Single base editing system for regulating and controlling selective expression of CaMKII-delta subtype

Abstract

The invention discloses a single base editing system for regulating and controlling selective expression of a CaMKII-delta subtype. The system is composed of a Cas9 protein mutant fused with base deaminase and sgRNA for recognizing a CaMKII-delta gene sequence. Wherein the sgRNA guides the fusion protein to realize single base substitution of a splicing donor of a target site intron 15 or a splicing ligand of intron 16 in the CaMKII-delta, the RNA splicing process of the target gene CaMKII-delta is effectively regulated and controlled, the CaMKII-delta 3 subtype is selectively expressed by the CaMKII-delta gene subjected to directional splicing regulation, and meanwhile, the CaMKII-delta 9 is eliminated. The invention further provides application of the base editing system in treating ischemic cardiomyopathy, cardiac CaMKII-delta subtype selective expression is regulated through single base mutation, myocardial damage caused by blood reperfusion is efficiently reversed, and the base editing system is expected to be applied to treatment of ischemic cardiomyopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a single-base editing system for regulating the selective expression of CaMKII-δ isoforms. Background Art

[0002] At present, reperfusion therapy is often used in clinical practice for ischemic cardiomyopathy, such as thrombolytic therapy or percutaneous coronary intervention, anti-cardiac remodeling drug therapy (such as angiotensin-converting enzyme inhibitors (ACEI), angiotensin II receptor antagonists (ARB)). However, these treatments cannot prevent myocardial damage caused by blood reperfusion. This damage may also lead to cardiac fibrosis, myocardial remodeling, arrhythmias and ultimately heart failure. At present, there is no effective treatment plan in clinical practice. It can only be treated with "ischemic post-conditioning" by using drugs such as adenosine receptor agonists, magnesium agents, statins, and angiotensin receptor antagonists. However, most drugs only delay myocardial damage but do not reduce the area of ​​myocardial infarction, and these drugs have not yet been widely used in clinical practice.

[0003] During cardiac reperfusion, CaMKII-δ, a central regulator of cardiac signaling and function, is a regulator of upstream signaling (Ca 2+ CaMKII-δ is an important hub for the regulation of cardiac function (overload, ROS upregulation) and downstream events (apoptosis, inflammation). CaMKII-δ is a single gene-multi-subtype family of multifunctional serine / threonine protein kinases. Among them, CaMKII-δ3 and CaMKII-δ9 are the main cardiac splice variants. CaMKII-δ3 promotes cell survival by participating in the co-activation pathway mediated by the transcription factor GATA binding protein 4 (GATA-4) and increasing the expression of the anti-apoptotic protein Bcl-2, while CaMKII-δ9 promotes cardiomyocyte death, hypertrophic cardiomyopathy and heart failure. Therefore, regulating the target protein mRNA splicing process to specifically produce the CaMKII-δ3 subtype that promotes cardiomyocyte survival and avoid the damage to the myocardium caused by the high-abundance CaMKII-δ9 subtype is a feasible strategy for the treatment of ischemic cardiomyopathy.

[0004] Single-base editing technology is a new type of gene modification technology further derived from the CRISPR / Cas system. It can be divided into cytosine base editors and adenine base editors according to the type of fused base modification enzymes. The principle is to fuse the inactivated dCas9 or nCas9 with only single-strand cutting activity with pyrimidine or purine deaminases that can act on single-stranded DNA (ssDNA), which has the ability to accurately realize base editing of target sites. At present, some reports have been reported that the use of base editing technology to inhibit CaMKII-δ activity can effectively reverse myocardial ischemia-reperfusion injury, but the activity of all CaMKII-δ protein subtypes is inhibited, and the specificity is not strong. Summary of the invention

[0005] In view of the shortcomings of the existing problems, the object of the present invention is to provide a single-base editing system for regulating the selective expression of CaMKII-δ isoforms.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] The present invention first provides a single-base editing system for regulating the selective expression of CaMKII-δ isoforms, wherein the single-base editing system is composed of a Cas9 protein mutant fused with a base deaminase and an sgRNA that recognizes the CaMKII-δ gene sequence; the Cas9 protein mutant is formed by a mutation in at least one of the two nuclease active regions of RuvC1 and HNH of the Cas9 protein, wherein at least one amino acid mutation in the WED domain and the PAM recognition domain of the Cas9 protein improves the editing efficiency and the editing interval.

[0008] In some embodiments, the Cas 9 protein used is from SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpt1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, RHAFnCas9, and KKH SaCas9.

[0009] In some specific embodiments, the coding sequence of the Cas9 protein mutant is shown in any one of SEQ ID NOs: 1 to 6.

[0010] In some more specific embodiments, the coding sequence of the Cas9 protein mutant fused with base deaminase is shown in SEQ ID NO:7.

[0011] In some more specific embodiments, the base editor is achieved by replacing the Cas9 protein in ABE8E with the Cas protein mutant niSpyMac encoded by the sequence SEQ ID NO:5.

[0012] In some embodiments, the sgRNA has the following characteristics:

[0013] i) The number of nucleotides in sgRNA is 17-24 nt;

[0014] ii) The nucleotide sequence of the sgRNA contains at least one of the splicing donor "AG" of intron 15 or the splicing acceptor "GT" of intron 16 in the target gene CaMKII-δ.

[0015] In some more specific embodiments, the nucleotide sequence of the sgRNA used is as follows:

[0016] sgRNA-1: 5′-GCACAGGAGTCAACTGAGAG-3′, as shown in SEQ ID NO: 8;

[0017] sgRNA-2: 5′-TGCACAGGAGTCAACTGAGA-3′, as shown in SEQ ID NO: 9;

[0018] sgRNA-3: 5′-CTGCACAGGAGTCAACTGAG-3′, as shown in SEQ ID NO: 10.

[0019] In some embodiments, the base deaminase comprises adenine deaminase and variants thereof or cytosine deaminase and variants thereof.

[0020] In some embodiments, the base deaminase used is from the APOBEC family and its variants and the TadA family and its variants.

[0021] In some more specific embodiments, the base deaminase is TadA-TadA*.

[0022] In the second aspect, the present invention also protects a recombinant vector containing any of the single-base editing systems described above.

[0023] In a third aspect, the present invention also protects a gene editing tool kit, which contains the single-base editing system described above or the recombinant vector described above.

[0024] In some embodiments, the gene editing tool kit may contain transcription reagents and the like that are used in conjunction with the gene editor.

[0025] In the fourth aspect, the present invention also protects the use of any of the single-base editing systems described above and the recombinant vectors described above in the preparation of gene editing products.

[0026] In a fifth aspect, the present invention also protects the use of any of the single-base editing systems described above in the preparation of products for treating ischemic cardiomyopathy.

[0027] In some embodiments, the product is a pharmaceutical product.

[0028] In some embodiments, the application is for non-disease diagnosis and treatment purposes, such as being used as a single-base editing system in basic research.

[0029] In some embodiments, the single-base editing system administration form is selected from any one of the following (B1)-(B3):

[0030] (B1) Cas9 protein mutants and sgRNA fused with base deaminase;

[0031] (B2) a recombinant plasmid vector containing a nucleotide sequence encoding an sgRNA and a nucleotide sequence encoding a Cas9 protein mutant fused with a base deaminase;

[0032] (B3) mRNA and sgRNA encoding Cas9 protein mutants fused with base deaminase.

[0033] In some embodiments, in (B2), the backbone plasmid of the recombinant plasmid vector is PX459 plasmid.

[0034] The single-base editing system of the present invention can regulate the selective expression of CaMKII-δ isoforms to promote the in vitro application of cardiomyocyte survival; and regulate the CaMKII-δ gene to selectively express the CaMKII-δ3 isoform by delivering a single-base editor, while eliminating CaMKII-δ9, promoting cardiomyocyte survival, and realizing the in vivo application of repairing damaged myocardial tissue, including but not limited to using polymers, lipid nanoparticles, recombinant proteins and viroids to carry delivery vectors containing base deaminase and Cas9 mutant fusion protein and sgRNA and deliver them to the site of cardiac injury through intravenous injection, inhalation or intramyocardial injection.

[0035] In some embodiments, the cell transfection administration form is RNA, protein or plasmid; in some embodiments, the transfection method is liposome transfection, cationic polymer method and virus-mediated method;

[0036] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP), a polymer, a recombinant protein, and a viroid;

[0037] In some embodiments, the administration route is intravenous injection, intramuscular injection, inhalation, oral administration, transdermal injection or intramyocardial injection.

[0038] The single-base editing system provided by the present invention regulates the selective expression of CaMKII-δ isoforms through cell transfection operations in the form of administration of DNA, RNA or protein and their combinations, thereby promoting the survival of cardiomyocytes; and regulates the selective expression of the CaMKII-δ isoform by delivering a single-base editor to the CaMKII-δ gene, while eliminating CaMKII-δ9, thereby promoting the survival of cardiomyocytes and repairing damaged myocardial tissue.

[0039] Beneficial Effects

[0040] The single-base editing system for regulating the selective expression of CaMKII-δ isoforms provided by the present invention has the following beneficial effects compared with the prior art:

[0041] The single-base editing system of the present invention achieves the replacement of the target site in the target gene CaMKII-δ sequence from A:T to G:C, effectively regulating the selective expression of the target gene CaMKII-δ isoform, so that the level of CaMKII-δ9 isoform is reduced and the level of CaMKII-δ3 isoform is increased.

[0042] At the same time, this single-base editing system achieves the goal of effectively treating ischemic cardiomyopathy in mice by regulating the selective expression of CaMKII-δ isoform.

[0043] The single-base editing system of the present invention has broad application prospects in the clinical treatment of ischemic cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the ABE8E-niSpyMac plasmid.

[0045] Figure 2 This is the agarose gel electrophoresis diagram of ABE8E-niSpyMac plasmid.

[0046] Figure 3 Denaturing agarose gel electrophoresis of ABE8E-niSpyMac mRNA and sgRNA.

[0047] Figure 4 Schematic diagram of the principle of using the single-base editing system to regulate the selective expression of CaMKII-δ isoforms to treat ischemic cardiomyopathy.

[0048] Figure 5 Sanger sequencing diagram of the target gene CaMKII-δ by two administration forms of ABE8E-niSpyMac plasmid and RNA.

[0049] Figure 6 This is the Sanger sequencing map of the target gene CaMKII-δ in the MIRI cell model of mouse cardiomyocytes HL-1 after single-base editing.

[0050] Figure 7 The bar graph shows the changes in RNA levels of CaMKII-δ3 and δ9 isoforms in mouse cardiomyocytes after single-base editing.

[0051] Figure 8 Western blot of CaMKII-δ3 and δ9 isoforms in mouse cardiomyocytes after single-base editing.

[0052] Fig. 9 Evaluation of myocardial repair in the MIRI cell model using mouse cardiomyocytes HL-1. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0054] Example 1: ABE8E-niSpyMac mRNA edits target sites in HL-1 cells

[0055] 1.1 Design and screening of sgRNA

[0056] With reference to the mouse genome sequence, according to the PAM characteristics of niSpyMac nuclease, the sgRNA sequences designed by CRISPOR (https: / / crispor.gi.ucsc.edu / ) were preliminarily screened, and the top three sgRNAs that met the theoretical requirements were selected according to the specificity scores provided by the system. The specific sequences are shown in Table 1. The sgRNAs were synthesized by General Biotechnology Co., Ltd.

[0057] The single-base editing efficiency of different sgRNA sequences for the target gene CaMKII-δ is shown in Table 1, among which sgRNA2 has the highest base editing efficiency of 61.6%.

[0058] Table 1 Single base editing efficiency of different sgRNA sequences for the target gene CaMKII-δ

[0059]

[0060] 1.2 Construction of plasmid (pABE) encoding ABE8E-niSpyMac

[0061] The inventors of the present application selected the Cas9 protein mutant niSpyMac (coding sequence such as SEQ ID NO: 5) for the target site of CaMKII-δ, and used it to construct a single-base editing system of the ABE8E-niSpyMac plasmid, which can achieve efficient site-specific replacement of A:T to G:C at sites near the PAM sequence of the mouse heart CaMKII-δ gene.

[0062] The ABE8E-niSpyMac plasmid expressing the single-base editing system was synthesized by General Biotechnology Co., Ltd. The plasmid was transformed into Escherichia coli, and the plasmid obtained by the Escherichia coli transformation in the previous step was extracted using the Novozymes plasmid extraction kit. The concentration and purity of the ABE8E-niSpyMac plasmid were further investigated by microquantitator and gel electrophoresis. The results are as follows Figure 2 shown.

[0063] 1.3 In vitro transcribed base editor ABE8E-niSpyMac mRNA molecule (mABE)

[0064] The ABE8E-niSpyMac plasmid was extracted from Escherichia coli and used as a template to amplify the target DNA fragment by polymerase chain reaction (PCR) technology. The primers used for amplification were:

[0065] The upstream primer was: 5′-TAAAACGACGGCCAGTGAAT-3′;

[0066] The downstream primer is: 5'-AGGAAACAGCTATGACCATG-3'.

[0067] The DNA fragments amplified above were in vitro transcribed using a T7 High Yield RNA Transcription Kit to obtain the mRNA molecules of the base editor. The in vitro transcription system is shown in Table 2:

[0068] Table 2. In vitro transcription reaction system for preparing ABE8E-niSpyMa mRNA

[0069]

[0070] The components were gently mixed and collected by brief centrifugation. They were incubated at 37°C for 2 h. The mRNA molecules were further purified, capped, and tailed to obtain mature mRNA molecules. The concentration and purity of the mRNA were further investigated by microquantitative instrument and gel electrophoresis. The results were as follows: Figure 3 shown.

[0071] 1.4 Transfection of mouse cardiomyocytes MIRI model

[0072] The day before transfection, HL-1 cells in the logarithmic growth phase were digested with trypsin, and the cells were counted and inoculated in 24-well plates. The MIRI cell model was constructed by oxygen deprivation, glucose deprivation, etc., so that the cell density during transfection reached about 80%, and the medium was changed 2 hours before transfection. Each well was transfected with 500ng of base editor mRNA and 500ng of sgRNA. D-Lin-MC3-DMA, distearoylphosphatidylcholine (DSPC), cholesterol and PEG-lipid were dissolved in ethanol respectively, and lipid nanoparticles were prepared by ethanol dilution method: mRNA and sgRNA were diluted to 0.2mg / mL in 25mM sodium acetate buffer (pH 4.0). Using an oscillating vortexer, the ethanol solution of lipids was mixed with the mRNA and sgRNA aqueous solution at a ratio of about 1:4 (volume / volume) (the ethanol solution of lipids was slowly added dropwise to the sodium acetate buffer solution of nucleic acids, and vortexed for 30s), and the external buffer was replaced with PBS by ultrafiltration. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. LNP-mABE was added to a 24-well plate, and the medium was replaced with complete culture medium containing 10% fetal bovine serum (FBS) after 6 h. Cell samples were collected 48 h after transfection.

[0073] The collected cardiomyocyte samples were used to extract the genomic DNA of the genome using the TIANamp Genomic DNA Kit Blood / Cell / Tissue Genomic DNA Extraction Kit of Tiangen Biochemical Co., Ltd. The extracted genomic DNA was used as a template to amplify the gene fragment containing the mutation site. The specific sequences of the primers are as follows:

[0074] Upstream primer: 5′-CATCCGTGACTAGGCAACCA-3′;

[0075] Downstream primer: 5′-CCCAAGCCAAAAAGCTCCCA-3′.

[0076] The PCR product was purified by a recovery kit (Axygen) and then Sanger sequenced to analyze the editing efficiency. The results are shown in Table 3. The editing efficiency of the base editor delivered in the form of mRNA was as high as 74.1%.

[0077] Table 3. Single base editing efficiency of the target gene CaMKII-δ by two administration forms of ABE8E-niSpyMac plasmid and RNA

[0078]

[0079] The collected cardiomyocyte samples were used to extract the genomic RNA using the SteadyPure universal RNA extraction kit of Eric Biotech Co., Ltd. The extracted genomic RNA was used as a template, and the Evo M-MLV reverse transcription premix kit was used to obtain cDNA by reverse transcription. After reverse transcription, the target sequence was amplified using the SYBR Green Pro Taq HS premix quantitative polymerase chain reaction (qPCR) kit. In addition, Table 4 shows the upstream primer and downstream primer sequences used to amplify the genes CaMKII-δ9, CaMKII-δ3 and GAPDH.

[0080] Table 4. Upstream and downstream primers used to amplify genes CaMKII-δ9, CaMKII-δ3 and GAPDH

[0081]

[0082] The base editing effect was evaluated at the RNA level by measuring the relative fluorescence intensity changes of evaluation indicators such as genes CaMKII-δ9 and CaMKII-δ3. Figure 8 As shown, the base editor delivered in the form of mRNA regulated the RNA level of CaMKII-δ9 to be approximately 2-fold lower than that of the control group, and the RNA level of CaMKII-δ3 was increased by approximately 3-fold.

[0083] The collected myocardial tissue samples were used to extract cytoplasmic protein and nuclear protein using the nuclear protein and cytoplasmic protein extraction kit (Biyuntian). The relative changes in the protein levels of genes CaMKII-δ9 (located in the cytoplasm) and CaMKII-δ3 (located in the nucleus) were determined using Western Blotting technology. The results are as follows Fig. 9 As shown, base editors delivered in the form of mRNA regulated the protein levels of CaMKII-δ9 and CaMKII-δ3 to be significantly reduced and increased compared with the control group, respectively.

[0084] Example 2 ABE8E-niSpyMac plasmid edits target sites in HL-1 cells

[0085] 2.1 Construction of plasmid encoding sgRNA-ABE8E-niSpyMac

[0086] The vector infrastructure was prepared using the PX459 plasmid, which included a nCas9 mutant (SEQ ID NO: 5) and an sgRNA expression element; wherein the nCas9 mutant was followed by a puromycin resistance gene (PURO) screening marker, which can be used for eukaryotic cell screening expression; the pCMV-ABE8E plasmid was used to obtain the nucleotide sequence expressing the deaminase TadA-TadA*, such as Figure 1As shown;

[0087] The recombinant plasmid PX459-U6-sgRNA-CBh-TadA-TadA*-niSpyMac was constructed by General Biotech Co., Ltd. and transformed into Escherichia coli. The plasmid obtained by the transformation of Escherichia coli in the previous step was extracted using the Novozymes plasmid extraction kit. The concentration and purity of the PX459-U6-sgRNA-CBh-TadA-TadA*-niSpyMac plasmid were investigated by microquantitative instrument and gel electrophoresis. The results are as follows: Figure 2 shown.

[0088] 2.2 Transfection of mouse cardiomyocytes MIRI model

[0089] The day before transfection, HL-1 cells in the logarithmic growth phase were digested with trypsin, and the cells were counted and inoculated in 24-well plates. The MIRI cell model was constructed by oxygen deprivation, glucose deprivation, etc., so that the cell density during transfection reached about 80%, and the medium was changed 2 hours before transfection. 1000ng of base editor plasmid was transfected in each well, D-Lin-MC3-DMA, DSPC, cholesterol and PEG-lipid were dissolved in ethanol respectively, and lipid nanoparticles were prepared by ethanol dilution method: the plasmid was diluted to 0.2mg / mL in 50mM sodium acetate buffer (pH 4.0). Using an oscillating vortexer, the ethanol solution of lipids and the aqueous solution of plasmids were mixed at a ratio of about 1:4 (volume / volume) (the ethanol solution of lipids was slowly added dropwise to the sodium acetate buffer solution of nucleic acids, and vortexed for 30s), and the external buffer was replaced with PBS by ultrafiltration. Finally, the lipid nanoparticles were filtered through a sterile filter with a pore size of 0.2μm. LNP-pABE was added to a 24-well plate, and the medium was replaced with complete culture medium containing 10% FBS after 6 h. Cell samples were collected 48 h after transfection.

[0090] Example 3 ABE8E-niSpyMac fusion protein edits target sites in HL-1 cells

[0091] 3.1 Extraction of ABE8E-niSpyMac fusion protein

[0092] The vector infrastructure was prepared with pET32a plasmid, which contained the nucleic acid sequence information of ABE8E-niSpyMac in the recombinant plasmid in Example 1. The recombinant plasmid was synthesized by General Biotechnology Co., Ltd. and transformed into competent cells of Escherichia coli expression strain DE3. The target expression strain was obtained by screening with ampicillin and tetracycline double resistance. The strain was inoculated into a liquid culture medium containing double resistance, cultured overnight at 37°C and 220rpm in a shaking incubator, and then transferred to 1L liquid culture medium containing the corresponding antibiotics at a ratio of 1:100, and cultured at 37°C and 220rpm until OD 600The value was 0.6 to 0.8. The bacterial cells were collected and disrupted, and a large amount of ABE8E-niSpyMac protein was quickly prepared by one-step nickel column affinity purification, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0093] 3.2 Transfection of mouse cardiomyocytes MIRI model

[0094] The day before transfection, HL-1 cells in the logarithmic growth phase were digested with trypsin, and the cells were counted and inoculated in 24-well plates. The MIRI cell model was constructed by oxygen deprivation, glucose deprivation, etc., so that the cell density during transfection reached about 80%, and the medium was changed 2h before transfection. The niSpyMac protein (10μM) was fully mixed with sgRNA (10μM) to assemble in vitro to form a 5μM ribonucleoprotein (RNP) complex. 2.5μL RNP complex was added to each well for transfection, D-Lin-MC3-DMA, DSPC, cholesterol and PEG-lipid were dissolved in ethanol respectively, and lipid nanoparticles were prepared by ethanol dilution method: RNP complex was diluted to 0.2mg / mL in 50mM sodium acetate buffer (pH 4.0). Using an oscillating vortexer, the ethanol solution of lipids was slowly added dropwise to the sodium acetate buffer solution of the RNP complex, vortexed for 30s, and the external buffer was replaced with PBS by ultrafiltration. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. LNP-RNP was added to a 24-well plate, and the medium was replaced with complete medium containing 10% FBS after 6 h. Cell samples were collected 48 h after transfection.

[0095] The collected cardiomyocyte samples were used to extract the genomic DNA of the genome using the TIANamp Genomic DNA Kit Blood / Cell / Tissue Genomic DNA Extraction Kit of Tiangen Biochemical Co., Ltd. The extracted genomic DNA was used as a template to amplify the gene fragment containing the mutation site. The amplified PCR product was purified by a recovery kit (Axygen) and then Sanger sequenced to analyze the editing efficiency. The results are shown in Table 3. The editing efficiency of the base editor delivered in the form of mRNA was as high as 74.1%.

[0096] The collected cardiomyocyte samples were used to extract the genomic RNA using the SteadyPure Universal RNA Extraction Kit of Eric Biotech Co., Ltd. The extracted genomic RNA was used as a template, and the Evo M-MLV reverse transcription premix kit was used to obtain cDNA by reverse transcription. After reverse transcription, the SYBR Green Pro Taq HS premix qPCR kit was used to amplify the target sequence. The upstream primer and downstream primer sequences for amplifying the genes CaMKII-δ9, CaMKII-δ3 and GAPDH are shown in Table 4. The base editing effect was evaluated at the RNA level by measuring the relative fluorescence intensity changes of evaluation indicators such as genes CaMKII-δ9 and CaMKII-δ3.

[0097] The collected myocardial tissue samples were used to extract cytoplasmic protein and nuclear protein using the nuclear protein and cytoplasmic protein extraction kit (Biyuntian). The relative changes in the protein levels of genes CaMKII-δ9 (located in the cytoplasm) and CaMKII-δ3 (located in the nucleus) were determined using Western Blotting technology.

[0098] Example 4 In vivo treatment of MIRI model mice using LNPs loaded with base editing tools

[0099] This example selected the MIRI disease model of C57BL / 6 mice, which was constructed by surgical ligation of coronary artery branches, but is not limited to this. For this model, we used adenine deaminase and nCas9 to design a base editing tool, targeting the target gene CaMKII-δ, and replaced the splicing donor "AG" of intron 15, causing "A" to mutate into "G" to skip exon 16, while retaining the expression of other isoforms of CaMKII-δ to the maximum extent without affecting the protein open reading frame.

[0100] Specifically, the base editing tool was constructed using the methods of Examples 1.3 and 1.4, wherein the designed sgRNA sequence for the MIRI mouse target site was: 5'-TGCACAGGAGTCAACTGAGA-3', and the obtained LNP expressing the MIRI mouse target site mRNA-sgRNA Delivery system.

[0101] Grouping: The MIRI model of C57BL / 6 mice was established by surgical ligation of coronary artery branches and the experimental group was given gene therapy, while the control group mice were given sham surgery.

[0102] Administration: Single intravenous injection of LNP mABE+sgRNA (mABE dose was 2 mg / kg), and the treatment period was 4 weeks. Control mice were simultaneously given an equal volume of sterile PBS;

[0103] Sampling and testing: The weight and behavioral changes of mice were recorded every 2 days after administration; every 1 week after administration, the cardiac function of mice was monitored by small animal ultrasound imaging system, and cardiac function parameters such as ejection fraction (EF,%) and shortening fraction (FS,%) were calculated; and myocardial blood perfusion was evaluated by ultrasound angiography. After 4 weeks of administration, the mice were euthanized and the weight and heart weight of each mouse were measured. The heart of each mouse was fixed and sliced, and H&E staining, TUNEL staining, Masson trichrome staining and Sirius red staining were performed. Based on the results of pathological staining of heart sections, the ischemic area, left ventricular anterior wall thickness, collagen volume and fibrosis area of ​​each heart were quantitatively analyzed, and the myocardial damage repair efficacy of the single-base editing system was comprehensively investigated. The results showed that the cardiac function of the mice in the drug group was significantly better than that of the control group, and the effective mutation of the target site reduced the area of ​​myocardial infarction and fibrosis.

[0104] The collected myocardial tissue samples were used to extract the genomic DNA using the TIANamp Genomic DNA Kit Blood / Cell / Tissue Genomic DNA Extraction Kit of Tiangen Biochemical Co., Ltd. The gene fragment containing the mutation site was amplified using the extracted genomic DNA as a template, and the amplified PCR product was purified by a recovery kit (Axygen) and then subjected to Sanger sequencing to analyze the editing efficiency.

[0105] The collected cell samples were used to extract the genomic RNA using the SteadyPure Universal RNA Extraction Kit of Eric Biotech Co., Ltd. The extracted genomic RNA was used as a template, and the Evo M-MLV reverse transcription premix kit was used to obtain cDNA by reverse transcription. After reverse transcription, the SYBR Green Pro Taq HS premix qPCR kit was used to amplify the target sequence. The upstream primer and downstream primer sequences for amplifying the genes CaMKII-δ9, CaMKII-δ3 and GAPDH are shown in Table 4. The base editing effect was evaluated at the RNA level by measuring the relative fluorescence intensity changes of evaluation indicators such as genes CaMKII-δ9 and CaMKII-δ3.

[0106] The collected myocardial tissue samples were used to extract cytoplasmic protein and nuclear protein using the nuclear protein and cytoplasmic protein extraction kit (Biyuntian). The relative changes in the protein levels of genes CaMKII-δ9 (located in the cytoplasm) and CaMKII-δ3 (located in the nucleus) were determined using Western Blotting technology.

[0107] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A single-base editing system, which consists of a Cas9 protein mutant fused with a base deaminase and a sgRNA that recognizes the CaMKII-δ gene sequence, wherein the Cas9 protein mutant is formed by a mutation in at least one of the two nuclease activity regions of the Cas9 protein, RuvC1 and HNH; Preferably, the Cas9 protein is selected from any one of SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpt1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, RHA FnCas9, and KKHSaCas9; Preferably, the coding sequence of the Cas9 protein mutant is shown in any one of SEQ ID NOs: 1 to 6; More preferably, the coding sequence of the Cas9 protein mutant fused with base deaminase is shown in SEQ ID NO:

7.

2. The single base editing system according to claim 1, characterized in that The sgRNA has the following characteristics: i) The number of nucleotides in the sgRNA is 17-24 nt; ii) The nucleotide sequence of the sgRNA contains at least one of the splicing donor "AG" of intron 15 or the splicing acceptor "GT" of intron 16 in the target gene CaMKII-δ; Preferably, the sgRNA is selected from any one of the following: sgRNA-1: 5′-GCACAGGAGTCAACTGAGAG-3′, as shown in SEQ ID NO: 8; sgRNA-2: 5′-TGCACAGGAGTCAACTGAGA-3′, as shown in SEQ ID NO: 9; sgRNA-3: 5′-CTGCACAGGAGTCAACTGAG-3′, as shown in SEQ ID NO:

10.

3. The single base editing system according to claim 1, characterized in that The base deaminase comprises adenine deaminase and variants thereof or cytosine deaminase and variants thereof, specifically TadA-TadA*.

4. A recombinant vector containing the single-base editing system according to any one of claims 1 to 3.

5. A base editing tool kit, characterized in that The tool kit contains the single base editing system according to any one of claims 1 to 3 or the recombinant vector according to claim 4; Preferably, the tool kit also contains reagents for use with the single-base editing system.

6. Use of the single-base editing system described in any one of claims 1-3 and the recombinant vector described in claim 4 in the preparation of gene editing products.

7. Use of the single-base editing system according to any one of claims 1 to 3 in the preparation of a product for treating ischemic cardiomyopathy; Preferably, the product is a medicine.

8. The use according to claim 7, characterized in that: The single-base editing system administration form is selected from any one of the following (B1)-(B3): (B1) Cas9 protein mutant and sgRNA fused with base deaminase; (B2) a recombinant vector containing a nucleotide sequence encoding sgRNA and a nucleotide sequence encoding a Cas9 protein mutant fused with a base deaminase, wherein the backbone vector of the recombinant vector is a PX459 plasmid; (B3) mRNA and sgRNA encoding Cas9 protein mutants fused with base deaminase.

9. The use according to claim 7, characterized in that: The drug delivery form is selected from one or more of polymers, lipid nanoparticles, recombinant proteins and viroids.

10. The use according to claim 7, characterized in that: The drug delivery route is selected from one or more of intravenous injection, intramuscular injection, inhalation, oral administration, transdermal injection or intramyocardial injection.

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