A single base editing system for regulating selective expression of CaMKII-delta isoform

Abstract

The application discloses a single-base editing system for regulating selective expression of a CaMKII-delta subtype, which comprises a Cas9 protein mutant fused with a base deaminase and sgRNA recognizing a CaMKII-delta gene sequence. The sgRNA guides the fusion protein to realize single-base substitution of a splicing donor of intron 15 or a splicing ligand of intron 16 in the CaMKII-delta, effectively regulates the RNA splicing process of the target gene CaMKII-delta, and selectively expresses the CaMKII-delta 3 subtype of the CaMKII-delta gene after directional splicing regulation, while eliminating the CaMKII-delta 9. The application also provides an application of the base editing system in treating ischemic cardiomyopathy, and the selective expression of the CaMKII-delta subtype in the heart is regulated through single-base mutation, so that myocardial injury caused by blood reperfusion can be effectively reversed, and the base editing system is expected to be applied to the treatment of ischemic cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a single-base editing system for regulating the selective expression of the CaMKII-δ isoform. Background Technology

[0002] Currently, reperfusion therapy is the primary treatment for ischemic cardiomyopathy in clinical practice. Treatments include thrombolysis, percutaneous coronary intervention, and anti-cardiac remodeling drugs (such as angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs)). However, these treatments do not prevent myocardial damage caused by reperfusion. This damage can lead to cardiac fibrosis, myocardial remodeling, arrhythmias, and ultimately heart failure. Currently, there is no effective treatment option; treatment involves "post-ischemic adaptation" using drugs such as adenosine receptor agonists, magnesium, statins, and angiotensin receptor blockers. However, most of these drugs only delay myocardial damage without reducing the infarct size, and their clinical application is not yet widespread.

[0003] During cardiac reperfusion, the central regulator of cardiac signaling and function, CaMKII-δ, is a linker to upstream signals (Ca... 2+ CaMKII-δ is a crucial link between overload, ROS upregulation, and downstream events (apoptosis, inflammation). It is a family of single-gene, multi-subtype, multifunctional serine / threonine protein kinases. CaMKII-δ3 and CaMKII-δ9 are the major cardiac splicing variants. CaMKII-δ3 promotes cell survival by participating in the GATA-4-mediated co-activation pathway 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 generate the CaMKII-δ3 subtype that promotes cardiomyocyte survival, while avoiding the myocardial damage caused by high abundance of the CaMKII-δ9 subtype, is a feasible strategy for treating ischemic cardiomyopathy.

[0004] Single-base editing technology is a novel gene modification technology derived from the CRISPR / Cas system. Depending on the type of base-modifying enzyme fused, it can be divided into cytosine base editors and adenine base editors. The principle involves fusing an inactivated dCas9 or an nCas9 with only single-strand cleavage activity with a pyrimidine or purine deaminase capable of acting on single-stranded DNA (ssDNA), enabling precise base editing at target sites. Currently, some studies have reported that inhibiting CaMKII-δ activity using base editing technology can effectively reverse myocardial ischemia-reperfusion injury; however, the activity of all CaMKII-δ protein isoforms is inhibited, indicating a lack of specificity. Summary of the Invention

[0005] To address the shortcomings of existing systems, the purpose of this invention is to provide a single-base editing system for selectively regulating the expression of the CaMKII-δ isotype.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] This invention first provides a single-base editing system for selectively regulating the expression of the CaMKII-δ isoform. The single-base editing system consists 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 mutating at least one of the nuclease active regions in the RuvC1 and HNH nuclease active regions of the Cas9 protein. In particular, mutations in at least one amino acid in the WED domain and PAM recognition domain of the Cas9 protein improve editing efficiency and improve the editing range.

[0008] In some embodiments, the Cas9 protein used is derived 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 as any one of SEQ ID NO: 1 to 6.

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

[0011] In some more specific embodiments, the base editor is implemented by replacing the Cas9 protein in ABE8E with the Cas protein mutant niSpyMac encoded by 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 splice donor “AG” of intron 15 or the splice 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 shown below:

[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 includes adenine deaminase and its variants or cytosine deaminase and its variants.

[0020] In some embodiments, the base deaminases used are derived 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] Secondly, the present invention also protects recombinant vectors containing any of the single-base editing systems described above.

[0023] Thirdly, the present invention also protects a gene editing toolkit containing the single-base editing system or the recombinant vector described above.

[0024] In some embodiments, the gene editing kit may contain transcription reagents or other materials that accompany the gene editor during use.

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

[0026] Fifthly, 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 not for disease diagnosis and treatment purposes, such as its use as a single-base editing system in basic research.

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

[0030] (B1) A Cas9 protein mutant fused with a base deaminase and sgRNA;

[0031] (B2) Recombinant plasmid vectors encoding nucleotide sequences of sgRNA and nucleotide sequences of Cas9 protein mutants encoding fusion base deaminases.

[0032] (B3) encodes the mRNA and sgRNA of the Cas9 protein mutant that is a fusion base deaminase.

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

[0034] The single-base editing system of the present invention can regulate the selective expression of the CaMKII-δ isoform to promote cardiomyocyte survival in vitro; and can regulate the selective expression of the CaMKII-δ gene CaMKII-δ3 isoform by delivering a single-base editor, while eliminating CaMKII-δ9, thereby promoting cardiomyocyte survival and achieving in vivo application for repairing damaged myocardial tissue. This includes, but is not limited to, delivery vectors such as polymers, lipid nanoparticles, recombinant proteins, and viruses carrying base deaminases and Cas9 mutant fusion proteins and sgRNA, which are delivered to the site of cardiac injury via intravenous injection, inhalation, or intramyocardial injection.

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

[0036] In some embodiments, the delivery carrier is a lipid nanoparticle (LNP), a polymer, a recombinant protein, or a virus-like substance;

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

[0038] The single-base editing system provided by this invention regulates the selective expression of the CaMKII-δ isoform through cell transfection via DNA, RNA, or protein and their combinations, thereby promoting cardiomyocyte survival; and regulates the selective expression of the CaMKII-δ gene CaMKII-δ3 isoform by delivering a single-base editor, while eliminating CaMKII-δ9, thereby promoting cardiomyocyte survival and repairing damaged myocardial tissue.

[0039] Beneficial effects

[0040] The present invention provides a single-base editing system for selectively regulating the expression of the CaMKII-δ isotype, which has the following advantages compared with the prior art:

[0041] The single-base editing system of this invention realizes the replacement of the target site in the CaMKII-δ sequence from A:T to G:C, effectively regulating the selective expression of the CaMKII-δ isotype of the target gene, resulting in a decrease in the level of the CaMKII-δ9 isotype and an increase in the level of the CaMKII-δ3 isotype.

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

[0043] The single-base editing system of this invention has broad clinical application prospects in the treatment of ischemic cardiomyopathy. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the ABE8E-niSpyMac plasmid.

[0045] Figure 2 Agarose gel electrophoresis image of the ABE8E-niSpyMac plasmid.

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

[0047] Figure 4 A schematic diagram illustrating the principle of using a single-base editing system to selectively express the CaMKII-δ subtype for the treatment of ischemic cardiomyopathy.

[0048] Figure 5 Sanger sequencing diagrams of the target gene CaMKII-δ for two administration methods: ABE8E-niSpyMac plasmid and RNA.

[0049] Figure 6 Sanger sequencing data for CaMKII-δ, a target gene in the MIRI cell model of mouse cardiomyocytes HL-1 after single-base editing.

[0050] Figure 7 A bar chart showing the changes in RNA levels of the CaMKII-δ3 and δ9 subtypes in mouse cardiomyocytes after single-base editing.

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

[0052] Figure 9 To evaluate myocardial repair in a mouse HL-1 cardiomyocyte MIRI cell model. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, all reagents or instruments used are considered to be conventional products that can be purchased on the market.

[0054] Example 1: Target site editing of ABE8E-niSpyMac mRNA in HL-1 cells

[0055] 1.1 Design and Screening of sgRNA

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

[0057] Table 1 shows the single-base editing efficiency of different sgRNA sequences on the target gene CaMKII-δ. Among them, sgRNA2 has the highest base editing efficiency, which is 61.6%.

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

[0059]

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

[0061] The inventors of this application selected the Cas9 protein mutant niSpyMac (coding sequence as shown in SEQ ID NO: 5) as the target site for CaMKII-δ and used it to construct a single-base editing system for the ABE8E-niSpyMac plasmid, which can achieve efficient site-specific substitution 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 *E. coli*, and the plasmid obtained from the *E. coli* transformation in the previous step was extracted using the Novizan plasmid extraction kit. The concentration and purity of the ABE8E-niSpyMac plasmid were further investigated using a micro-quantitative analyzer and gel electrophoresis. The results are as follows: Figure 2 As shown.

[0063] 1.3 mRNA molecule of the in vitro transcription base editor ABE8E-niSpyMac (mABE)

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

[0065] The upstream primer is: 5'-TAAAACGACGGCCAGTGAAT-3';

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

[0067] The DNA fragments obtained from the above amplification were transcribed in vitro using the T7 High Yield RNA Transcription Kit to obtain base editor mRNA molecules. 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 briefly centrifuged. The mixture was incubated at 37°C for 2 hours. Further post-processing, including mRNA purification, capping, and tailing, yielded mature mRNA molecules. The concentration and purity of the mRNA were then further assessed using a micro-quantitative analyzer and gel electrophoresis. The results are as follows: Figure 3 As shown.

[0071] 1.4 Transfection of mouse cardiomyocyte MIRI model

[0072] One day before transfection, HL-1 cells in logarithmic growth phase were digested with trypsin. After cell counting, they were seeded into 24-well plates. A MIRI cell model was constructed using oxygen deprivation and glucose deprivation to achieve a cell density of approximately 80% at transfection. The medium was changed 2 hours before transfection. 500 ng of base editor mRNA and 500 ng of sgRNA were transfected into each well. D-Lin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and PEG-lipids were dissolved in ethanol, and lipid nanoparticles were prepared using the ethanol dilution method: mRNA and sgRNA were diluted to 0.2 mg / mL in 25 mM sodium acetate buffer (pH 4.0). Using a vortex mixer, the ethanol solution of lipids was mixed with the aqueous solutions of mRNA and sgRNA at a ratio of approximately 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 30 seconds). The mixture was then ultrafiltered, with PBS used instead of external buffer. Finally, the lipid nanoparticles were filtered through a sterile filter with 0.2 μm pores. LNP-mABE was added to 24-well plates, and after 6 h, the medium was replaced with complete medium containing 10% fetal bovine serum (FBS). Cell samples were collected 48 h after transfection.

[0073] Genomic DNA was extracted from the collected cardiomyocyte samples using the TIANamp Genomic DNA Kit (Blood / Cell / Tissue Genomic DNA Extraction Kit) from Tiangen Biotech Co., Ltd. Using the extracted genomic DNA as a template, gene fragments containing mutation sites were amplified. The specific primer sequences are shown below:

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

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

[0076] The PCR products were purified using a recovery kit (Axygen) and then subjected to Sanger sequencing to analyze the editing efficiency. The results are shown in Table 3. The editing efficiency of the base editor, which was 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 methods of ABE8E-niSpyMac plasmid and RNA.

[0078]

[0079] Genomic RNA was extracted from collected cardiomyocyte samples using the SteadyPure Universal RNA Extraction Kit from Eric Biotechnology Co., Ltd. Using the extracted genomic RNA as a template, cDNA was obtained by reverse transcription using the Evo M-MLV Reverse Transcription Premix Kit. The target sequences were then amplified using the SYBR Green Pro Taq HS Premixed Quantitative Polymerase Chain Reaction (qPCR) Kit. Table 4 shows the upstream and downstream primer sequences used for amplifying genes CaMKII-δ9, CaMKII-δ3, and GAPDH.

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

[0081]

[0082] The effectiveness of base editing was evaluated at the RNA level by measuring the relative fluorescence intensity changes of evaluation indicators such as CaMKII-δ9 and CaMKII-δ3. The results are as follows: Figure 8 As shown, the base editor, which delivers mRNA, reduced the RNA level of CaMKII-δ9 by about 2 times compared to the control group, while increasing the RNA level of CaMKII-δ3 by about 3 times.

[0083] Cytoplasmic and nuclear proteins were extracted from collected myocardial tissue samples using a nuclear protein and cytoplasmic protein extraction kit (Beyotime). Western blotting was used to determine the relative changes in the protein levels of genes CaMKII-δ9 (located in the cytoplasm) and CaMKII-δ3 (located in the nucleus). The results are as follows: Figure 9 As shown, the base editor, which delivers mRNA, significantly reduced and increased the protein levels of CaMKII-δ9 and CaMKII-δ3, respectively, compared to the control group.

[0084] Example 2: Target site editing using the ABE8E-niSpyMac plasmid in HL-1 cells.

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

[0086] The basic vector structure was prepared using the PX459 plasmid, which includes an nCas9 mutant (SEQ ID NO: 5) and an sgRNA expression element; the nCas9 mutant is followed by a puromycin resistance gene (PURO) selection marker, which can be used for eukaryotic cell selection expression; the expression deaminase TadA-TadA* nucleotide sequence was obtained using the pCMV-ABE8E plasmid, as follows: Figure 1As shown;

[0087] The recombinant plasmid PX459-U6-sgRNA-CBh-TadA-TadA*-niSpyMac was synthesized by General Biotechnology Co., Ltd. and implanted into E. coli. The plasmid obtained from the E. coli transformation in the previous step was extracted using the Novizan plasmid extraction kit. The concentration and purity of the PX459-U6-sgRNA-CBh-TadA-TadA*-niSpyMac plasmid were assessed using a micro-quantitative analyzer and gel electrophoresis. The results are as follows: Figure 2 As shown.

[0088] 2.2 Transfection of mouse cardiomyocyte MIRI model

[0089] One day before transfection, HL-1 cells in logarithmic growth phase were digested with trypsin, counted, and seeded into 24-well plates. A MIRI cell model was constructed using oxygen and glucose deprivation to achieve a cell density of approximately 80% at transfection. The medium was changed 2 hours before transfection. 1000 ng of base editor plasmid was transfected into each well. D-Lin-MC3-DMA, DSPC, cholesterol, and PEG-lipids were dissolved separately in ethanol, and lipid nanoparticles were prepared using the ethanol dilution method: the plasmid was diluted to 0.2 mg / mL in 50 mM sodium acetate buffer (pH 4.0). Using a vortex mixer, the ethanol solution of lipids was mixed with the aqueous plasmid solution at approximately a 1:4 (volume / volume) ratio (the ethanol solution of lipids was slowly added dropwise to the sodium acetate buffer solution of nucleic acids, and vortexed for 30 s). Ultrafiltration was performed using PBS instead of external buffer. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter. LNP-pABE was added to a 24-well plate, and after 6 hours the medium was replaced with complete medium containing 10% FBS. Cell samples were collected 48 hours after transfection.

[0090] Example 3: Target site editing of the ABE8E-niSpyMac fusion protein in HL-1 cells.

[0091] 3.1 Extraction of ABE8E-niSpyMac fusion protein

[0092] The basic structure of the vector was prepared using pET32a plasmid, which contained the nucleic acid sequence information of ABE8E-niSpyMac from the recombinant plasmid in Example 1. This recombinant plasmid was synthesized by General Biotechnology Co., Ltd. and transformed into competent cells of E. coli expression strain DE3. The target expression strain was obtained by screening with ampicillin and tetracycline antibiotics. The strain was inoculated into liquid medium containing the antibiotics and cultured overnight at 37°C and 220 rpm. Then, it was transferred at a ratio of 1:100 to 1 L of liquid medium containing the corresponding antibiotics and cultured at 37°C and 220 rpm until OD. 600The values ​​ranged from 0.6 to 0.8. Bacterial cells were collected and lysed, and large quantities of ABE8E-niSpyMac protein were rapidly prepared using a one-step affinity purification method with a nickel column. The protein was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0093] 3.2 Transfection of mouse cardiomyocyte MIRI model

[0094] One day before transfection, HL-1 cells in logarithmic growth phase were digested with trypsin, counted, and seeded into 24-well plates. A MIRI cell model was constructed using oxygen and glucose deprivation to achieve a cell density of approximately 80% at transfection. The medium was changed 2 hours before transfection. The niSpyMac protein (10 μM) was thoroughly mixed with sgRNA (10 μM) to assemble a 5 μM ribonucleoprotein (RNP) complex in vitro. 2.5 μL of the RNP complex was added to each well for transfection. D-Lin-MC3-DMA, DSPC, cholesterol, and PEG-lipids were dissolved in ethanol, and lipid nanoparticles were prepared using the ethanol dilution method: the RNP complex was diluted to 0.2 mg / mL in 50 mM sodium acetate buffer (pH 4.0). Using a vortex mixer, the ethanol solution of lipids was slowly added dropwise to the sodium acetate buffer solution of the RNP complex, vortexed for 30 seconds, and then ultrafiltered with PBS instead of the external buffer. Finally, the lipid nanoparticles were filtered through a sterile filter with 0.2 μm pores. LNP-RNP was added to 24-well plates, and after 6 h, the medium was replaced with complete medium containing 10% FBS. Cell samples were collected 48 h after transfection.

[0095] Genomic DNA was extracted from collected cardiomyocyte samples using the TIANamp Genomic DNA Kit (Blood / Cell / Tissue Genomic DNA Extraction Kit) from Tiangen Biotech Co., Ltd. Using the extracted genomic DNA as a template, gene fragments containing mutation sites were amplified. The amplified PCR products were purified using a recovery kit (Axygen) and then subjected to Sanger sequencing. Editing efficiency was analyzed, and the results are shown in Table 3. The base editor, delivered in mRNA form, achieved an editing efficiency as high as 74.1%.

[0096] Genomic RNA was extracted from collected cardiomyocyte samples using the SteadyPure Universal RNA Extraction Kit from Eric Biotechnology Co., Ltd. Using the extracted genomic RNA as a template, cDNA was obtained through reverse transcription using the Evo M-MLV Reverse Transcription Premix Kit. Following reverse transcription, the target sequences were amplified using the SYBR Green Pro Taq HS Premixed qPCR Kit. The upstream and downstream primer sequences used for amplifying 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] Cytoplasmic and nuclear proteins were extracted from collected myocardial tissue samples using a nuclear protein and cytoplasmic protein extraction kit (Beyotime). Western blotting was used to determine the relative changes in the protein levels of genes CaMKII-δ9 (located in the cytoplasm) and CaMKII-δ3 (located in the nucleus).

[0098] Example 4: In vivo treatment of MIRI model mice using LNP loaded with a base editing tool.

[0099] This embodiment uses the C57BL / 6 mouse MIRI disease model, which is constructed by surgical ligation of coronary artery branches, but is not limited to this method. For this model, we designed a base editing tool using adenine deaminase and nCas9, targeting the CaMKII-δ gene. We mutated the intron 15 splice donor "AG" to "G", thus skipping exon 16, maximizing the preservation of expression of other CaMKII-δ isoforms without affecting the protein's open reading frame.

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

[0101] Grouping: The MIRI model of C57BL / 6 mice was established by surgically ligating coronary artery branches and used as the experimental group to receive 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 Detection: Mouse weight and behavioral changes were recorded every 2 days after drug administration. Cardiac function was monitored weekly using a small animal ultrasound imaging system, and cardiac function parameters such as ejection fraction (EF, %) and fractional shortening (FS, %) were calculated. Myocardial perfusion was assessed using contrast-enhanced ultrasound. Four weeks after drug administration, mice were euthanized, and the weight and heart weight of each mouse were measured. Hearts from each mouse were fixed, sectioned, and stained with H&E, TUNEL, Masson trichrome, and Sirius red. Based on the pathological staining results of the heart sections, the ischemic area, left ventricular anterior wall thickness, collagen volume, and fibrosis area of ​​each heart were quantitatively analyzed to comprehensively evaluate the myocardial injury repair efficacy of the single-base editing system. Results showed that the cardiac function of the drug-treated group was significantly better than that of the control group, and effective mutations at the target site reduced the area of ​​myocardial infarction and fibrosis.

[0104] Genomic DNA was extracted from collected myocardial tissue samples using the TIANamp Genomic DNA Kit (Blood / Cell / Tissue Genomic DNA Extraction Kit) from Tiangen Biotech Co., Ltd. Using the extracted genomic DNA as a template, gene fragments containing mutation sites were amplified. The amplified PCR products were purified using a recovery kit (Axygen) and then subjected to Sanger sequencing to analyze the editing efficiency.

[0105] Genomic RNA was extracted from collected cell samples using the SteadyPure Universal RNA Extraction Kit from Eric Biotechnology Co., Ltd. Using the extracted genomic RNA as a template, cDNA was obtained through reverse transcription using the Evo M-MLV Reverse Transcription Premix Kit. Following reverse transcription, the target sequences were amplified using the SYBR Green Pro Taq HS Premixed qPCR Kit. The upstream and downstream primer sequences used for amplifying 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] Cytoplasmic and nuclear proteins were extracted from collected myocardial tissue samples using a nuclear protein and cytoplasmic protein extraction kit (Beyotime). Western blotting was used to determine the relative changes in the protein levels of genes CaMKII-δ9 (located in the cytoplasm) and CaMKII-δ3 (located in the nucleus).

[0107] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A single-base editing system comprising a Cas9 protein mutant fused with a base deaminase and an sgRNA recognizing the CaMKII-δ gene sequence, wherein the Cas9 protein mutant fused with the base deaminase comprises a Cas9 protein mutant encoding the sequence shown in SEQ ID NO: 5 and an adenine deaminase TadA-TadA* whose nucleotide sequence is obtained from the pCMV-ABE8E plasmid; the Cas9 protein mutant is generated by mutation of at least one nuclease active region in the RuvC1 and HNH nuclease active regions of the Cas9 protein; the Cas9 protein mutant is niSpyMac; and the nucleotide sequence of the sgRNA contains the splicing donor "AG" for intron 15 of the target gene CaMKII-δ; and the sgRNA is selected from any 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.

2. A recombinant vector containing the single-base editing system of claim 1.

3. A base editing toolkit, characterized in that, The toolbox contains the single-base editing system of claim 1 or the recombinant vector of claim 2.

4. The base editing toolkit according to claim 3, characterized in that, The kit also contains reagents that are used with the single-base editing system.

5. The application of the single-base editing system of claim 1 and the recombinant vector of claim 2 in the preparation of gene editing reagents.

6. The use of the single-base editing system of claim 1 in the preparation of a medicament for treating ischemic cardiomyopathy.

7. The application according to claim 6, characterized in that, The single-base editing system is administered via a method selected from any one of the following (B1)-(B3): (B1) A Cas9 protein mutant fused with a base deaminase and sgRNA; (B2) A recombinant vector encoding the nucleotide sequence of sgRNA and the nucleotide sequence of a Cas9 protein mutant encoding a fusion base deaminase; (B3) The mRNA and sgRNA of the Cas9 protein mutant encoding a fusion base deaminase.

8. The application according to claim 7, characterized in that, The backbone vector of the recombinant vector is the PX459 plasmid.

9. The application according to claim 6, characterized in that, The drug delivery method is selected from one or more of polymers, lipid nanoparticles, recombinant proteins, and viruses.

10. The application according to claim 6, characterized in that, The drug delivery route is selected from one or more of the following: intravenous injection, intramuscular injection, inhalation, oral administration, percutaneous or intramyocardial injection.

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