Miniaturized single-base editor for specifically removing CaMKII from heart and application of miniaturized single-base editor

Abstract

The invention discloses a miniaturized single-base editor for heart specific removal of CaMKII and application of the miniaturized single-base editor, sgRNA and an adenine single-base editor (ABE) are delivered into a human body by using AAV, a compound composed of the sgRNA and the ABE edits a 5 'splicing site (5' SS) of a No.7 intron of a CAMK2A / CAMK2B / CAMK 2D / CAMK2G gene, normal splicing of mRNA is destroyed, and thus heart specific gene silencing is achieved. According to the miniaturized single-base editor for specifically removing CaMKII from the heart and the application of the miniaturized single-base editor, a compact Cas9 is utilized to construct a single AAV-ABE vector, a heart specific promoter is combined, and a gene editing vector for efficiently and specifically knocking out CAMK2D in the heart is obtained through sgRNA screening.

Description

Technical Field

[0001] The present invention relates to the technical field of single-base editors, and in particular to a miniaturized single-base editor for cardiac-specific CaMKII clearance and applications thereof. Background Art

[0002] In recent years, with the in-depth study of the molecular mechanism of heart disease and the development of gene therapy, some studies have attempted to treat heart disease by regulating gene expression or correcting gene mutations through gene therapy, and many advances have been made. The principles of gene therapy are mainly divided into gene enhancement, gene silencing and gene editing. Gene enhancement refers to increasing the expression of beneficial genes, gene silencing refers to shutting down the expression of harmful genes, and gene editing refers to more precise replacement of single bases or fragments in genes.

[0003] The advent of CRISPR / Cas9 technology provides a more convenient gene editing tool. The CRISPR / Cas9 system consists of two parts: single-stranded guide RNA (sgRNA) and Cas9 protein. sgRNA and Cas9 form a complex, which is located to the target site near the PAM sequence on the genome under the guidance of sgRNA, and then Cas9 exerts double-strand cutting activity to cause DNA breaks. The use of non-homologous end joining repair (NHEJ) in cells can cause insertion or deletion mutations, causing DNA frameshift, thereby achieving gene knockout. The use of homologous recombination repair (HDR) can achieve repair that depends on exogenous DNA templates and can be used to accurately edit specific sequences. NHEJ can cause unpredictable insertion or deletion mutations, resulting in unpredictable risks; HDR is not suitable for therapeutic applications because of its low efficiency.

[0004] Recently, researchers have developed a more accurate adenine base editor (ABE) based on CRISPR / Cas9, which can achieve precise replacement of A·T>G·C base pairs. ABE is composed of a fusion of Cas9 nickase (nCas9) and adenine deaminase (TadA). Under the guidance of sgRNA, it locates to the target site of the genome. TadA deaminates adenine A at the target site into hypoxanthine I. Subsequently, hypoxanthine I will be treated as guanine G during DNA replication or repair, thereby achieving the replacement of A·T>G·C base pairs. Single base editing technology provides a more accurate and safer gene editing tool.

[0005] At present, single-base editors are mainly used to correct single-base mutations or mutations in protein post-translational modification sites. However, due to the characteristics of Cas9 itself, the background of the sgRNA target sequence, and the limitations of chromatin accessibility, the editing success rate is not high. On the one hand, ABE is limited by the PAM sequence and may not be able to design sgRNA in the target area. On the other hand, it is limited by the bystander effect, and adenines in adjacent sites may be edited. In addition, factors such as the degree of chromatin openness will also affect the editing efficiency. Therefore, compared to single base conversion, ABE can design a series of sgRNAs for screening at the start codon site and splicing site of the gene, and finally obtain an optimal sgRNA, which has a higher success rate for gene silencing.

[0006] In addition, as an important part of gene therapy, gene delivery vectors are responsible for delivering exogenous genes to target cells in the body. Delivery vectors must meet the advantages of high safety and good targeting. At present, two types of delivery vectors, nanolipid particles (LNP) and adeno-associated virus (AAV), have entered clinical application, but only AAV can achieve cardiac delivery, so AAV is widely used as a cardiac delivery vector in current clinical and research. However, due to the limited load of AAV, it can only carry a genome of ~4.7kb, and gene cargo that exceeds its load limit cannot be delivered by a single AAV. The current solution is to split the gene cargo into two halves through split-intein technology and deliver them through two AAVs respectively.

[0007] The size of the adenine single-base editor (5.4kb) constructed based on the early SpCas9 has exceeded the limit and can only be delivered by dual AAV. This method requires two AAVs to enter the same cell to form a complete ABE, which theoretically requires a larger AAV dose. In clinical applications, there are strict restrictions on AAV doses for safety reasons. Therefore, the development of small single-base editors and the use of single AAV delivery are expected to solve this problem. At present, studies have been conducted on the development of ABE vectors that can be loaded by a single AAV based on several compact Cas9s, including nSauriCas9, nNme2Cas9, nCjCas9, and nSaKKHCas9. Among them, the PAM sequence (NNGG) of nSauriCas9 is relatively concise and very similar to the PAM sequence (NGG) of SpCas9, so it has great application potential.

[0008] CAMKII is an important calcium / calmodulin-dependent kinase, which is mainly expressed in the δ subtype in the heart and is encoded by the CAMK2D gene. Studies have shown that CaMKIIδ is involved in the calcium release and contraction process in cardiomyocytes, and plays an important role in diseases such as myocardial disease and heart failure. Myocardial cell ischemia caused by myocardial infarction can cause abnormal activation of CaMKIIδ, including activation methods such as oxidation, autophosphorylation, and nitrosylation. Abnormal activation of CaMKIIδ is associated with the occurrence of arrhythmias, cardiomyopathy, myocardial infarction, heart failure, etc. Therefore, CAMK2D is considered to be a key therapeutic target for heart disease, and it is expected to intervene in the pathophysiological process of myocardial infarction by regulating its activity or expression through gene therapy, thereby improving the prognosis of patients. Summary of the invention

[0009] To solve the above problems, the present invention provides a miniaturized single-base editor for cardiac-specific CaMKII elimination and its application. A single AAV-ABE vector is constructed using compact Cas9 and combined with a cardiac-specific promoter. Through sgRNA screening, a gene editing vector for efficient and specific knockout of CAMK2D in the heart is obtained.

[0010] To achieve the above object, the present invention provides an sgRNA, comprising:

[0011] (1) sgRNA7, the nucleotide sequence of sgRNA7 is shown in SEQ ID NO.1-SEQ ID NO.16;

[0012] (2) scaffold RNA, the nucleotide sequence of the scaffold RNA is shown in SEQ ID NO.17 or SEQ ID NO.18;

[0013] The 3′ end of sgRNA7 is directly connected to the 5′ end of scaffold RNA to form a complete sgRNA molecule.

[0014] Preferably, the target sequence of sgRNA7 is located at the junction of exon 7 and intron 7 of CAMK2A, CAMK2B, CAMK2D or CAMK2G gene of human, mouse, rat or pig.

[0015] A sgRNA / Cas9 ribonucleoprotein complex, comprising:

[0016] (1) sgRNA as described above;

[0017] (2) A gene editing protein comprising one or more of an adenine single base editor constructed by a nSauriCas9 nickase mutant or an adenine single base editor constructed by a nSpCas9 nickase mutant;

[0018] The amino acid sequence of the nSauriCas9 gap enzyme mutant is shown in SEQ ID NO.19, and the amino acid sequence of the nSpCas9 gap enzyme mutant is shown in SEQ ID NO.20.

[0019] Preferably, the adenine single-base editor is formed by connecting a Cas9 nickase mutant to an adenine deaminase TadA8e or TadA9.

[0020] An application of the sgRNA / Cas9 ribonucleoprotein complex as described above in the preparation of a gene therapy drug, wherein the sgRNA / Cas9 ribonucleoprotein complex can knock out CAMK2A and / or CAMK2B and / or CAMK2G and / or CAMK2D genes of humans, mice, rats or pigs.

[0021] An application of the sgRNA / Cas9 ribonucleoprotein complex as described above in the preparation of a drug for treating cardiomyopathy. The sgRNA / Cas9 ribonucleoprotein complex can enhance myocardial protection and prevent and treat ischemic heart disease.

[0022] A plasmid vector for delivering and expressing a single base editor, comprising:

[0023] (1) one or more genes driven by the U6 promoter, wherein the gene expression driven by the U6 promoter is as described above by the sgRNA;

[0024] (2) one or more protein-encoding genes, wherein the protein-encoding genes express gene editing proteins such as the gene editing proteins in the sgRNA / Cas9 ribonucleoprotein complex described above.

[0025] An AAV vector for delivering and expressing a single base editor, the vector being composed of a single or two AAVs, comprising:

[0026] (1) one or more genes driven by the U6 promoter, where the gene expression driven by the U6 promoter is as described above by the sgRNA;

[0027] (2) one or more genes driven by a cardiomyocyte-specific promoter, wherein the cardiomyocyte-specific promoter is selected from Tnnt2 promoter or Myl2 promoter, the Tnnt2 promoter sequence is shown in SEQ ID NO.21, and the Myl2 promoter sequence is shown in SEQ ID NO.22;

[0028] The cardiomyocyte-specific promoter drives a gene encoding a protein, and the gene encoding the protein expresses a gene editing protein in the ribonucleoprotein complex described above;

[0029] (3) AAV capsid with cardiotropism.

[0030] Preferably, the AAV capsid is one or more of AAV2i8, AAV1, AAV5, AAV6, AAV8, AAV9, AAVMYO, and MyoAAV.

[0031] The present invention provides a miniaturized single-base editor for cardiac-specific CaMKII clearance and its application, which has the following beneficial effects:

[0032] (1) Compared with single base conversion, the success rate of using ABE for gene silencing is higher. The present invention designs a series of sgRNAs for screening based on the start codon site and splicing site of the gene, and finally obtains an optimal sgRNA;

[0033] (2) The present invention uses several compact Cas9s to construct single AAV-ABE vectors, and by comparing with dual AAV-ABE vectors, it is demonstrated that the editing efficiency of single AAV-ABE is significantly better than that of dual AAV-ABE;

[0034] (3) The combination of cardiac-tropic AAV9 and cardiac-specific promoter will still leak in the liver. The present invention screened out a cardiac-specific sgRNA based on the difference in the open regions of cardiac and liver chromatin.

[0035] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :Part A shows the volume of four compact Cas9s and a schematic diagram of a single AAV-ABE vector; Part B shows the sgRNA design site and the screening results in the N2a cell line (n=6), na means that the site cannot be designed with sgRNA; Part C shows the sequences of human (Hs) and mouse (Mm) sgRNA1, sgRNA6, and sgRNA7, in which non-homologous bases are marked in red; Part D shows the editing efficiency of human sgRNA1, sgRNA6, and sgRNA7 in the 293T cell line (n=3);

[0037] Figure 2:Part A shows the editing efficiency of sgRNA1, sgRNA6, and sgRNA7 in various organs of mice (heart, liver, spleen, lung, kidney, brain, and skeletal muscle) (n=4 or 5); Part B shows the ratio of editing efficiency between heart and liver and the editing efficiency of all adenine sites in the sgRNA7 targeting sequence (n=5); Part C shows the peaks of mouse heart and liver ATAC-seq and DNase-seq in IGV and the ratio of heart and liver signals after quantifying the average signal intensity using pyBigWig, n=2 for newborn mouse ATAC-seq data, n=4 for adult mouse ATAC-seq data, n=2 for newborn mouse DNase-seq data, *P<0.05, **P<0.01, ***P<0.001;

[0038] Figure 3 :Part A is the fragment at the junction of exon 7 and intron 7 after RT-PCR amplification and agarose gel electrophoresis results; Part B is Sanger sequencing of the amplified products of RT-PCR, and the arrow points to the edited site; Part C is RNA-seq analysis of the reads connecting exon 7 and exon 8, the reads connecting exon 7 and intron 7, and the total mRNA expression changes of CAMK2D (n=3); Part D is the detection of CaMKIIδ protein level changes by western blot and quantitative analysis of the bands (n=5); *P<0.05, **P<0.01, ***P<0.001;

[0039] Figure 4 :Part A is a schematic diagram of single AAV and dual AAV vectors; Part B is the “dose-effect” relationship curve between the total AAV dose and editing efficiency (n=4); Part C is a western blot to detect the changes in CaMKIIδ protein levels (n=4); mixed effects ANOVA, *Padj<0.05, **Paj<0.01, ***Padj<0.001;

[0040] Figure 5 : Dynamic changes of ejection fraction (EF) monitored by echocardiography in mice treated with single AAV-ABE and mice not treated with AAV; mixed effects ANOVA, *Padj<0.05, **Padj<0.01, ***Padj<0.001. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0042] Embodiment 1

[0043] First, a single AAV-ABE vector was constructed using nSaKKHCas9, nNme2Cas9, nSauriCas9, and nCjCas9. Next, 18 sgRNAs were designed for the start codon site and intron 5′ splicing donor site of the mouse CAMK2D gene, and the sgRNAs were cloned into a single AAV-ABE vector. Next, the results of in vitro cell line screening showed that SauriABE performed best, so SauriABE's sgRNA1, sgRNA6, and sgRNA7 were selected for in vivo experiments and the sgRNAs with the highest editing efficiency in the heart and the highest knockout efficiency for CaMKIIδ were screened. The following steps are included:

[0044] (1) Construction of a single AAV-ABE vector

[0045] Plasmids pAAV-EFS-SauriABE8e-bGH-U6-sgRNA-Bs mBI (#189925), pAAV-EFS-SaKKHABE8e-bGH-U6-sgRNA-BsmBI (#189923), CMV-Nme2ABE8e (#189928), and CMV-CjABE8e (#189929) were purchased from Addgene.

[0046] Nme2 and scaffold RNA sequences were amplified using CMV-Nme2ABE8e as a template and cloned into pAAV-EFS-SaKKHABE8e-bGH-U6-sgRNA-BsmBI using a seamless cloning kit to obtain pAAV-EFS-Nme2ABE8e-bGH-U6-sgRNA-Bs mBI.

[0047] Cj and scaffold RNA sequences were amplified using CMV-CjABE8e as a template and cloned into pAAV-EFS-SaKKHABE8e-bGH-U6-sgRNA-BsmBI using a seamless cloning kit to obtain pAAV-EFS-CjABE8e-bGH-U6-sgRNA-BsmBI.

[0048] The structures of four single AAV-ABE plasmid vectors SaKKHABE, SauriABE, Nme2ABE, and CjCas9 are shown in the figure. Figure 1 As shown in A.

[0049] (2) Design of sgRNA

[0050] The mouse CAMK2D gene sequence published in the NCBI database was searched to screen the start codon site and the protospacer adjacent motif (PAM) site near the 3′ end of the complementary chain of the 5′ splice donor site of introns 2-10. Figure 1 As shown in B, a total of 7 sgRNAs were designed for SaKKHABE, a total of 5 sgRNAs were designed for Nme2ABE, a total of 6 sgRNAs were designed for SauriABE, and a total of 0 sgRNAs were designed for CjCas9.

[0051] The 7 sgRNAs designed for SaKKHABE include: SaKKH-sgRNA1, SaKKH-sgRNA2, SaKKH-sgRNA4, SaKKH-sgRNA5, SaKKH-sgRNA7, SaKKH-sgRNA8, and SaKKH-sgRNA9, and their nucleotide sequences are shown in SEQ ID NO.23 to SEQ ID NO.29, respectively.

[0052] The five sgRNAs designed for Nme2ABE include: Nme2-sgRNA1, Nme2-sgRNA4, Nme2-sgRNA7, Nme2-sgRNA8, and Nme2-sgRNA9, and their nucleotide sequences are shown in SEQ ID NO.30 to SEQ ID NO.34, respectively.

[0053] The six sgRNAs designed for SauriABE include: Sauri-sgRNA1, Sauri-sgRNA3, Sauri-sgRNA6, Sauri-sgRNA7, Sauri-sgRNA9, and Sauri-sgRNA10, and their nucleotide sequences are shown in SEQ ID NO.35-SEQ ID NO.40, respectively.

[0054] The restriction site sequence AAAC was inserted into the 5′ end of the upstream annealing primer of the sgRNA, and the restriction site sequence CACC was inserted into the 5′ end of the downstream annealing primer. The designed sgRNA sequence was handed over to a third-party company for synthesis.

[0055] (3) Cloning sgRNA into a single AAV-ABE plasmid

[0056] After obtaining a single AAV-ABE plasmid vector and the 18 sgRNA sequences designed in step (2), the 18 sgRNAs need to be inserted into the multiple cloning sites of the corresponding plasmid vectors respectively. The specific implementation method is as follows:

[0057] 5 μL of each of the upstream and downstream annealing primers of the sgRNA were mixed and gradient annealed to form sgRNA double strands. The annealing program was as follows: (1) 95°C, 5 min, (2) 95°C, 2 min, (3) 90°C, 2 min, (4) 85°C, 2 min, (5) 80°C, 2 min, (6) 75°C, 2 min, (7) 70°C, 2 min, (8) 65°C, 2 min, (9) 60°C, 2 min, (10) 55°C, 2 min, (11) 50°C, 2 min, (12) 45°C, 2 min, (13) 40°C, 2 min, (14) 35°C, 2 min, (15) 30°C, 2 min, (16) 25°C, 2 min, (17) 20°C, storage.

[0058] Next, the single AAV-ABE plasmid vector was linearized using restriction endonuclease BsmBI. The restriction enzyme system is shown in Table 1 below:

[0059] Table 1

[0060] Reaction system Volume (μL) Plasmid vector 1 BbI 0.5 10×Buffer 3 <![CDATA[ddH2O]]> 25.5 Total volume 30

[0061] The reaction procedure is: incubate at 37°C for 1 hour. The linearized single AAV-ABE plasmid vector is subjected to agarose gel electrophoresis using 1.2% agarose, the target fragment size band is cut out, gel recovery is performed using a gel recovery kit, and the concentration is determined.

[0062] The annealed products of the 18 sgRNAs were ligated into the linearized single AAV-ABE plasmid vector using T4 DNA ligase. The ligation system is shown in Table 2 below:

[0063] Table 2

[0064] Reaction system Volume (μL) Linearized vector 5 sgRNA annealing product 1 T4 DNA ligase 0.5 5×Buffer 2 <![CDATA[ddH2O]]> 1.5 Total volume 10

[0065] The reaction procedure is: 25°C, 10 minutes. Take 5 μL of the ligation product to transform 50 μL of competent cells E. coli Stbl3, add 500 μL of LB medium, shake at 37°C for 1 hour, centrifuge at 5000 rpm for 1 minute, blow the competent cell pellet, evenly spread it on the LB plate containing ampicillin, and place it in a 37°C bacterial incubator for overnight culture. Several colonies were picked for Sanger sequencing identification, and the monoclonal colonies with positive identification results were expanded and cultured. The plasmids were extracted using an endotoxin-free plasmid extraction kit to obtain single AAV-ABE plasmids inserted with sgRNA, namely pAAV-EFS-SaKKHABE8e-U6-Camk2d sgRNA (7), pAAV-EFS-Nme2ABE8e-U6-Camk2d sgRNA (5), and pAAV-EFS-SauriABE8e-U6-Camk2d sgRNA (6).

[0066] (4) In vitro screening of sgRNA

[0067] The 18 plasmids obtained in step (3) were transfected into Neuro2a cells respectively, and the medium was changed after 8 hours. The cells were collected after 48 hours, and genomic DNA was extracted using a genomic DNA extraction kit for amplicon sequencing (AMP-Seq) analysis. The specific implementation method is as follows:

[0068] Primers for high-throughput sequencing library construction of the target site were designed, wherein the nucleotide sequence of the upstream primer F is shown in SEQ ID NO.41-SEQ ID NO.50, and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO.51-SEQ ID NO.60. Next, the target DNA fragment was amplified using genomic DNA as a template, and the PCR reaction system is shown in Table 3 below:

[0069] Table 3

[0070] Reaction system Volume (μL) Genomic DNA 5 Upstream primer F 2 Downstream primer R 2 2xTaqMix 20 <![CDATA[ddH2O]]> 11 Total volume 40

[0071] The reaction procedure is: (1) 94°C, 3 minutes, (2) 94°C, 30 seconds, (3) 60°C, 30 seconds, (4) 72°C, 30 seconds, wherein steps (2)-(4) are repeated for 30 cycles, (5) 72°C, 5 minutes, (6) 4°C, stored. The amplified product was subjected to agarose gel electrophoresis using 1.5% agarose, the target fragment size band was cut out, and the gel recovery kit was used for gel recovery. The library after gel recovery was handed over to a third-party company for high-throughput sequencing of the amplified product. The sequencing results were analyzed using CRISPResso2 and plotted using GraphpadPrism software. Figure 1As shown in B, the results show that Sauri-sgRNA1, Sauri-sgRNA6, and Sauri-sgRNA7 have higher editing efficiency.

[0072] (5) Design and verify human sgRNA

[0073] The sgRNA adapted to SauriABE was designed for the start codon site of the human CAMK2D gene and the 5′ splice site of introns 6 and 7, where:

[0074] Homo sapiens-CAMK2D-sgRNA1, the sequence of which is shown in SEQ ID NO.61;

[0075] Homo sapiens-CAMK2D-sgRNA6, the sequence of which is shown in SEQ ID NO.62;

[0076] Homo sapiens-CAMK2D-sgRNA7, the sequence of which is shown in SEQ ID NO.3.

[0077] like Figure 1 As shown in C, the sequences of sgRNA1, sgRNA6, and sgRNA7 of human (Hs) and mouse (Mm) origin are compared, in which non-homologous bases are marked in red. It can be found that sgRNA1, sgRNA6, and sgRNA7 of human and mouse origin have high homology.

[0078] The human sgRNA was verified in the 293T cell line. The specific implementation method is as described in steps (3)-(4) of this embodiment. The results are as follows: Figure 1 As shown in D, human sgRNA1, sg RNA6, and sgRNA7 have high editing efficiency in 293T cells.

[0079] (6) In order to enable Sauri-sgRNA1, Sauri-sgRNA6, and Sauri-sgRNA7 to specifically target cardiomyocytes in vivo, the EFS promoter of the pAAV-EFS-SauriABE8e-U6-Camk2dsgRNA plasmid vector was replaced with the cardiomyocyte-specific Myl2 promoter. The specific implementation method is as follows:

[0080] The Myl2 promoter sequence was amplified using mouse genomic DNA as a template, and the Myl2 promoter sequence is shown in SEQ ID NO. 22. The plasmid pAAV-EFS-SauriABE8e-U6-Camk2d sgRNA was linearized using restriction endonucleases NheI and SacI. The Myl2 promoter was then cloned into the linearized plasmid using a seamless cloning kit to obtain the pAAV-Myl2-SauriABE8e-U6-Camk2d sgRNA plasmid.

[0081] This method was used to obtain the pAAV-Myl2-SauriABE8e-U6-Camk2d sgRNA plasmid vector containing Sauri-sgRNA1, Sauri-sgRNA6, and Sauri-sgRNA A7.

[0082] (7) Preparation of AAV9 virus

[0083] After replacing the promoter, the pAAV-Myl2-SauriABE8e-U6-Camk2d sgRNA plasmid vector containing Sauri-sgRNA1, Sauri-sgRNA6, and Sauri-sgRNA7 was then used for AAV9 virus packaging preparation. The specific implementation method is as follows:

[0084] ① Prepare HEK293 cells. For each plasmid to be packaged, 10 dishes of HEK293 cells should be prepared. The cell density should reach 70-80% on the day of transfection.

[0085] ② 2 hours before transfection, change to fresh culture medium (10% FBS, 1% Pen-Strep in DMEM), add 140μg plasmid vector, 140μg AAV9-Rep / Cap plasmid and 320μg pHelper to 20mL Opti-MEM, mix well, add 2.4mL PEI (1mg / mL) and mix again, let stand at room temperature for 15 minutes. Add 2mL PEI-DNA mixture to each plate and shake well;

[0086] ③ After 12 hours, the culture medium was replaced with serum-free medium (DMEM supplemented with 1% Pen-Strep);

[0087] ④ After continuing to culture for 72 hours, harvest the cells, transfer the cells and the culture medium to a sterile centrifuge tube, and centrifuge at 2000 rpm for 5 minutes at 4°C. Resuspend the cell pellet in 7 mL of lysis buffer to a final volume of about 10 mL, add 1 μL of universal nuclease (≥250 units / μL) to the cell suspension, and transfer it to a 50 mL centrifuge tube;

[0088] ⑤ Lyse the cells using the following freeze-thaw cycles: -80°C for 30 minutes, 37°C for 30 minutes, -80°C for 30 minutes, and 37°C for 30 minutes. After thawing at 37°C, centrifuge at 4000 rpm for 30 minutes at 4°C, and spread the supernatant on a gradient of iodixanol for ultracentrifugation to separate AAV virus particles;

[0089] ⑥ In a rotary concentrator with a molecular weight cutoff of 100 kDa, the extracted AAV-containing layer (about 4 mL) was mixed with 10 mL PBS + 0.001% non-ionic surfactant Pluronic F-68 and centrifuged at 5000 × g for 30 minutes at 4°C. The effluent was removed, and then 15 mL of new PBS containing F68 (0.001% F68 in PBS) was added and the washing was repeated 3 times;

[0090] ⑦ The AAV titer was determined by real-time fluorescence quantitative PCR (RT-qPCR), and the purified AAV virus solution was packaged and stored in a -80°C refrigerator.

[0091] Through the above steps, AAV-Myl2-SauriABE8e-U6-Camk2d sgRNA loaded with Sauri-sgRNA1, Sauri-sgRNA6, and Sauri-sgRNA7 respectively can be obtained.

[0092] (8) In vivo screening of sgRNA

[0093] In order to verify the effectiveness of Sauri-sgRNA1, Sauri-sgRNA6, and Sauri-sgRNA7 in vivo, the prepared AAV9 was injected into mice, and amplicon sequencing analysis and western blot detection were performed. The specific implementation method is as follows:

[0094] C57BL / 6 mice were divided into four groups after birth. One group was used as the control group. The other three groups were injected subcutaneously with AAV-Myl2-SauriABE8e-U6-Camk2dsgRNA (1×10 11 vg). The mice were fed for 7 days. After 7 days, fresh heart, liver, spleen, lung, kidney, brain, and skeletal muscle tissues were collected from the mice. Genomic DNA from all organs was extracted using a genomic DNA extraction kit and amplicon sequencing (AMP-Seq) analysis was performed. The specific implementation method is as described in step (3) of this embodiment.

[0095] The results showed that the editing efficiencies of Sauri-sgRNA1, Sauri-sgRNA6, and Sauri-sgRNA A7 in the heart were 6.57%, 14.26%, and 22.40%, respectively. Despite the use of a cardiac-specific promoter, Sauri-sgRNA1 and Sauri-sgRNA6 still had a certain degree of editing in the liver, at 2.45% and 4.33%, respectively. Although Sauri-sgRNA7 showed the highest editing efficiency in the heart, it produced almost no editing in the liver (e.g. Figure 2 Moreover, among all adenines covered by Sauri-sgRNA7, A7 at the target site has the highest editing efficiency (as shown in Figure 2A). Figure 2 (shown in B)

[0096] Next, we analyzed the high-throughput sequencing (ATAC-seq) data of chromatin accessibility in different mouse organs and DNase-seq data in the public database NCBI. Figure 2 As shown in C, the peaks of mouse heart and liver ATAC-seq and DNase-seq are viewed in IGV, and the displayed area is ±200bp around the sgRNA target site; pyBigWig is used to quantify the average signal intensity and calculate the ratio of heart to liver signals.

[0097] The results showed that the chromatin regions targeted by Sauri-sgRNA1 and Sauri-sgRNA6 were open in both the heart and liver, and the chromatin region targeted by Sauri-sgRNA7 was open in the heart but closed in the liver (e.g. Figure 2 This result explains the organ selectivity of Sauri-sgRNA7.

[0098] Conclusion: Sauri-sgRNA7 not only has the highest editing efficiency but also has cardiac specificity.

[0099] (9) Verification of editing effects at the cDNA level

[0100] Total RNA was extracted from the heart tissue obtained in step (8) using a total RNA extraction kit, and RT-PCR was performed on the control group and the Sauri-sgRNA7 group using gene-specific primers to amplify the fragment at the junction of exon 7 and intron 7.

[0101] Gene-specific primers:

[0102] RTprimer, the nucleotide sequence of which is shown in SEQ ID NO.63;

[0103] PCRprimer-Fwd, the nucleotide sequence of which is shown in SEQ ID NO.64;

[0104] PCRprimer-Rvs, the nucleotide sequence of which is shown in SEQ ID NO.65.

[0105] After Sauri-sgRNA7 edits the 5′ splice site of intron 7, intron 7 will be connected to exon 7 and a stop codon will appear in advance. The fragment at the junction of exon 7 and intron was amplified by RT-PCR. The results of agarose gel electrophoresis are as follows Figure 3 As shown in A, the Sauri-sgRNA7 group has obvious bands. The amplified product was subjected to Sanger sequencing, and the results are as follows Figure 3 As shown in B, the 5′ splice site of intron 7 of Sauri-sgRNA7 group was successfully edited.

[0106] Conclusion: Sauri-sgRNA7 verified successful editing at the cDNA level.

[0107] (10) Verify the effect of Sauri-sgRNA7 in knocking out CaMKIIδ

[0108] In order to explore the changes in the mRNA level of Camk2d and the protein level of CaMKIIδ after the 5′ splicing donor site of intron 7 was destroyed by Sauri-sgRNA7, RNA-seq analysis and western blot analysis were performed on the heart tissue obtained in step (8). The specific implementation method is as follows:

[0109] After obtaining RNA, reverse transcription reaction was performed to reverse transcribe RNA into cDNA, and RNA library was prepared using library construction kit, including end modification, linker connection and PCR amplification. PE150 double-end sequencing was performed using Illumina Nova-seq high-throughput sequencing platform. FastQC was used to control the quality of sequencing data, and Cutadapt was used to trim the adapters, low-quality bases and filter out short fragments. STAR was used to map the quality-controlled data to the mouse reference genome mm10, and featureCounts was used to calculate the number of genes mapped to each gene to quantify gene expression levels. The results are shown in Figure 3 As shown in middle C, after using Sauri-sgRNA7 to disrupt the 5′ splice donor site of intron 7, the connection between exon 7 and exon 8 was significantly reduced, the connection between exon 7 and intron 7 was significantly increased, and the total CAMK2D mRNA level was significantly reduced.

[0110] Collect tissue samples into grinding tubes, add RIPA lysis buffer mixed with protease inhibitors, and then use a tissue grinder to break the tissue. After stirring and lysing on ice for 30 minutes, centrifuge at 12000rpm for 15 minutes at 4°C, collect the supernatant and use a BCA kit to determine the protein concentration. Mix the sample with 5×SDS loading buffer and denature it. Load the denatured sample into a polyacrylamide gel for protein electrophoresis separation and then transfer it to a PVDF membrane. Block the membrane with 5% skim milk at room temperature for 1 hour, then incubate with specific primary antibodies (Anti-CAMK2D, 1:2000; Anti-GAPDH, 1:2000) at 4°C overnight. Then rinse three times with TBST for 10 minutes each time, and incubate the secondary antibody (HRP-Gt-Anti-Ms, 1:10000; HRP-Gt-Anti-Rb, 1:10000) at room temperature. Continue to rinse three times with TBST for 10 minutes each time. The substrate was incubated with ECL chemiluminescent solution and the results were captured using an imaging system. Figure 3 As shown in D, consistent with the changes in mRNA levels, CaMKIIδ protein was significantly reduced after disruption of the 5' splice donor site of intron 7 using Sauri-sgRNA7.

[0111] Conclusion: The use of Sauri-sgRNA7 can not only achieve cardiac-specific editing, but also effectively knock out CaMKIIδ in the heart.

[0112] (11) Comparison of single AAV vector and dual AAV vector

[0113] In order to compare single AAV with dual AAV vectors, three dual AAV vectors were constructed, e.g. Figure 4 As shown in A, they are: V1, carrying a split SauriABE and a copy of U6-sgRNA7; V2, carrying a split SauriABE and two copies of U6-sgRNA7; V3, carrying a split SpCas9-ABE and two copies of U6-sgRNA7.

[0114] A total dose of 2.5×10 10 , 5×10 10 , 10×10 10 , 20×10 10 , 40×10 10The mice were injected with a dose gradient of vg and kept for 7 days. After 7 days, fresh heart tissues were collected from the mice and genomic DNA from all organs was extracted using a genomic DNA extraction kit for amplicon sequencing (AMP-Seq) analysis. The specific implementation method is as described in step (4) of this embodiment. A “dose-effect” relationship curve was drawn based on the AAV dose and editing efficiency. The results are shown in Figure 4 As shown in B, when the total AAV dose is the same, the editing efficiency of single AAV is significantly higher than that of dual AAV.

[0115] Next, the level of CaMKIIδ protein in the heart was detected by western blot, and the specific implementation method was as described in step (10) of this embodiment. Figure 4 As shown in middle C, the CaMKIIδ protein in the single AAV group was significantly reduced with the increase of AAV dose.

[0116] Conclusion: The editing efficiency of single AAV-ABE vector is significantly better than that of dual AAV-ABE vector

[0117] (12) Investigate the therapeutic effect on myocardial infarction in mice

[0118] Based on the above experiments, it was confirmed that the use of AAV9 to deliver adenine base editors and Sauri-sgRNA7 into mice can effectively knock out CaMKIIδ in the heart. Next, the therapeutic effect of this therapy on the mouse myocardial infarction model will be verified.

[0119] First, adult male C57BL / 6 mice were selected, and a mouse myocardial infarction model was established by ligating the left anterior descending branch of the ventricle. Echocardiography analysis was performed two days before and two days after the animal surgery to screen out mice with significant cardiac function decline caused by myocardial infarction surgery and similar disease severity. Three days after myocardial infarction in the mice in the treatment group, AAV was injected through the tail vein, and the therapeutic effect was observed. At 10, 20, and 30 days after myocardial infarction, echocardiography was used to continuously monitor the cardiac function of the mouse model to observe the effect of AAV treatment on the trajectory of cardiac function changes. Figure 5 As shown, the results showed that compared with the control group mice that did not receive AAV treatment, the cardiac function of the AAV-treated myocardial infarction mice was significantly restored one week after administration, and this improvement effect could last for at least one month.

[0120] Conclusion: Knocking out CaMKIδ in the heart by delivering SauriABE and Sauri-sgRNA7 using a single AAV can effectively alleviate the phenotype of myocardial infarction mice and enhance myocardial protection.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A sgRNA, characterized in that include: sgRNA7, the nucleotide sequence of sgRNA7 is shown in SEQ ID NO.1-SEQ ID NO.16; Scaffold RNA, the nucleotide sequence of the scaffold RNA is shown in SEQ ID NO.17 or SEQ ID NO.18; The 3′ end of sgRNA7 is directly connected to the 5′ end of scaffold RNA to form a complete sgRNA molecule.

2. An sgRNA according to claim 1, characterized in that: The target sequence of sgRNA7 is located at the junction of exon 7 and intron 7 of CAMK2A, CAMK2B, CAMK2D or CAMK2G gene of human, mouse, rat or pig.

3. A sgRNA / Cas9 ribonucleoprotein complex, characterized in that include: The sgRNA according to claim 1 or 2; A gene editing protein, comprising one or more of an adenine single base editor constructed by a nSauriCas9 nickase mutant or an adenine single base editor constructed by a nSpCas9 nickase mutant; The amino acid sequence of the nSauriCas9 gap enzyme mutant is shown in SEQ ID NO.19, and the amino acid sequence of the nSpCas9 gap enzyme mutant is shown in SEQ ID NO.

20.

4. The sgRNA / Cas9 ribonucleoprotein complex according to claim 3, characterized in that: The adenine single-base editor is formed by connecting a Cas9 nickase mutant to adenine deaminase TadA8e or TadA9.

5. Use of the sgRNA / Cas9 ribonucleoprotein complex as claimed in claim 3 or 4 in the preparation of a gene therapy drug, characterized in that: The sgRNA / Cas9 ribonucleoprotein complex can knock out the CAMK2A and / or CAMK2B and / or CAMK2G and / or CAMK2D genes of humans, mice, rats or pigs.

6. Use of the sgRNA / Cas9 ribonucleoprotein complex according to claim 3 or 4 in the preparation of a drug for treating cardiomyopathy, characterized in that: sgRNA / Cas9 ribonucleoprotein complex can enhance myocardial protection and prevent ischemic heart disease.

7. A plasmid vector for delivering and expressing a single-base editor, characterized in that: include: One or more genes driven by a U6 promoter, wherein the gene expression driven by the U6 promoter is the sgRNA as described in claim 1-2; One or more protein-encoding genes, wherein the protein-encoding gene expresses the gene editing protein in the sgRNA / Cas9 ribonucleoprotein complex as described in claim 3 or 4.

8. An AAV vector for delivering and expressing a single base editor, characterized in that: The vector is composed of a single or dual AAV vector and includes: One or more genes driven by a U6 promoter, wherein the gene expression driven by the U6 promoter is the sgRNA as described in claim 1 or 2; One or more genes driven by a cardiomyocyte-specific promoter, wherein the cardiomyocyte-specific promoter is selected from Tnnt2 promoter or Myl2 promoter, the Tnnt2 promoter sequence is shown in SEQ ID NO.21, and the Myl2 promoter sequence is shown in SEQ ID NO.22; The cardiomyocyte-specific promoter drives a protein-encoding gene, and the protein-encoding gene expresses the gene editing protein in the ribonucleoprotein complex as described in claim 3 or 4; AAV capsids with cardiotropism.

9. An AAV vector for delivering and expressing a single base editor according to claim 8, characterized in that: The AAV capsid is one or more of AAV2i8, AAV1, AAV5, AAV6, AAV8, AAV9, AAVMYO, and MyoAAV.

10. Use of the AAV vector according to claim 8 or 9 in the preparation of a gene therapy drug, characterized in that: Use in knocking out CAMK2A and / or CAMK2B and / or CAMK2G and / or CAMK2D genes in humans, mice, rats or pigs.

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