitor cells, and A > G replacement is targeted and introduced in an editing window.

The invention discloses a construction method for introducing A > G substitution to a target site in a human hematopoietic stem/progenitor cell, which comprises the following steps:

(1) preparing ABE-SPRY protein;

(2) designing an sgRNA;

(3) synthesizing sgRNA;

(4) mixing the sgRNA and the ABE-SPRY protein according to the molar ratio (1-3):1 and electrically transducing the sgRNA and the ABE-SPRY protein into the human hematopoietic stem/progenitor cells;

(5) the cells after electroporation were cultured in CD34⁺Extracting genome DNA of the cell obtained in the step when the cell is electrically transformed for 96 hours in an EDM-1 differentiation system for 7 days, and determining mutation efficiency by Sanger sequencing or deep sequencing through PCR amplification;

(6) after the mutation is determined, EDM-2 stage differentiation is carried out for 4 days, and EMD-3 stage differentiation is carried out for 7 days. After the differentiation is finished, RNA is extracted and is reversely transcribed into cDNA, and the expression of the mutant protein mRNA is detected by gel electrophoresis.

The hematopoietic stem/progenitor cells used in the present invention include beta-diji IVS2-654C, in addition to hematopoietic stem/progenitor cells of healthy humans>T genotype patient hematopoietic stem/progenitor cells (CD 34)⁺HSPCs). Electrotransfering the chemically modified sgRNA and ABE-SPRY protein mixture to healthy human hematopoietic stem/progenitor cells or beta-thalassemia IVS2-654C>T hematopoietic stem/progenitor cells capable of efficiently introducing A at the gene target site of healthy human hematopoietic stem/progenitor cells>G substitution at beta-geoslean IVS2-654C>Genes for T hematopoietic stem/progenitor cellsThe target site efficiently restores the aberrant splicing mutation site, thereby restoring the expression of the beta-globin gene.

The invention can repair the beta-thalassemia IVS2-654C > T dependent on transfusion type by using gene editing technology, has high editing efficiency and can efficiently modify the hematopoietic stem/progenitor cell persistent balance hematopoietic system.

Example 1 efficient introduction of A > G substitutions at target sites in healthy human hematopoietic stem/progenitor cells guided by sgRNAs of NGG PAM and sgRNAs of non-NGG PAM, respectively (in electrotransport delivery mode for example)

The healthy human hematopoietic stem/progenitor cells used in this example were derived from healthy human peripheral blood.

1. Design of sgrnas

Designing sgRNA of non-NGG PAM at the normal site intergenic region NACT-2 site, wherein the serial number is sgNACT-2(PAM is GGCT), and the target sequence is as follows:

sgNACT-2：

(SEQ ID No.5)；

designing an sgRNA of NGG PAM in the normal site BCL11A gene enhancer region, wherein the number of the sgRNA is sg1620(PAM is GGG), and the target sequence is as follows:

sg1620：

(SEQ ID No.6)；

wherein, the length of the above 2 target sequences is 20bp, A in the ABE-SPRY editing window is positioned at the position marked by the bold transverse line in the above sequences.

2. Preparation of sgRNA, ABE7.10 protein and ABE-SPRY protein

Chemically modifying and synthesizing 2 sgRNAs in the step 1, and simultaneously preparing ABE7.10 and ABE-SPRY protein;

the amino acid sequence of ABE7.10 is described in detail in "Koblan, L.W., et al," Improving cycle and adenosine base estimates by expression optimization and antibiotic recovery. Nat Biotechnol,2018.36(9): p.843-846. ";

the ABE-SPRY protein is a combined product of the eighth generation TadA-Tad 8e obtained by phage assisted evolution and nCas9-SPRY obtained by structural modification, namely ABE8 e-SPRY;

the ABE-SPRY protein is formed by connecting adenosine deaminase and endonuclease, wherein the adenosine deaminase is positioned at the N end of the endonuclease;

the amino acid sequence of the adenosine deaminase comprises a sequence shown in SEQ ID No.1, the DNA coding sequence of the adenine nucleoside deaminase is shown in SEQ ID No.3,

the amino acid sequence of the endonuclease comprises a sequence shown in SEQ ID No.2, and the DNA coding sequence of the endonuclease is shown in SEQ ID No. 4.

3. Obtaining recombinant cells by electrotransformation

Mixing any one of the sgRNAs in the step 2 with ABE7.10 protein or ABE-SPRY protein according to a certain ratio (1: 2), incubating at room temperature for 10min to respectively obtain four complexes (RNPs) of the sgRNA and the ABE7.10 protein or ABE-SPRY protein, and respectively electrotransfering any one of the four RNPs to a healthy human CD34+ hematopoietic stem/progenitor cell (the dosage ratio of the complexes of ABE7.10 or ABE-SPRY and sgRNA to the cells is 30 mu g of the complex: 1 × 10⁴Individual cells) and mixing the electrotransformation liquid according to the proportion of the electrotransformation kit by taking the healthy human hematopoietic stem/progenitor cells without any RNP as blank Control (CK), wherein the number of the electrotransformation cells is not more than 10⁵Suspending the cells in the electrotransfer solution after cell centrifugation, slightly mixing the cells with the incubated RNP uniformly, transferring the cells to an electrotransfer cup, avoiding air bubbles in the operation process, performing electrotransfer (Lonza-4D electrotransfer) by using a CD34 cell electrotransfer program EO-100, standing and incubating the cells for 5min at room temperature after confirming the successful electrotransfer, re-centrifuging to remove ABE7.10 protein or ABE-SPRY protein and electrotransfer solution, and obtaining CD34⁺After suspending the cells by EDM-1 culture medium, adding the cells into a cell culture plate for differentiation culture at 37 ℃ to obtain the recombinant hematopoietic stem/progenitor cells, wherein RNP is guided to a target site by sgRNA, and A is introduced into an editing window of ABE7.10 or ABE-SPRY at the target site>And G is replaced.

4. Identification results

(1) Sanger sequencing to identify mutations in genomic DNA

The method comprises the following steps: after the in-vitro differentiation culture of the recombinant hematopoietic stem/progenitor cells prepared in the step 3 is carried out for 3-4 days, collecting a proper amount of cells, extracting genome DNA, and detecting the mutation efficiency by Sanger sequencing after PCR amplification, wherein primer information used by PCR is as follows:

the primer pair 1 for detecting the NACT-2 site mutation has the following sequences:

HEK293_Site4-F：5’-GTGGAGACAGACCACAAGCA-3’(SEQ ID NO.12)；

HEK293_Site4-R：5’-GGATCAGAAGCCCTAAGCGG-3’(SEQ ID No.13)；

the sequences of the primer pair 2 for detecting the site mutation of the enhancer region of the BCL11A gene are as follows:

58-checksimilar-F：5’-AGCATCACAACAGGCAGAGAAT-3’(SEQ ID No.14)；

58-checksimilar-R：5’-GGGAACACAGATCCTAACACAGT-3’(SEQ ID No.15)。

as a result: as shown in fig. 6, the RNP formed by the ABE7.10 protein and either of the two sgrnas had little change in the base a (dashed box position) within the editing window of the target site, and the peak height of the base G at this site was very low or even invisible, which means that introduction of a > G was very inefficient and almost ineffective; RNP formed by ABE-SPRY and sgNACT-2 can have base G peaks at the peaks of the base A at the +6A and the +8A in an editing window of a target site at the same time, wherein the base G peak at the +6A is remarkable, and the peak height of the base G accounts for up to 70% of the total peak height of A, G of the two bases, so that the RNP formed by ABE-SPRY and sgNACT-2 efficiently introduces A > G mutation at the site; RNP formed by ABE-SPRY and sg1620 can have base G peaks at the peaks of the bases A at +4A, +7A and +9A in an editing window of a target site, wherein the base G peaks at +4A and +7A are remarkable, and the peak heights of the bases G at +4A and +7A account for A, G total peak heights of the two bases respectively to be 30% and 50%, which indicates that the RNP formed by ABE-SPRY and sg1620G efficiently introduces A > G mutation at the two positions.

(2) Deep sequencing for identifying mutations in genomic DNA

The method comprises the following steps: after the in vitro differentiation culture of the recombinant hematopoietic stem/progenitor cells prepared in the step 3 is carried out for 3-4 days, a proper amount of cells are collected, genome DNA is extracted, and the mutation efficiency is detected by deep sequencing after PCR amplification, wherein the primer information used by PCR is as follows:

the primer pair 3 for detecting the NACT-2 site mutation has the following sequences:

NACT-2-DS-F1：5’-ggagtgagtacggtgtgcCGGGGACGAGGGAAATTTGAA-3’(SEQ ID No.16)；

NACT-2-DS-R1：5’-gagttggatgctggatggTCTCCGTTCGGGTTGAAAGG-3’(SEQ ID No.17)；

the primer pair 4 for detecting the site mutation of the enhancer region of the BCL11A gene has the following sequences:

DeepSPCR-F-try：5’-ggagtgagtacggtgtgcGCCAGAAAAGAGATATGGCATC-3’(SEQ ID No.18)；

DeepSPCR-R-try：5’-gagttggatgctggatgg AGAGAGCCTTCCGAAAGAGG-3’(SEQ ID No.19)；

wherein the sequence represented by lower case letters is a sequencing company universal linker sequence.

As a result: as shown in fig. 2, ABE7.10 introduced a > G with very low efficiency at the region targeted by sgNACT-2 and sg1620, almost 0; ABE-SPRY efficiently introduces A to G mutation (A > G) at +6A and +8A positions of an sgNACT-2 targeting region, wherein the efficiencies are 67.4% and 14.8% respectively; ABE-SPRY efficiently introduced the A to G mutation at positions +4A, +7A, +9A of the sg1620 targeting region (A > G) with efficiencies of 25.7%, 38.8%, 4.8%, respectively.

Example 2 repair of pathogenic sites in beta-thalassemia IVS2-654C > T deficient hematopoietic stem/progenitor cells (delivery by electroporation for example)

The β -thalassemia IVS2-654C > T-deficient hematopoietic stem/progenitor cells used in this example were from patients heterozygous for β -thalassemia IVS 2-654.

1. Design of sgrnas

Designing sgRNAs of non-NGG PAM within 20bp range including the abnormal splicing mutation IVS2-654C > T sites, numbering IVS2-654-sg1 to IVS2-654-sg5 respectively, and targeting target sequences are respectively:

IVS2-654-sg1:

(SEQ ID No.7, PAM is TCA);

IVS2-654-sg2:

(SEQ ID No.8, PAM ATC);

IVS2-654-sg3:

(SEQ ID No.9, PAM TAT);

IVS2-654-sg4:

(SEQ ID No.10, PAM TTA);

IVS2-654-sg5:

(SEQ ID No.11, PAM for ATT);

wherein, the lengths of the above 5 target sequences are all 20bp, and IVS2-654C > T mutation sites (complementary strand is A, and is located at the position marked by bold transverse line in the above sequences) are respectively located at the 3 rd, 4 th, 5 th, 6 th and 7 th positions of the target sequences.

2. Preparation of sgRNA and ABE-SPRY protein

A total of 5 sgrnas in step 1 were synthesized by chemical modification, and ABE-SPRY proteins were prepared simultaneously.

3. Obtaining recombinant cells by electrotransformation

Mixing any one sgRNA in the step 2 with ABE-SPRY protein according to a certain ratio (1: 2), incubating at room temperature for 10min to respectively obtain five sgRNAs and ABE-SPRY protein complexes (RNPs), and respectively converting any one of the five RNPs to beta-thalassemia IVS2-654C>T-deficient hematopoietic stem/progenitor cells (30 μ g of complex of ABE-SPRY and sgRNA to cell ratio of 1X 10⁴Individual cells) and is administered as beta-diji IVS2-654C without any RNP introduced>T-deficient hematopoietic stem/progenitor cells were used as a blank control (P), while healthy human hematopoietic stem/progenitor cells into which no RNP had been introduced were used as a Control (CK). Mixing the electrotransfer liquid according to the proportion of the electrotransfer kit, wherein the number of electrotransfer cells is not more than 10⁵After cell centrifugation, cell suspension was resuspended in electrotransfer solution and gently mixed with the incubated RNP and transferred to an electrotransfer cup, where air bubbles were avoided during the procedure, which was performed using the CD34 cell electrotransfer procedure EO-100Performing electrotransfer (Lonza-4D electrotransfer instrument), standing and incubating the cells for 5min at room temperature after confirming the success of electrotransfer, and centrifuging again to remove ABE-SPRY protein and electrotransfer solution, CD34⁺After suspending cells by EDM-1 culture medium, adding a cell culture plate for differentiation culture at 37 ℃ to obtain recombinant cells, namely the recombinant hematopoietic stem/progenitor cells, and completing the repair of pathogenic mutation sites of the defective hematopoietic stem/progenitor cells.

4. Identification results

(1) Sanger sequencing to identify mutations in genomic DNA

The method comprises the following steps: and (3) carrying out in-vitro differentiation culture on the recombinant hematopoietic stem/progenitor cells prepared in the step (3) for 3-4 days, collecting a proper amount of cells, extracting genome DNA, and carrying out Sanger sequencing detection on mutation efficiency after PCR amplification.

Wherein, the primer sequences used in the PCR are as follows:

IVS-654F-PCR：5’-CACATATTGACCAAATCAGGG-3’(SEQ ID No.20)；

IVS-654R-PCR：5’-CTTTGCCAAAGTGATGGGCCA-3’(SEQ ID No.21)。

as a result: as shown in fig. 3, the complementary strand of the disease-causing mutation site C > T is G > a (black box line), the peak height of base a in β -dipoor IVS2-654C > T deficient hematopoietic stem/progenitor cells (blank control, P) without any introduced RNP is significantly higher than G, which is disease-causing, whereas the peak height ratio of G relative to a in patient cells edited by RNP formed by IVS2-654-sg2, IVS2-654-sg3, IVS2-654-sg4 and ABE-SPRY in the above five sgrnas is increased compared with patients, wherein the peak height ratio of G relative to a is increased by IVS2-654-sg2 and IVS2-654-sg4 introduced a > G (i.e., equivalent to introduction of T > C in the complementary strand), thereby the effect of repairing IVS2-654C > T mutations on the complementary strand is more significant.

(2) Deep sequencing for identifying mutations in genomic DNA

The method comprises the following steps: and (3) carrying out in-vitro differentiation culture on the recombinant hematopoietic stem/progenitor cells prepared in the step (3) for 3-4 days, collecting a proper amount of cells, extracting genome DNA, and carrying out deep sequencing detection on mutation efficiency after PCR amplification.

Wherein, the primer sequences used in the PCR are as follows:

654deepseq F2：5’-ggagtgagtacggtgtgcAGCAGAATGGTAGCTGGATTGT-3’(SEQ ID No.22)；

654deepseq R2：5’-gagttggatgctggatggTCATGCCTCTTTGCACCATTC-3’(SEQ ID No.23)；

wherein the sequence represented by lower case letters is a sequencing company universal linker sequence.

As a result: as shown in fig. 4, the unedited healthy human hematopoietic stem/progenitor cell (control, CK) had a base C (complementary strand G) ratio at the target site of 100%, and the unedited patient hematopoietic stem/progenitor cell (control, P) had a base C (complementary strand G) ratio at the mutation site of 54%; the base C (complementary strand G) ratio at the target site of the hematopoietic stem/progenitor cells of the patients is improved after the RNP formed by the ABE-SPRY and any one of the five sgRNAs (the numbers are 1, 2, 3, 4 and 5 in figure 4), and the ratio is 58%, 67%, 61%, 64% and 58%; wherein, the ratio of C (complementary strand is G) after RNP repair formed by IVS2-654-sg2 and IVS2-654-sg4 is obviously improved, and is respectively improved by 13% and 10%.

(3) EDM-2 and EDM-3 differentiation culture

After Sanger sequencing determines that the target site mutation is successful, the differentiation can be continued in an EDM-2 culture medium. At this stage, the cells can be greatly expanded, after the EDM-2 differentiation stage is finished, the cells are continuously differentiated into EDM-3, and after the in vitro differentiation is finished for 18 days, RNA is extracted and inverted into cDNA, and the cDNA is used for detection in the following steps (4) and (5).

(4) PCR amplification of exons of HBB

The method comprises the following steps: amplifying the differentiated HBB exon by PCR, and verifying whether the pathogenic site can be normally spliced after mutation; the primer sequences used in the PCR are as follows:

654-exon1-F：5’-TGAGGAGAAGTCTGCCGTTAC-3’(SEQ ID No.24)；

654-exon3-R：5’-CACCAGCCACCACTTTCTGA-3’(SEQ ID No.25)。

as a result: as shown in fig. 5, compared with unedited patient hematopoietic stem/progenitor cells (control, P) and unedited healthy human hematopoietic stem/progenitor cells (control, CK), the intensity ratio of the abnormally spliced band (468bp) to the normally spliced band (395bp) in the patient hematopoietic stem/progenitor cells after RNP editing by ABE-SPRY and any one of the above five sgrnas (numbered 1, 2, 3, 4, 5 in fig. 5) is reduced, i.e., the intron has a certain ratio to restore normal splicing after the pathogenic site is edited, wherein IVS2-654-sg4 has the best effect on restoring normal splicing.

(5) qPCR detection of globin expression

The method comprises the following steps: after the pathogenic site of the hematopoietic stem/progenitor cells of the patient is edited in the step 3 and the erythrocyte is differentiated in 18 days, taking the complete cDNA to carry out qPCR, detecting the expression condition of the HBB gene (coding beta-globin) and the HBA gene (coding alpha-globin) and calculating the ratio of the two; the primer sequences used in qPCR are as follows:

HBB_RT_F：5’-CAGTGCAGGCTGCCTATC-3’(SEQ ID No.26)；

HBB_RT_R：5’-ATACTTGTGGGCCAGGGCAT-3’(SEQ ID No.27)；

HBA_RT_F：5’-GCCCTGGAGAGGATGTTC-3’(SEQ ID No.28)；

HBA_RT_R：5’-TTCTTGCCGTGGCCCTTA-3’(SEQ ID No.29)。

as a result: as shown in fig. 6, compared with unedited patient hematopoietic stem/progenitor cells (control, P), the mRNA expression ratio of the gene HBB encoding β -globin and the gene HBA encoding α -globin in the patient hematopoietic stem/progenitor cells edited by RNP formed by ABE-SPRY and four of the above five sgrnas (numbered 1, 2, 3, 4, 5 in fig. 6) was increased, which could eliminate erythrotoxicity caused by excessive HBA levels to various degrees and effectively relieve thalassemia symptoms.

Because PAM limitation can be overcome, besides the ABE-SPRY repairs IVS2-654C > T thalassemia types, the invention can also introduce A > G replacement at other sites of genome, and guided by sgRNA of NGG or non-NGG, the A in the ABE-SPRY editing window is mutated into G (namely the introduced change of the corresponding complementary strand is T > C); particularly, the ABE-SPRY is used for realizing high-efficiency A > G replacement at a target site guided by sgRNA of non-NGG, which is difficult to realize by applying an adenine editing system before the invention.

For example, the ABE-SPRY system is used to repair the aberrant splicing of introns caused by HBB (beta-globin gene) IVS 1-110G > A mutations; sgRNAs designed for repair of the disease mutation site are:

IVS1-110-sgRNA1:

(SEQ ID No.26, PAM TAG);

IVS1-110-sgRNA2:

(SEQ ID No.27, PAM TTA);

IVS1-110-sgRNA3:

(SEQ ID No.28, PAM CTT);

IVS1-110-sgRNA4:

(SEQ ID No.29, PAM in CCT);

IVS1-110-sgRNA5:

(SEQ ID No.30, PAM CCC);

IVS1-110-sgRNA6:

(SEQ ID No.31, PAM for ACC);

IVS1-110-sgRNA7:

(SEQ ID No.32, PAM CAC);

the lengths of the above 7 target sequences are all 20bp, wherein the A of the G > A mutation to be repaired is positioned at the marked position of the bold transverse line in the above sequences and is respectively positioned at the 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th and 9 th positions of the target sequences.

Then for example, ABE-SPRY system is used for repairing the globin synthesis defect caused by HbB exon region HbE G > A; sgRNAs designed for repair of the disease mutation site are:

HbE-sgRNA1:

(SEQ ID No.33, PAM GTA);

HbE-sgRNA2:

(SEQ ID No.34, PAM for GGT);

HbE-sgRNA3:

(SEQ ID No.35, PAM TGG);

HbE-sgRNA4:

(SEQ ID No.36, PAM TTG);

HbE-sgRNA5:

(SEQ ID No.37, PAM GTT);

HbE-sgRNA6:

(SEQ ID No.38, PAM for GGT);

HbE-sgRNA7:

(SEQ ID No.39, PAM AGG);

the lengths of the above 7 target sequences are all 20bp, wherein the A of the G > A mutation to be repaired is positioned at the bold transverse line mark in the above sequences and is respectively positioned at the 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th and 9 th positions of the target sequences.

Those not described in detail in this specification are well within the skill of the art. The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Sequence listing

<110> Shanghai Bodhisae Biotech Co., Ltd, university of east China

<120> adenine single base editing product without PAM restriction, method and application

<130> JH-CNP201681

<160> 39

<170> PatentIn version 3.5

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Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr

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Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val

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Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

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Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

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Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Arg Thr Arg Leu

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Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

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Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

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Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

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His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

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His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

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Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

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Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

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Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

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Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

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Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

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Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

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Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

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Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

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Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

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Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

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Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

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Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

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Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

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Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

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Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

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Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

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Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

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Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

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Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

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Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

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Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

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Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

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Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

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Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

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Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

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Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

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Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

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Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

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His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

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Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

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Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

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Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

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Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

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His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

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Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

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Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

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Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

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Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

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Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

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Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

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Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

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Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

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Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

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Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

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Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

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Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

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Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

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Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

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Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

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Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

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Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

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Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

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Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

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Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

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Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

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Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val

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Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

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Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

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Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

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Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

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Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Lys

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Gln Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

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Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

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Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

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Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala Pro Arg Ala

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Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr Arg Ser

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Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

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tctgaggtgg agttttccca cgagtactgg atgagacatg ccctgaccct ggccaagagg 60

gcacgcgatg agagggaggt gcctgtggga gccgtgctgg tgctgaacaa tagagtgatc 120

ggcgagggct ggaacagagc catcggcctg cacgacccaa cagcccatgc cgaaattatg 180

gccctgagac agggcggcct ggtcatgcag aactacagac tgattgacgc caccctgtac 240

gtgacattcg agccttgcgt gatgtgcgcc ggcgccatga tccactctag gatcggccgc 300

gtggtgtttg gcgtgaggaa ctccaaaaga ggcgccgcag gctccctgat gaacgtgctg 360

aattaccccg gcatgaatca ccgcgtcgaa attaccgagg gaatcctggc agatgaatgt 420

gccgccctgc tgtgcgattt ctaccggatg cctagacagg tgttcaatgc tcagaagaag 480

gcccagagct ccattaat 498

<210> 4

<211> 4104

<212> DNA

<213> Artificial sequence

<223> Endonuclease

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atggacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg 60

atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 120

cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 180

cggacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 240

tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 300

ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 360

aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 420

aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 480

atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540

gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 600

atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 660

cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 720

ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780

gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840

cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 900

ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 960

atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 1020

cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 1080

ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 1140

gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 1200

aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac 1260

gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 1320

gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 1380

agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa 1440

gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1500

aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560

tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 1620

agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 1680

gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 1740

tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 1800

atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 1860

ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 1920

cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 1980

aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 2040

gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2100

agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 2160

cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 2220

gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2280

atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 2340

atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 2400

gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 2460

gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 2520

atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 2580

gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 2640

aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2700

accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 2760

ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 2820

actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 2880

aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 2940

taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 3000

taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 3060

atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 3120

aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 3180

cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3240

gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3300

cagacaggcg gcttcagcaa agagtctatc cggcccaaga ggaacagcga taagctgatc 3360

gccagaaaga aggactggga ccctaagaag tacggcggct tcctgtggcc caccgtggcc 3420

tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 3480

aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 3540

tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3600

tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc caagcagctg 3660

cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 3720

cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 3780

cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 3840

atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3900

cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccag actgggagcc 3960

cctcgggcct tcaagtactt tgacaccacc atcgacccca agcagtacag gagcaccaaa 4020

gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 4080

gacctgtctc agctgggagg tgac 4104

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<223> sgNACT-2

<400> 5

gggtcagacg tccaaaacca 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<223> sg1620

<400> 6

tttatcacag gctccaggaa 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS2-654-sg1

<400> 7

ttaccttaac ccagaaatta 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS2-654-sg2

<400> 8

attaccttaa cccagaaatt 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS2-654-sg3

<400> 9

tattacctta acccagaaat 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS2-654-sg4

<400> 10

ctattacctt aacccagaaa 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS2-654-sg5

<400> 11

gctattacct taacccagaa 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<223> HEK293_Site4-F

<400> 12

gtggagacag accacaagca 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<223> HEK293_Site4-R

<400> 13

ggatcagaag ccctaagcgg 20

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence

<223> 58-checksimilar-F

<400> 14

agcatcacaa caggcagaga at 22

<210> 15

<211> 23

<212> DNA

<213> Artificial sequence

<223> 58-checksimilar-R

<400> 15

gggaacacag atcctaacac agt 23

<210> 16

<211> 39

<212> DNA

<213> Artificial sequence

<223> NACT-2-DS-F1

<400> 16

ggagtgagta cggtgtgccg gggacgaggg aaatttgaa 39

<210> 17

<211> 38

<212> DNA

<213> Artificial sequence

<223> NACT-2-DS-R1

<400> 17

gagttggatg ctggatggtc tccgttcggg ttgaaagg 38

<210> 18

<211> 40

<212> DNA

<213> Artificial sequence

<223> DeepSPCR-F

<400> 18

ggagtgagta cggtgtgcgc cagaaaagag atatggcatc 40

<210> 19

<211> 38

<212> DNA

<213> Artificial sequence

<223> DeepSPCR-R-try

<400> 19

gagttggatg ctggatggag agagccttcc gaaagagg 38

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<223> IVS-654F-PCR

<400> 20

cacatattga ccaaatcagg g 21

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<223> IVS-654R-PCR

<400> 21

ctttgccaaa gtgatgggcc a 21

<210> 22

<211> 40

<212> DNA

<213> Artificial sequence

<223> 654 deepseq F2

<400> 22

ggagtgagta cggtgtgcag cagaatggta gctggattgt 40

<210> 23

<211> 39

<212> DNA

<213> Artificial sequence

<223> 654 deepseq R2

<400> 23

gagttggatg ctggatggtc atgcctcttt gcaccattc 39

<210> 24

<211> 21

<212> DNA

<213> Artificial sequence

<223> 654-exon1-F

<400> 24

tgaggagaag tctgccgtta c 21

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<223> 654-exon3-R

<400> 25

caccagccac cactttctga 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS1-110-sgRNA1

<400> 26

ttagtctatt ttcccaccct 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS1-110-sgRNA2

<400> 27

attagtctat tttcccaccc 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS1-110-sgRNA3

<400> 28

tattagtcta ttttcccacc 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS1-110-sgRNA4

<400> 29

ctattagtct attttcccac 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS1-110-sgRNA5

<400> 30

cctattagtc tattttccca 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS1-110-sgRNA6

<400> 31

gcctattagt ctattttccc 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<223> IVS1-110-sgRNA7

<400> 32

tgcctattag tctattttcc 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<223> HbE-sgRNA1

<400> 33

gtaaggccct gggcaggttg 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<223> HbE-sgRNA2

<400> 34

ggtaaggccc tgggcaggtt 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<223> HbE-sgRNA3

<400> 35

tggtaaggcc ctgggcaggt 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence

<223> HbE-sgRNA4

<400> 36

gtggtaaggc cctgggcagg 20

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<223> HbE-sgRNA5

<400> 37

ggtggtaagg ccctgggcag 20

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence

<223> HbE-sgRNA6

<400> 38

tggtggtaag gccctgggca 20

<210> 39

<211> 29

<212> DNA

<213> Artificial sequence

<223> HbE-sgRNA7

<400> 39

ttggtggtaa ggccctgggc aggttggta 29

Claims (10)

1. A PAM-restriction-free fusion protein for adenine single base editing, which is characterized by comprising the following two parts: adenine nucleoside deaminase and endonuclease,

the amino acid sequence of the adenosine deaminase comprises a sequence shown in SEQ ID No.1 or a sequence which has more than 80 percent of homology with the sequence shown in SEQ ID No.1, preferably, the sequence shown in SEQ ID No.1,

the amino acid sequence of the endonuclease comprises a sequence shown in SEQ ID No.2 or a sequence which has homology of more than 80% with the sequence shown in SEQ ID No.2, preferably, the sequence shown in SEQ ID No. 2.

2. The fusion protein of claim 1, wherein the two portions of the fusion protein are linked in the order: the adenosine deaminase is positioned at the N end or the C end of the endonuclease, and preferably, the adenosine deaminase is positioned at the N end of the endonuclease.

3. A nucleic acid molecule capable of encoding the fusion protein of claim 1 or 2, said nucleic acid molecule comprising a DNA molecule and/or an RNA molecule comprising a eukaryotic mRNA and/or a viral RNA,

preferably, the DNA coding sequence of the adenine nucleoside deaminase in the fusion protein is shown in SEQ ID No.3 or a sequence with more than 80% homology with the sequence shown in SEQ ID No.3,

preferably, the DNA coding sequence of the endonuclease in the fusion protein is shown as SEQ ID No.4 or a sequence with more than 80% homology with the sequence shown as SEQ ID No. 4.

4. A recombinant vector comprising the nucleic acid molecule of claim 3, wherein the recombinant vector comprises a viral vector, and/or a non-viral vector; the viral vector comprises an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector, and/or an oncolytic viral vector, and the non-viral vector comprises a cationic high molecular polymer, a liposome, and/or a plasmid vector.

5. A recombinant cell or a recombinant bacterium comprising the fusion protein of claim 1 or 2, or the nucleic acid molecule of claim 3, or the recombinant vector of claim 4;

preferably, the recombinant cell is a hematopoietic stem/progenitor cell or an erythroid progenitor cell, more preferably, the hematopoietic stem/progenitor cell is CD34⁺The hematopoietic stem/progenitor cells of (a),

preferably, the recombinant cell is of mammalian origin, more preferably of human origin.

6. A single base gene editing system comprising the fusion protein of claim 1 or 2, and/or the nucleic acid molecule of claim 3, and/or the recombinant vector of claim 4, and/or the recombinant cell or recombinant bacterium of claim 5, and/or a sgRNA that directs the fusion protein to perform single base gene editing of a genetic target site in a cell;

preferably, the cells are hematopoietic stem/progenitor cells or erythroid progenitor cells;

more preferably, the gene target site is IVS2-654C > T mutation site of HBB gene, the target sequence of sgRNA includes at least one of SEQ ID No.7-11, more preferably, at least one of SEQ ID No.8, 9, 10,

and/or the genetic target site is the IVS 1-110G > A mutation site of HBB gene, the target sequence of sgRNA comprises at least one of SEQ ID Nos. 26, 27, 28, 29, 30, 31 and 32,

and/or the gene target site is a Hb E G > A mutation site of HBB gene, and the target sequence of the sgRNA comprises at least one of SEQ ID Nos. 33, 34, 35, 36, 37, 38 and 39.

7. Use of the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant vector according to claim 4, the recombinant cell or recombinant bacterium according to claim 5, the single-base gene editing system according to claim 6 for the preparation of a gene editing product, a disease treatment and/or prevention product, an animal model or a new plant variety;

preferably, the cell to which the gene editing product is directed is a hematopoietic stem/progenitor cell or an erythroid progenitor cell, more preferably, the hematopoietic stem/progenitor cell is CD34⁺The hematopoietic stem/progenitor cells of (a),

preferably, the target site edited by the gene editing product is a genome site without PAM nearby,

the cell is of mammalian, more preferably, human,

the disease comprises beta-thalassemia, more preferably, the beta-thalassemia is caused by a mutation comprising IVS2-654C > T, IVS 1-110G > A, and/or Hb E G > A.

8. A repair method for aberrant splicing of introns in cells due to HBB (β -globin gene) IVS2-654C > T mutation, said IVS2-654C > T mutation introducing an additional splice donor site resulting in aberrant splicing of introns,

the method comprises the following steps: introducing the fusion protein of claim 1 or 2 and a sgRNA thereof into a cell, the sgRNA directing the fusion protein to perform A > G and/or T > C editing of the mutation site of IVS2-654C > T in the HBB to restore normal splicing,

preferably, the cell is a hematopoietic stem/progenitor cell or an erythroid progenitor cell, more preferably, the hematopoietic stem/progenitor cell is CD34⁺The hematopoietic stem/progenitor cells of (a),

preferably, the cell is of mammalian origin, more preferably, of human origin,

preferably, the target sequence of the sgRNA includes 15-25bp sequence including the mutation site of IVS2-654C > T, preferably, 17-22bp, more preferably, 20bp, more preferably, the mutation site of IVS2-654C > T is located at position 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, more preferably, 3, 4, 5, 6, or 7, more preferably, 4, 5, or 6, more preferably, the target sequence of the sgRNA includes at least one of SEQ ID Nos. 7-11, more preferably, at least one of SEQ ID Nos. 8, 9, 10,

preferably, the sgRNA comprises a chemical modification of a base,

preferably, the sequence of the additional splice donor site is AAGGTAATA,

preferably, a complex comprising the fusion protein and the sgRNA is introduced into the cell by means of electrotransformation.

9. A repair method for aberrant splicing of introns in cells due to HBB (β -globin gene) IVS 1-110G > A mutation, said IVS 1-110G > A mutation introducing an additional splice donor site resulting in aberrant splicing of introns,

the method comprises the following steps: introducing the fusion protein of claim 1 or 2 and a sgRNA thereof into a cell, the sgRNA directing the fusion protein to perform A > G and/or T > C editing of the mutation site of IVS 1-110G > A in the HBB to restore normal splicing,

preferably, the cell is a hematopoietic stem/progenitor cell or an erythroid progenitor cell, more preferably, the hematopoietic stem/progenitor cell is CD34⁺The hematopoietic stem/progenitor cells of (a),

preferably, the cell is of mammalian origin, more preferably, of human origin,

preferably, the target sequence of the sgRNA includes 15-25bp sequence including the mutation site of IVS 1-110G > a, preferably, 17-22bp, more preferably, 20bp, more preferably, the mutation site of IVS 1-110G > a is located at position 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, more preferably, 3, 4, 5, 6, 7, 8, or 9, more preferably, the target sequence of the sgRNA includes at least one of SEQ ID nos. 26, 27, 28, 29, 30, 31, and 32;

preferably, the sgRNA comprises a chemical modification of a base,

preferably, a complex comprising the fusion protein and the sgRNA is introduced into the cell by means of electrotransformation.

10. A method for repairing a defective synthesis of globin caused by a mutation site Hb E G > A of HBB gene in a cell,

the method comprises the following steps: introducing the fusion protein of claim 1 or 2 and sgRNAs thereof into a cell, the sgRNAs directing the fusion protein to A > G and/or T > C editing of the Hb E G > A mutation site of the HBB gene to restore normal globin synthesis,

preferably, the cell is a hematopoietic stem/progenitor cell or an erythroid progenitor cell, more preferably, the hematopoietic stem/progenitor cell is CD34⁺The hematopoietic stem/progenitor cells of (a),

preferably, the cell is of mammalian origin, more preferably, of human origin,

preferably, the target sequence of the sgRNA includes 15-25bp sequence including the Hb E G > A mutation site of the HBB gene, preferably, 17-22bp, more preferably, 20bp, more preferably, the Hb E G > A mutation site of the HBB gene is located at position 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, more preferably, 3, 4, 5, 6, 7, 8, or 9 of the target sequence of the sgRNA, more preferably, the target sequence of the sgRNA includes at least one of SEQ ID Nos. 33, 34, 35, 36, 37, 38, and 39,

preferably, the sgRNA comprises a chemical modification of a base,

preferably, a complex comprising the fusion protein and the sgRNA is introduced into the cell by means of electrotransformation.

Images