SgRNA, gene editing system and use thereof

Abstract

This invention relates to the field of gene editing technology, and in particular to an sgRNA, a gene editing system, and their uses. The sgRNA comprises a target sequence targeting the PCSK9 gene or a target sequence targeting the APOC3 gene. The nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprises a sequence as shown in any one of SEQ ID Nos. 1-22; the nucleotide sequence encoding the target sequence targeting the APOC3 gene comprises a sequence as shown in any one of SEQ ID Nos. 23-36. Through systematic screening and experimental verification, this invention has obtained sgRNAs capable of efficiently and specifically targeting both the PCSK9 and APOC3 genes. Based on the screened sgRNAs, this invention has developed a dual-target gene editing system capable of simultaneously targeting both the PCSK9 and APOC3 genes. This provides a reliable core component for developing gene-editing-based therapies for lipid metabolism diseases, and has significant clinical significance and application prospects.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to an sgRNA, a gene editing system, and its uses. Background Technology

[0002] In the field of gene editing therapy, precise intervention targeting specific disease-causing genes has become a highly promising treatment strategy. Gene editing technology also shows significant potential for metabolic diseases such as dyslipidemia.

[0003] Previous studies have used a disposable GalNAc-LNP delivery system to load adenine base editor (ABE) targeting the PCSK9 gene, enabling efficient in vivo editing that reduces serum PCSK9 and low-density lipoprotein cholesterol (LDL-C) levels by approximately 80% and 62%, respectively. This approach has entered the VERVE-101 / 102 human clinical trial phase and is suitable for the prevention of hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD).

[0004] On the other hand, the world's first case of gene editing therapy for hypertriglyceridemia (TG) has also been achieved: by delivering CS-121 (tBE base editor) targeting the APOC3 gene in a single dose, a significant decrease in TG levels was observed in patients with hyperchylomicronemia after a single administration, with no obvious adverse reactions. This provides a new treatment possibility for patients with high TG and related pancreatitis risk.

[0005] However, clinically common dyslipidemia often presents as mixed hyperlipidemia, where both LDL-C and triglycerides are elevated simultaneously. Existing single-target intervention strategies (inhibiting only PCSK9 or APOC3) can partially correct the lipid profile, but they struggle to simultaneously reduce both key pathogenic lipids, leaving patients still facing residual cardiovascular risk or the risk of acute pancreatitis. Delivering two separate editing systems would require increased dosages, posing safety risks. Therefore, developing a dual-target gene editing system capable of simultaneously and efficiently editing both PCSK9 and APOC3 is crucial for comprehensive lipid management.

[0006] Currently, technological development in this field mainly focuses on optimizing the base editor itself and developing novel editors, while research on sgRNAs that guide the precise localization of the editor is relatively insufficient. As one of the core components of the editing system, the sequence design of sgRNA directly affects the efficiency, specificity, and final protein knockout effect of gene editing, and is a key factor determining the success or failure of treatment.

[0007] Designing sgRNAs with both high editing efficiency and high protein knockout activity for specific gene loci remains a significant challenge: on the one hand, the activity of sgRNAs is affected by multiple factors such as their sequence characteristics, chromatin accessibility of the target site, and local genome structure, making prediction and screening difficult; on the other hand, the optimal sgRNA design rules are often specific for different genes or even different loci of the same gene, requiring extensive systematic screening and experimental verification.

[0008] Therefore, developing and identifying sgRNAs suitable for human treatment that can efficiently guide editors to edit the PCSK9 and APOC3 genes and achieve high-level protein knockout is a key prerequisite and an urgent technical problem to be solved in advancing the clinical translation of dual-target lipid editing therapy. Summary of the Invention

[0009] In view of the shortcomings of the prior art described above, in order to solve the problems of the lack of sgRNA that can efficiently and specifically target the PCSK9 or APOC3 gene, and the lack of dual-target gene editing systems that can simultaneously reduce LDL-C and TG levels, the purpose of this application is to provide an sgRNA, a gene editing system and its uses to solve the problems in the prior art.

[0010] To achieve the above and other related objectives, this application first provides an sgRNA, wherein the sgRNA comprises a target sequence targeting the PCSK9 gene or a target sequence targeting the APOC3 gene; the nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprises a sequence as shown in any one of SEQ ID No. 1 to 22; and the nucleotide sequence encoding the target sequence targeting the APOC3 gene comprises a sequence as shown in any one of SEQ ID No. 23 to 36.

[0011] This application further provides a gene editing system selected from any of the following forms:

[0012] a) A single-target gene editing system: comprising a base editor and an sgRNA; said sgRNA comprising a target sequence targeting the PCSK9 gene or a target sequence targeting the APOC3 gene; the nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprising a sequence as shown in any one of SEQ ID No. 1-22; the nucleotide sequence encoding the target sequence targeting the APOC3 gene comprising a sequence as shown in any one of SEQ ID No. 23-36;

[0013] b) A dual-target gene editing system: comprising a base editor and two sgRNAs, namely sgRNA (1) and sgRNA (2); wherein sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 1 to 22; wherein sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 23 to 36.

[0014] This application subsequently provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned sgRNA.

[0015] This application then provides an expression vector containing the above-described nucleic acid molecules.

[0016] This application then provides a cell in which the above-mentioned nucleic acid molecules or the above-mentioned expression vectors are integrated into the genome of the cell.

[0017] This application also provides a composition comprising the above-described sgRNA, or the above-described gene editing system, or the above-described nucleic acid molecule, or the above-described expression vector, and a delivery vector.

[0018] In addition, this application provides a gene editing method, including delivering the above-mentioned sgRNA, or the above-mentioned gene editing system, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned composition to cells.

[0019] This application concludes by providing the use of the above-described sgRNA, gene editing system, nucleic acid molecule, expression vector, cell, and composition in the preparation of any of the following products:

[0020] 1) Cells and / or animals modified with the PCSK9 gene and / or APOC3 gene;

[0021] 2) Cells and / or animals with PCSK9 gene knockout or knockdown and APOC3 gene knockout;

[0022] 3) Products for the prevention and / or treatment of diseases related to the PCSK9 gene and / or APOC3 gene.

[0023] Compared with the prior art, the beneficial effects of this application are as follows:

[0024] 1) Through systematic screening and experimental verification, this invention has obtained sgRNAs that can efficiently and specifically target the PCSK9 and APOC3 genes. These sgRNA sequences, when designed for practical applications, can significantly improve the editing efficiency of target sites and the final protein knockout level, providing a reliable core component for developing gene-editing-based therapies for lipid metabolism diseases.

[0025] 2) Based on the sgRNAs screened above, this invention develops a dual-target gene editing system that can simultaneously target the PCSK9 and APOC3 genes. The two sgRNAs in this system can share the same base editor for delivery, enabling simultaneous knockdown of two key disease-related genes in a single treatment. This not only avoids the problems associated with delivering two separate systems, such as increased vector and editor dosages and potential safety risks, but also simplifies the manufacturing process, laying the foundation for the development of gene-editing drugs.

[0026] 3) The dual-target editing system of this invention can simultaneously reduce the levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) in plasma through a single intervention. This is expected to more comprehensively correct the lipid imbalance in patients with mixed hyperlipidemia, thereby potentially reducing their residual cardiovascular risk and pancreatitis risk more effectively, which has significant clinical significance and application prospects. Attached Figure Description

[0027] Figure 1 This shows the knockout effect of PCSK9 protein after single-target editing in Example 2.

[0028] Figure 2 This shows the knockout effect of APOC3 protein after single-target editing in Example 2.

[0029] Figure 3 This refers to the editing efficiency of the liver dual-target PCSK9 / APOC3 in Example 5.

[0030] Figure 4 The percentage of plasma PCSK9 / APOC3 protein knockdown in Example 5.

[0031] Figure 5 This represents the decrease in plasma LDL-C from baseline on day 28 of administration in Example 5.

[0032] Figure 6 This represents the decrease in plasma TG from baseline on day 28 after administration in Example 5. Detailed Implementation

[0033] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0034] The first aspect of this application provides an sgRNA, said sgRNA comprising a target sequence targeting the PCSK9 gene or a target sequence targeting the APOC3 gene; the nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprises a sequence as shown in any one of SEQ ID No. 1 to 22; the nucleotide sequence encoding the target sequence targeting the APOC3 gene comprises a sequence as shown in any one of SEQ ID No. 23 to 36.

[0035] In some specific embodiments, the nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprises the sequence shown in any one of SEQ ID No. 1, 5, 7, 14, 18, 22.

[0036] In some specific embodiments, the nucleotide sequence encoding the target sequence targeting the APOC3 gene comprises a sequence as shown in any one of SEQ ID No. 24, 25, 32, 36.

[0037] In some specific implementations, the sgRNA also includes a scaffold sequence.

[0038] Preferably, the nucleotide sequence encoding the scaffold sequence comprises the sequence shown in SEQ ID No. 37.

[0039] In some specific embodiments, the sgRNA has three phosphorothioamide (PS) bonds introduced at each of the 5′ and 3′ ends, and a 2′-O-methyl (2′-OMe) modification introduced at the 2′-hydroxyl position to increase nuclease tolerance and reduce immunogenicity.

[0040] A second aspect of this application provides a gene editing system selected from any of the following:

[0041] a) A single-target gene editing system: comprising a base editor and an sgRNA; said sgRNA comprising a target sequence targeting the PCSK9 gene or a target sequence targeting the APOC3 gene; the nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprising a sequence as shown in any one of SEQ ID No. 1 to 22; the nucleotide sequence encoding the target sequence targeting the APOC3 gene comprising a sequence as shown in any one of SEQ ID No. 23 to 36.

[0042] b) A dual-target gene editing system: comprising a base editor and two sgRNAs, namely sgRNA (1) and sgRNA (2); wherein sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 1 to 22; wherein sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 23 to 36.

[0043] In some specific embodiments, in the single-target gene editing system, the base editor is an adenine base editor, the sgRNA contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 1, 4, 5, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21.

[0044] Preferably, in the single-target gene editing system, the base editor is an adenine base editor, the sgRNA contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 1, 5, 7, 14, and 18.

[0045] In some specific embodiments, in the single-target gene editing system, the base editor is a cytosine base editor, the sgRNA contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 2, 3, 6, 9, 10, 11, 13, 14, 15, 16, 17, 22.

[0046] Preferably, in the single-target gene editing system, the base editor is a cytosine base editor, the sgRNA contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 14 or 22.

[0047] In some specific embodiments, in the single-target gene editing system, the base editor is an adenine base editor, the sgRNA contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 23, 25, 26, 27, 32, 33.

[0048] Preferably, in the single-target gene editing system, the base editor is an adenine base editor, the sgRNA contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 25 and 32.

[0049] In some specific embodiments, in the single-target gene editing system, the base editor is a cytosine base editor, the sgRNA contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 24, 25, 28, 29, 30, 31, 32, 34, 35, 36.

[0050] Preferably, in the single-target gene editing system, the base editor is a cytosine base editor, the sgRNA contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 24, 25, 32, and 36.

[0051] In some specific embodiments, in the dual-target gene editing system, the base editor is an adenine base editor; the sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains any one of the sequences shown in SEQ ID No. 1, 4, 5, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21; the sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains any one of the sequences shown in SEQ ID No. 23, 25, 26, 27, 32, 33.

[0052] Preferably, in the dual-target gene editing system, the base editor is an adenine base editor; the sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 1, 5, 7, 14, 18; the sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 25, 32.

[0053] In some specific embodiments, in the dual-target gene editing system, the base editor is a cytosine base editor; the sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains any one of the sequences shown in SEQ ID No. 2, 3, 6, 9, 10, 11, 13, 14, 15, 16, 17, 22; the sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains any one of the sequences shown in SEQ ID No. 24, 25, 28, 29, 30, 31, 32, 34, 35, 36.

[0054] Preferably, in the dual-target gene editing system, the base editor is a cytosine base editor; the sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 14 or 22; the sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 24, 25, 32, or 36.

[0055] In this application, "adenine base editor" refers to a fusion protein capable of deaminated adenine (A) at specific target sites on DNA to convert it to hypoxanthine (I) without causing DNA double-strand breaks, thereby achieving efficient and targeted conversion of A·T base pairs to G·C base pairs during cell repair. This includes, but is not limited to, ABE8e, CE-ABE8e, ABE9, ABE7.10, and their functional variants and derivatives based on different Cas proteins (such as SpCas9, SaCas9, Cas12b, etc.).

[0056] Preferably, the adenine base editor is selected from ABE8e or CE-ABE8e.

[0057] Among them, CE-ABE8e is the base editor disclosed in the patent document with publication number "CN117568313A".

[0058] In this application, "cytosine base editor" refers to a fusion protein capable of deaminating cytosine (C) at specific target sites on DNA to convert it to uracil (U) without causing DNA double-strand breaks, thereby achieving efficient and targeted conversion of C·G base pairs to T·A base pairs during cell repair. Examples include, but are not limited to, AncBE4max and CE-A3A. 1048-1063 、 BE4max, Target-AID, and their functional variants and derivatives based on different Cas proteins (such as SpCas9, SaCas9, Cas12b, etc.).

[0059] Preferably, the cytosine base editor is selected from AncBE4max or CE-A3A. 1048-1063 .

[0060] Among them, CE-A3A 1048-1063 The base editor disclosed in the patent document with publication number "CN111172133B".

[0061] In the second aspect, when the gene editing system is actually prepared, formulated, or administered, its base editor and sgRNA can exist and be combined in various physical forms, depending on the production process, delivery vector type, and route of administration.

[0062] The base editor may exist in, but is not limited to, the following forms: protein form (i.e., polypeptides or proteins obtained after in vitro expression and purification, which can be pre-assembled with sgRNA into ribonucleoprotein complexes RNP); messenger RNA form (i.e., in vitro transcribed mRNA encoding base editor proteins, which, after entering the cell, is translated to produce functional editor proteins); DNA plasmid form (i.e., circular DNA expression vectors carrying base editor coding sequences, which need to be transcribed and translated in the cell); viral vector form (i.e., base editor expression cassettes integrated into the genome of recombinant viruses, such as AAV and lentiviral vectors, which achieve long-term expression through viral infection); and non-viral linearized DNA form (i.e., linear expression cassettes used for transient expression).

[0063] Accordingly, the sgRNA may exist in, but is not limited to, the following forms: chemically synthesized mature sgRNA (which can be directly co-delivered with a protein or RNP complex); in vitro transcribed sgRNA (which can be co-delivered with mRNA); DNA plasmid form (i.e., plasmid DNA carrying the sgRNA expression cassette, which needs to be transcribed into mature sgRNA in the cell); viral vector form (i.e., sgRNA expression cassette integrated into the viral genome); and linearized DNA expression cassette (such as PCR amplification products, used for transient expression).

[0064] Based on the above, the combined forms of the gene editing system include, but are not limited to: ribonucleoprotein complexes (base editor protein pre-assembled with chemically synthesized sgRNA); mRNA and sgRNA co-preparations (mRNA encoding a base editor mixed with chemically synthesized or in vitro transcribed sgRNA for delivery); single plasmid systems (the same plasmid vector simultaneously carries a base editor expression cassette and an sgRNA expression cassette); dual plasmid systems (base editor expression plasmids and sgRNA expression plasmids prepared separately and then co-delivered); viral vector systems (single or mixed viral vectors carrying a base editor expression cassette and an sgRNA expression cassette, such as an AAV dual vector system or an integrated AAV vector); mRNA and viral vector hybridization systems (e.g., delivering a base editor in mRNA form and delivering an sgRNA expression cassette in an AAV vector); DNA expression cassette and RNP hybridization systems, etc.

[0065] All of the above-mentioned forms can be adapted to the type of delivery vector (such as lipid nanoparticles, viral particles, electroporation systems, hydrodynamic injection, etc.) and all fall within the protection scope of the gene editing system described in this application.

[0066] Preferably, when the gene editing system is a combination of mRNA and sgRNA, the mass ratio of sgRNA to mRNA encoding the base editor is 1:1 to 5.

[0067] A third aspect of this application provides a nucleic acid molecule comprising a nucleotide sequence encoding the sgRNA described in the first aspect.

[0068] In some specific embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a base editor in the gene editing system described in the second aspect.

[0069] A fourth aspect of this application provides an expression vector containing the nucleic acid molecule described in the third aspect.

[0070] In this application, "expression vector" refers to a recombinant DNA molecule that can autonomously replicate or integrate into the host genome and contains regulatory elements necessary to guide the transcription and / or translation of a foreign gene within a specific host cell. This includes, but is not limited to, the following types: plasmid vectors, such as the pUC series, pcDNA3.1 series, and pLVX vectors; viral vectors, such as vectors constructed based on lentiviruses, adeno-associated viruses (AAVs, such as serotypes AAV8 and AAV9), and adenoviruses; and linearized DNA templates for in vitro transcription.

[0071] The fifth aspect of this application provides a cell in which the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect is integrated into the genome of the cell.

[0072] In the fifth aspect, "cell" refers to a class of cells used as bioreactors or expression hosts, which, after artificial modification, carry the nucleic acid molecules described in the third aspect or the expression vector described in the fourth aspect, and are capable of efficiently transcribing, translating, or assembling the sgRNA described in the first aspect or the gene editing system described in the second aspect of this application. This includes, but is not limited to: industrial expression cell lines used for the large-scale production of base editor proteins and / or sgRNA and / or gene editing systems (such as BL21(DE3) Escherichia coli for protein purification, the HEK293 suspension-adapted cell line for mammalian protein production, and the CHO cell line).

[0073] The sixth aspect of this application provides a composition comprising the sgRNA described in the first aspect, or the gene editing system described in the second aspect, or the nucleic acid molecule described in the third aspect, or the expression vector and delivery vector described in the fourth aspect.

[0074] In this application, "delivery vector" refers to an artificial assembly system or naturally derived vector capable of encapsulating, packaging, or conjugating the sgRNA described in the first aspect, the gene editing system described in the second aspect, the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect, to help them cross biological barriers, target specific tissues or cells, and promote their entry into the cytoplasm or cell nucleus for functional delivery. This includes, but is not limited to, lipid nanoparticles (such as GalNAc-LNP), viral vectors (such as adeno-associated virus vectors AAV8, AAV9, AAVDJ, lentiviral vectors, adenovirus vectors), non-lipid nanomaterials (such as PLGA polymer nanoparticles, gold nanoparticles, exosomes, cell-penetrating peptide-modified delivery systems), virus-like particles, and conjugates (such as GalNAc-sgRNA conjugates, antibody fragment-nucleic acid conjugates).

[0075] In some specific embodiments, the delivery carrier is a lipid nanoparticle.

[0076] Preferably, the lipid nanoparticles are lipid nanoparticles that target the liver.

[0077] In this application, the "liver-targeting lipid nanoparticles" refer to a class of nanoscale lipid carrier systems that are specifically designed to efficiently deliver the sgRNA described in the first aspect, the gene editing system described in the second aspect, the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect to liver tissue, including but not limited to: GalNAc-LNP, DLin-MC3-DMA-based LNP, ALC-0315-based LNP, SM-102-based LNP, cKK-E12-based LNP, and TT3-based LNP.

[0078] More preferably, the lipid nanoparticles shown are GalNAc-LNP.

[0079] In some specific embodiments, the composition further includes pharmaceutically acceptable excipients.

[0080] In this application, "pharmaceuticalally acceptable excipient" refers to a pharmaceutical excipient or additive required for the formulation of gene-editing drug preparations that is biocompatible with the active ingredient (including sgRNA, base editor protein or their encoding nucleic acid, and delivery vector) and does not cause excessive toxicity, irritation, allergic reactions or other side effects to subjects. For drug formulations based on gene editing systems, the excipients include, but are not limited to: ionizable cationic lipids (such as DLin-MC3-DMA, SM-102, ALC-0315), phospholipids (such as distearylphosphatidylcholine DSPC), cholesterol, and PEGylated lipids (such as PEG2000-DMG) required for lipid nanoparticle delivery systems; buffer salts (such as phosphate buffer, Tris-HCl), sodium chloride, sucrose, mannitol, and poloxamer 188 required for viral vector formulations; lyophilization protectants (such as sucrose, trehalose, and mannitol), fillers (such as mannitol and glycine), and pH adjusters (such as sodium hydroxide and hydrochloric acid) required for non-viral vector lyophilized formulations; and isotonic adjusters (such as sodium chloride and glucose), stabilizers (such as human serum albumin and polysorbate 80), preservatives (such as benzyl alcohol, suitable for multi-dose formulations) and water for injection required for injectable formulations. The excipient should be suitable for the intended route of administration (including intravenous injection, subcutaneous injection, portal vein injection, etc.) and dosage form (including lipid nanoparticle injection, viral vector suspension, lyophilized powder injection, etc.), and maintain the stability and bioactivity of the active ingredient under storage and use conditions.

[0081] The seventh aspect of this application provides a gene editing method, comprising delivering to cells the sgRNA described in the first aspect, or the gene editing system described in the second aspect, or the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect, or the composition described in the sixth aspect.

[0082] In some specific implementations, when the editing target is the splice site region of the PCSK9 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in any one of SEQ ID No. 1-6, 8-21.

[0083] Preferably, when the editing target is the splice site region of the PCSK9 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in any one of SEQ ID No. 1, 5, 14, and 18.

[0084] In some specific implementations, when the editing target is the exon 3 region of the PCSK9 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in SEQ ID No. 7.

[0085] In some specific implementations, when the editing target is the exon 7 region of the PCSK9 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in SEQ ID No. 22.

[0086] In some specific implementations, when the editing target is the start codon region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in SEQ ID No. 23.

[0087] In some specific implementations, when the target of editing is the secretory signal peptide region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in SEQ ID No. 24.

[0088] In some specific implementations, when the editing target is the splice site region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes any one of the sequences shown in SEQ ID No. 25-27, 32-33, and 35.

[0089] Preferably, when the editing target is the splice site region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in any one of SEQ ID No. 25 and 32.

[0090] In some specific implementations, when the editing target is exon 1 region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in any one of SEQ ID No. 28-31.

[0091] In some specific implementations, when the editing target is the exon 2 region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in any one of SEQ ID No. 34 or 36.

[0092] Preferably, when the editing target is the exon 2 region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA contains the sequence shown in SEQ ID No. 36.

[0093] This application provides, in its eighth aspect, the use of the sgRNA described in the first aspect, the gene editing system described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the cell described in the fifth aspect, and the composition described in the sixth aspect in the preparation of any of the following products:

[0094] 1) Cells and / or animals modified with the PCSK9 gene and / or APOC3 gene;

[0095] 2) Cells and / or animals with PCSK9 gene knockout or knockdown and APOC3 gene knockout;

[0096] 3) Products for the prevention and / or treatment of diseases related to the PCSK9 gene and / or APOC3 gene.

[0097] In some specific implementations, the PCSK9 gene-related diseases include hypercholesterolemia and atherosclerotic cardiovascular disease.

[0098] In some specific implementations, the APOC3 gene-related diseases include hypertriglyceridemia, hyperchylomicronemia, and pancreatitis.

[0099] The ninth aspect of this application provides a treatment method for any of the following diseases: hypercholesterolemia, atherosclerotic cardiovascular disease, hypertriglyceridemia, hyperchylomicronemia, and pancreatitis, comprising administering an effective dose of the sgRNA described in the first aspect, or the gene editing system described in the second aspect, or the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect, or the composition described in the sixth aspect to the subject.

[0100] In this application, "effective dose" refers to the amount of the sgRNA described in the first aspect, the gene editing system described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the composition described in the sixth aspect, which is sufficient to produce a detectable or quantifiable target biological effect or clinical therapeutic effect in the subject. For gene editing therapy targeting hyperlipidemia and related diseases, the "effective dose" refers to an amount sufficient to achieve one or more of the following effects: significantly reducing serum PCSK9 protein levels; significantly reducing serum low-density lipoprotein cholesterol (LDL-C) levels; significantly reducing serum apolipoprotein C3 (APOC3) protein levels; significantly reducing serum triglyceride (TG) levels; reducing the risk of cardiovascular events or acute pancreatitis in the subject; or delaying, alleviating, controlling, or preventing the progression of mixed hyperlipidemia and its complications. This dose should be sufficient to produce the expected therapeutic effect, but not sufficient to cause serious toxicity or adverse events disproportionate to the clinical benefit. The specific effective dose depends on a variety of factors, including but not limited to: the type and performance of the selected delivery vector, the route of administration, the subject's weight, age, sex, health status, disease severity, genetic background, PCSK9 and APOC3 gene sequence polymorphisms, individual sensitivity to gene editing therapy, prior treatment history, and combination therapy. For example, for a combination of base editor mRNA and sgRNA delivered via lipid nanoparticles, the effective dose range can be expressed as 0.01 mg to 10 mg of mRNA per kilogram of body weight; for adeno-associated virus vectors, the effective dose range can be expressed as 1 × 10⁻⁶ mg per kilogram of body weight. 11 Up to 1×10 14 Vector genome copy number; for directly delivered base editor protein-sgRNA ribonucleoprotein complexes, the effective dose range can be expressed as 0.001 mg to 5 mg of protein per kilogram of body weight. The effective dose can be reasonably determined by those skilled in the art through routine dose escalation trials or based on existing clinical research data.

[0101] In some specific implementations, the object is a mammal.

[0102] Preferably, the mammal is a human, monkey, or ape.

[0103] The present invention will be further illustrated by the following examples, but these examples do not limit the scope of the invention.

[0104] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of this invention may be used to implement this invention.

[0105] Example 1: Design and preparation method of single-target sgRNA

[0106] This experiment obtained the reference genome sequences (GRCh38 / hg38) of PCSK9 (Gene ID255738) and APOC3 (Gene ID345) from NCBI Gene.

[0107] Twenty-two candidate sgRNAs were designed targeting the splicing site and catalytic domains (exon 3 and exon 7) of the PCSK9 gene. Their target sequences and the functional regions of the PCSK9 gene they cover are shown in Table 1.

[0108] Fourteen candidate sgRNAs were designed targeting the start codon, exons, splice sites, and secretory signal peptide regions of the APOC3 gene. Their target sequences and the functional regions of the APOC3 gene they cover are shown in Table 2.

[0109] Each sgRNA consists of a target sequence and a CRISPR / Cas9 sgRNA scaffold. It is synthesized using a standard S. pyogenes Cas9 scaffold (sequence shown in SEQ ID No. 37), with three phosphothioamide (PS) bonds introduced at the 5′ and 3′ ends, and a 2′-O-methyl (2′-OMe) modification introduced at the 2′-hydroxyl position to increase nuclease tolerance and reduce immunogenicity.

[0110] SEQ ID No. 37:

[0111] 5′-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT-3′

[0112] All sgRNAs were chemically synthesized by Genscript Biotech Inc. (Beijing), with a purity ≥95% (HPLC detection) and molecular weight confirmed by MALDI-TOF.

[0113] Table 1: Single-target PCSK9-sgRNA sequences and related information

[0114]

[0115] Table 2: Single-target APOC3-sgRNA sequences and related information

[0116]

[0117] Example 2: In vitro validation of single-target sgRNA-mediated editing

[0118] 2.1 Preparation of base editor

[0119] Simultaneously, two widely used base editor variants—ABE8e (amino acid sequence shown in SEQ ID No. 38) and AncBE4max (amino acid sequence shown in SEQ ID No. 39)—were selected as representative base editors for efficient sgRNA screening. The ABE8e plasmid was derived from Addgene (Plasmid, #138489), and the AncBE4max plasmid was derived from Addgene (Plasmid #112094). Both were linearized using BspQI (NEB, catalog number R0712S), followed by in vitro transcription using the MEGAscript™ T7 transcription kit (Thermo Fisher, catalog number AM1333), yielding high-purity, translationally active mRNAs (RNA Integrity > 9.0).

[0120] SEQ ID No. 38

[0121]

[0122] SEQ ID No.39

[0123]

[0124] 2.2 Cell Transfection and Editing Identification

[0125] PCSK9 or APOC3 are both genes that are highly expressed in the liver. Therefore, HepG2 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-510) were selected as the editing target in this embodiment.

[0126] Thirty-six sgRNAs were mixed with their corresponding editor mRNAs at a mass ratio of 1:1 and encapsulated using lipid nanoparticles (LNPs) disclosed in patent number 202310557830.3 to obtain LNP1~LNP36 delivery compositions with a total RNA content of 0.3 mg / mL. The LNP encapsulation steps are as follows: 1) Take sgRNA and corresponding gene editor mRNA and mix them in sodium citrate buffer (0.1M, pH4) at a mass ratio of 1:1 to obtain sgRNA-mRNA mixture (i.e., aqueous phase); 2) Use the lipid formulation disclosed in patent CN202310557830.3 (ionizable lipid:DSPC:cholesterol:DMG-PEG2000=50:10:38.5:1.5, molar ratio) to dissolve in anhydrous ethanol to prepare lipid phase; 3) Use a microfluidic device to mix the two phases at a total flow rate of 12 mL / min (aqueous phase:lipid phase=3:1, volume ratio) and collect the initially encapsulated LNP; 4) After dilution (PBS pH 7.4) and ultrafiltration tube (AmiconUltra-15, 100 kDa), centrifuge twice (100, 0×g, 10min) to remove free RNA and ethanol, and then centrifuge again (100, 0×g, 10min) to concentrate.

[0127] HepG2 cells (SCSP-510) were seeded at 70% confluence in 24-well plates, with 3.5 µL of LNP (approximately 1 µg RNA) added to each well. Genomic DNA was extracted after 48 h. Sanger sequencing analysis was performed on target sites using EditR software to calculate A→G (ABE) or C→T (CBE) editing rates. Simultaneously, GUIDE-seq was used to capture off-target sites across the entire genome.

[0128] Experimental results showed that among the 22 target sgRNAs of PCSK9, the editing efficiency of sgRNA1, 5, 7, 14, 18, and 22 was ≥60% (see Table 3), and the off-target rate was ≤0.07%, which was lower than the 0.1% limit. Among the 14 target sgRNAs of APOC3, the editing efficiency of sgRNA2, 3, 10, and 14 was ≥64% (see Table 4), and the off-target rate was ≤0.06%, which was lower than the 0.1% limit.

[0129] 2.3 Determination of protein knockdown ratio

[0130] Since PCSK9 and APOC3 are secreted into the cell supernatant after being expressed in hepatocytes, the supernatant protein concentrations before transfection (baseline) and 72 h after transfection were measured using the Human PCSK9 ELISA Kit (Abcam, ab209884) and the Human APOC3 ELISA Kit (Sigma-Aldrich, RAB0618).

[0131] The protein knockdown percentage is calculated using the formula (post-edit concentration - baseline concentration) / baseline concentration × 100%.

[0132] Experimental results showed that in the PCSK9-sgRNA editing groups (1, 5, 7, 14, 18, and 22), the concentration of PCSK9 protein in the cell supernatant decreased by 71%-89% (average approximately 80%), which was statistically significant (p<0.001, see [reference needed]). Figure 1 In the APOC3-sgRNA editing groups (2, 3, 10, and 14), the APOC3 protein concentration decreased by 76%-82% (average approximately 79.7%), which was also statistically significant (p<0.001, see [link]). Figure 2 ).

[0133] 2.4 Summary

[0134] This embodiment achieved a base editing rate of ≥60% in HepG2 cells by combining two mainstream base editors, ABE8e and AncBE4max, with an off-target rate controlled below ≤0.07%, meeting the patent's safety requirements. The protein knockdown rate caused by editing was in the range of 70%-90%, further validating the functionality of in vitro editing and providing reliable technical support for subsequent in vivo dual-target editing experiments.

[0135] Table 3: In vitro editing effects mediated by single-target PCSK9-sgRNA

[0136]

[0137] Table 4: In vitro editing effects mediated by single-target APOC3-sgRNA

[0138]

[0139] Example 3: In vivo editing validation of single-target PCSK9

[0140] 3.1 Editor and sgRNA Preparation

[0141] To verify the synergistic editing effect of gRNA combined with other base editors, this embodiment uses the patented base editor variant CE-A3A. 1048-1063 In vivo editing tests were performed using CE-ABE8e (CN111172133B, amino acid sequence as shown in SEQ ID No. 40) and CE-ABE8e (CN117568313A, amino acid sequence as shown in SEQ ID No. 41). Both editors were transcribed in vitro using the MEGAscript™ T7 Ultra Kit to obtain high-purity, translationally active mRNA.

[0142] SEQ ID NO: 40

[0143]

[0144] SEQ ID NO: 41

[0145]

[0146] Six PCSK9-sgRNAs with an editing rate ≥62% after in vitro screening were mixed with their corresponding editor mRNAs at a mass ratio of 1:1. The RNA was encapsulated using liver-targeting GalNac-modified lipid nanoparticles (LNPs) to obtain delivery compositions with a total RNA concentration of 0.5 mg / mL, named LNP43~LNP48 (corresponding to Table 5).

[0147] 3.2 Animal Models and Grouping

[0148]

[0149] Human PCSK9 single transgenic mouse B6-hPCSK9 (Strain NO. T015765) was selected as the in vivo editing target for PCSK9, provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., with an age of 8 weeks and a weight of 25±2g. Twenty-one mice were randomly divided into 7 groups (n=3 per group), and the specific drug administration regimens are as follows:

[0150] On day 7 post-injection, each mouse was euthanized, and liver tissue was harvested and homogenized. Genomic DNA was extracted, and the target region was amplified by PCR, followed by Sanger sequencing. The whole-liver C→T (CBE) or A→G (ABE) editing rate was analyzed using the EditR online database.

[0151] 3.3 Experimental results (see Table 5)

[0152] The results showed that the whole liver editing rate in all treatment groups was ≥58%, which was significantly higher than that in the control group (<1%), and p<0.001 (n=3).

[0153] 3.4 Significance

[0154] In vivo experiments confirmed:

[0155] 1) The highly efficient PCSK9-sgRNA delivered via GalNAc-LNP, combined with other base editor variants CE-A3A1048-1063 / CE-ABE8e, can achieve >58% base editing in the liver.

[0156] 2) This delivery system maintains high transfection efficiency and low off-target risk in complex physiological environments (off-target detection (GUIDE-seq) up to 0.06%).

[0157] Table 5: In vivo editing efficiency of single-target PCSK9-sgRNA

[0158]

[0159] Example 4: In vivo editing validation of single-target APOC3

[0160] 4.1 Editor and sgRNA Preparation

[0161] Also using CE-A3A 1048-1063 Alternatively, in vitro transcribed mRNAs of CE-ABE8e were used. Four APOC3-sgRNAs with an editing rate ≥64% after in vitro screening were selected and mixed with editor mRNAs at a mass ratio of 1:1. The mixtures were encapsulated using GalNAc-LNP to obtain delivery compositions with a total RNA concentration of 0.5 mg / mL, named LNP49-LNP52 (corresponding to Table 6).

[0162] 4.2 Animal Models and Grouping

[0163] Human APOC3 single transgenic mice B6-hAPOC3-Tg (Strain NO. T055510) were selected as the in vivo editing target for single-target APOC3, provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., with an age of 8 weeks and a weight of 25±2g. Fifteen mice were randomly divided into 5 groups (n=3 per group), and the specific drug administration regimens are as follows:

[0164]

[0165] 4.3 Experimental results (see Table 6)

[0166] The results showed that the editing rate of all treatment groups was ≥54%, which was significantly higher than that of the control group (<1%), with a statistical significance of p<0.001 (n=3).

[0167] 4.4 Significance

[0168] In vivo experiments confirmed:

[0169] 1) The selected APOC3-sgRNA, combined with the base editor variants CE-A3A1048-1063 / CE-ABE8e, achieved >54% base editing in the liver.

[0170] 2) The composition also maintains high transfection efficiency and low off-target risk in vivo (maximum off-target rate of 0.05%).

[0171] Table 6: In vivo editing efficiency of single-target APOC3-sgRNA

[0172]

[0173] Example 5: In vivo editing validation of dual-target PCSK9 / APOC3

[0174] 5.1 Reagents and Delivery Systems

[0175] To evaluate the synergistic editing effect of dual-target sgRNAs in vivo, this implementation case uses CE-A3A... 1048-1063 Alternatively, CE-ABE8e can be used as the editor mRNA. From the screened high-efficiency sgRNAs, one targeting PCSK9 and one targeting APOC3 (each with a single target editing rate ≥62%) are selected for dual-target combined editing. The editor mRNA, the selected PCSK9-sgRNA, and the APOC3-sgRNA are mixed at a mass ratio of 1:1:1 and encapsulated using GalNAc-LNP to obtain a delivery composition with a total RNA concentration of 0.5 mg / mL, named LNP53~LNP55 respectively (Table 7).

[0176] Table 7: Composition of the dual-target delivery composition

[0177]

[0178] 5.2 Animal Models and Grouping

[0179] Mice were selected from the human PCSK9 / APOC3 double transgenic mouse strain B6-hPCSK9 / hAPOC3 (Strain NO. T070910), provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd., at 8 weeks of age and weighing 25±2g. Twelve mice were randomly divided into 4 groups (n=3 per group), and the specific drug administration regimens are as follows:

[0180]

[0181] Baseline sampling: Blood was drawn before injection, plasma was collected by centrifugation and stored at -80°C as a baseline for PCSK9 and APOC3 protein levels and blood lipid concentration.

[0182] 5.3 DNA Editing Efficiency Assessment (Day 7)

[0183] One mouse was randomly selected from each group and euthanized, and liver tissue was collected. Genomic DNA was extracted, and the PCSK9 and APOC3 dual-target regions were amplified by PCR, followed by Sanger sequencing. The C→T (CBE) or A→G (ABE) editing rate was analyzed using EditR. Figure 3Experimental results showed that the 0.5 mg / kg low-dose dual-target delivery composition exhibited base editing efficiency similar to that of single-target delivery in model mice. Specifically, the average editing efficiency of the PCSK9 / APOC3 dual-target group was: 68% / 62% in the LNP53 group, 71% / 66% in the LNP54 group, and 70% / 59% in the LNP55 group. There was no significant difference in efficiency between the groups and the single-target delivery group (P>0.05), confirming that dual-target delivery can achieve single-target levels at low doses.

[0184] 5.4 Assessment of protein knockout ratio (day 28)

[0185] 1) Sampling: Blood was collected from the remaining 4 mice in each group, and plasma was collected by centrifugation.

[0186] 2) ELISA detection: The concentrations of the two proteins in plasma were measured using the corresponding PCSK9 (catalog number: ab209884) and APOC3 (catalog number: RAB0618) ELISA kits.

[0187] 3) Calculation formula: Protein knockout ratio = (post-edited plasma protein content - baseline protein content) / baseline protein content * 100%.

[0188] 4) Experimental results: Statistical analysis (p<0.001) showed that the treatment group had significantly lower levels of both target proteins compared to the control group. Figure 4 ).

[0189] 5.5 Assessment of blood lipid regulation

[0190] Detection method:

[0191] 1) The concentration of low-density lipoprotein (LDL-C) in plasma was determined using the Mouse LDL-Cholesterol Assay Kit (Crystal Chem, catalog number: 79980).

[0192] 2) Plasma triglyceride (TG) concentration was determined using the Mouse Triglyceride Assay Kit (Crystal Chem, catalog number: 79981).

[0193] Experimental results:

[0194] 1) Plasma LDL-C: The treatment group showed a significant decrease compared to the control group, with a reduction of approximately 80%–82%. Figure 5 ).

[0195] 2) Plasma TG: The treatment group showed a significant decrease compared to the control group, with a reduction of approximately 64%–66%. Figure 6 ).

[0196] The above results indicate that dual-target PCSK9 / APOC3 editing can significantly inhibit plasma LDL-C and TG levels, validating its potential therapeutic effect in lipid regulation.

[0197] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. An sgRNA, said sgRNA comprising a target sequence targeting the PCSK9 gene or a target sequence targeting the APOC3 gene; The nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprises any one of the sequences shown in SEQ ID No. 1 to 22; The nucleotide sequence encoding the target sequence targeting the APOC3 gene comprises the sequence shown in any one of SEQ ID No. 23-36.

2. The sgRNA of claim 1, wherein, The nucleotide sequence encoding the target sequence targeting the PCSK9 gene comprises any one of the sequences shown in SEQ ID No. 1, 5, 7, 14, 18, or 22; The nucleotide sequence encoding the target sequence targeting the APOC3 gene includes any one of the sequences shown in SEQ ID No. 24, 25, 32, and 36.

3. The sgRNA of claim 1, wherein, The sgRNA further comprises a scaffold sequence; preferably, the nucleotide sequence encoding the scaffold sequence comprises the sequence shown in SEQ ID No.

37.

4. A gene editing system selected from any of the following: a) Single-target gene editing system: contains a base editor and an sgRNA; The sgRNA contains a target sequence targeting the PCSK9 gene or a target sequence targeting the APOC3 gene; the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 1 to 22; the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 23 to 36. b) Dual-target gene editing system: contains a base editor and two sgRNAs, namely sgRNA (1) and sgRNA (2). The sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains a sequence as shown in any one of SEQ ID No. 1 to 22; The sgRNA (2) contains a target sequence that targets the APOC3 gene, and the nucleotide sequence encoding the target sequence that targets the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 23 to 36.

5. The gene editing system of claim 4, wherein, The single-target gene editing system is selected from any of the following forms: I. The base editor is an adenine base editor, and the sgRNA contains a target sequence targeting the PCSK9 gene. The nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains any one of the sequences shown in SEQ ID No. 1, 4, 5, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21. II. The base editor is a cytosine base editor, the sgRNA contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains any one of the sequences shown in SEQ ID No. 2, 3, 6, 9, 10, 11, 13, 14, 15, 16, 17, 22. III. The base editor is an adenine base editor, and the sgRNA contains a target sequence targeting the APOC3 gene. The nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 23, 25, 26, 27, 32, and 33. IV. The base editor is a cytosine base editor, and the sgRNA contains a target sequence targeting the APOC3 gene. The nucleotide sequence encoding the target sequence targeting the APOC3 gene contains any one of the sequences shown in SEQ ID No. 24, 25, 28, 29, 30, 31, 32, 34, 35, and 36. The dual-target gene editing system is selected from any of the following forms: i. The base editor is an adenine base editor; The sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains any one of the sequences shown in SEQ ID No. 1, 4, 5, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21. The sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains any one of the sequences shown in SEQ ID No. 23, 25, 26, 27, 32, 33; ii. The base editor is a cytosine base editor; The sgRNA (1) contains a target sequence targeting the PCSK9 gene, and the nucleotide sequence encoding the target sequence targeting the PCSK9 gene contains any one of the sequences shown in SEQ ID No. 2, 3, 6, 9, 10, 11, 13, 14, 15, 16, 17, 22. The sgRNA (2) contains a target sequence targeting the APOC3 gene, and the nucleotide sequence encoding the target sequence targeting the APOC3 gene contains a sequence as shown in any one of SEQ ID No. 24, 25, 28, 29, 30, 31, 32, 34, 35, 36.

6. The gene editing system of claim 5, wherein, the adenine base editor is selected from ABE8e or CE-ABE8e; the cytosine base editor is selected from AncBE4max or CE-A3A 1048-1063 .

7. The gene editing system of claim 4, wherein, The combination of sgRNA and base editor in the gene editing system can be selected from any of the following: ribonucleoprotein complex, mRNA and sgRNA co-preparation, single plasmid system, dual plasmid system, viral vector system, mRNA and viral vector hybridization system, and DNA expression cassette and RNP hybridization system.

8. A biomaterial, said biomaterial being selected from any of the following forms: A. A nucleic acid molecule comprising a nucleotide sequence encoding the sgRNA as described in any one of claims 1 to 3; preferably, the nucleic acid molecule further comprising a nucleotide sequence encoding a base editor in the gene editing system as described in any one of claims 4 to 7; B. An expression vector containing the nucleic acid molecule described in A; C. A cell whose genome integrates the nucleic acid molecule described in A or contains the expression vector described in B.

9. A composition comprising the sgRNA as described in any one of claims 1 to 3, or the gene editing system as described in any one of claims 4 to 7, or the biomaterial as described in claim 8, and a delivery vector; preferably, the delivery vector is a lipid nanoparticle.

10. A gene editing method comprising delivering to cells the sgRNA as described in any one of claims 1 to 3, or the gene editing system as described in any one of claims 4 to 7, or the biological material as described in claim 8, or the composition as described in claim 9.

11. The editing method as described in claim 10, characterized in that, When the editing target is the splice site region of the PCSK9 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes any one of the sequences shown in SEQ ID No. 1~6, 8~21; Alternatively, when the editing target is the exon 3 region of the PCSK9 gene, the nucleotide sequence encoding the target sequence in the sgRNA contains the sequence shown in SEQ ID No. 7; Alternatively, when the editing target is the exon 7 region of the PCSK9 gene, the nucleotide sequence encoding the target sequence in the sgRNA contains the sequence shown in SEQ ID No. 22; Alternatively, when the editing target is the start codon region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA contains the sequence shown in SEQ ID No. 23; Alternatively, when the target of editing is the secretory signal peptide region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in SEQ ID No. 24; Alternatively, when the editing target is the splice site region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes any one of the sequences shown in SEQ ID No. 25~27, 32~33, and 35; Alternatively, when the target of editing is exon 1 region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in any one of SEQ ID No. 28-31; Alternatively, when the editing target is the exon 2 region of the APOC3 gene, the nucleotide sequence encoding the target sequence in the sgRNA includes the sequence shown in either SEQ ID No. 34 or 36.

12. The use of the sgRNA as described in any one of claims 1 to 3, the gene editing system as described in any one of claims 4 to 7, the biomaterial as described in claim 8, and the composition as described in claim 9 in the preparation of any of the following products: 1) Cells and / or animals modified with the PCSK9 gene and / or APOC3 gene; 2) Cells and / or animals with PCSK9 gene knockout or knockdown and APOC3 gene knockout; 3) Products for the prevention and / or treatment of diseases related to the PCSK9 gene and / or APOC3 gene.

13. The use as described in claim 12, characterized in that, The diseases associated with the PCSK9 gene include hypercholesterolemia and atherosclerotic cardiovascular disease; the diseases associated with the APOC3 gene include hypertriglyceridemia, hyperchylomicronemia, and pancreatitis.

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