Grna targeting AGT gene and use thereof

Abstract

Provided are a gRNA targeting an AGT gene and a use thereof, and in particular, provided is a gRNA targeting an exon-intron splicing junction region of the AGT gene. The gRNA designed to target the exon-intron splicing junction region of the AGT gene (e.g., an intron 1 acceptor, an intron 2 donor / acceptor, and an intron 4 donor / acceptor) can effectively inhibit the expression of the AGT gene.

Description

gRNA targeting AGT gene and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to application No. CN2024100877106, filed on January 22, 2024, entitled “gRNA targeting AGT gene and its application”, which patent application (including any sequence listing and drawings) is incorporated herein by reference in its entirety.

[0003] References to electronic sequence listings

[0004] The present disclosure contains an electronic sequence listing which is incorporated herein by reference in its entirety. Where the sequence is an RNA sequence, T in the sequence should be considered as U. Technical Field

[0005] The present disclosure relates to the field of gene editing, and in particular, to gRNA targeting the AGT gene and its application. Background Art

[0006] Hypertension is a major risk factor for ischemic heart disease, stroke, and chronic kidney disease, and is the leading preventable cause of cardiovascular death worldwide. Despite the availability of effective treatment options, nearly half of patients with hypertension do not achieve guideline-recommended blood pressure targets, partly due to physicians' failure to initiate or intensify antihypertensive therapy and poor patient adherence to prescribed daily oral medications. Even when blood pressure appears to be well managed based on intermittent office measurements, suboptimal control may still occur due to significant variability in blood pressure over the diurnal cycle and over time.

[0007] The renin-angiotensin-aldosterone system (RAAS) plays a central role in blood pressure regulation. Angiotensinogen (AGT) has been shown to be a therapeutic target for hypertension. Specifically reducing hepatic angiotensinogen messenger RNA (mRNA) levels, thereby reducing angiotensinogen production, has been clinically proven to effectively lower blood pressure (Desai AS, Webb DJ, Taubel J, Casey S, Cheng Y, Robbie GJ, Foster D, Huang SA, Rhyee S, Sweetser MT, Bakris GL. Zilebesiran, an RNA Interference Therapeutic Agent for Hypertension. N Engl J Med. 2023 Jul 20; 389(3): 228-238. doi: 10.1056 / NEJMoa2208391. PMID: 37467498.). AGT is the only precursor of all angiotensin peptides. Therefore, inhibiting RAAS by genetically knocking out AGT can theoretically limit compensatory angiotensin activation associated with inhibiting angiotensin-converting enzyme or blocking angiotensin receptors, thereby achieving a better blood pressure-lowering effect.

[0008] There are currently a small number of AGT small molecule inhibitors under development on the market, but small molecule inhibitors have the problems of requiring long-term administration, prone to drug resistance, and poor targeting. It is generally believed that compared with small molecule chemical drugs, antibody drugs, recombinant proteins, and siRNA drugs, gene therapy (including CRISPR gene editing therapy) can achieve long-lasting and stable therapeutic effects through genetic manipulation, which can reduce adverse reactions caused by long-term administration, is convenient to treat, and the cost of long-term treatment is relatively low. In addition, it is relatively easier to achieve a balance between therapeutic effect and safety by editing regulatory elements in non-coding regions for treatment.

[0009] Genome editing is a genetic engineering technique that uses artificial nucleases to modify specific locations in an organism's genome. CRISPR / Cas9 technology, in particular, has attracted widespread attention from researchers due to its ease of use, high efficiency, and universal applicability. With the development of CRISPR / Cas9, single-base editing technologies based on it, including cytosine base editing systems and adenine base editing systems, have been developed to guide targeted base substitutions within target genes. Adenine base editing (ABE) primarily utilizes a fusion protein composed of the Cas protein and adenosine deaminase. Under the guidance of a single guide RNA (sgRNA), adenine base editing deaminates the target base A within the base editing active window to form hypoxanthine I. This base is then gradually replaced with G after DNA repair and replication, ultimately resulting in a targeted A-to-G substitution (A>G). Since its development, base editing technology has been successfully applied to a variety of organisms, including plants and animals, due to its high efficiency, independence from double-strand breaks, and the absence of donor DNA.

[0010] The principle of ABE gene knockout lies in its editing of the splicing junctions in the gene. This region refers to the sequence on both sides of the cut and rejoining sites. The junction on the left side of the intron is called the donor, and the one on the right side of the intron is called the acceptor. All introns in the structural genes of the cell nucleus (i.e., genes encoding polypeptides) have a common sequence of GU...AG at the exon-intron junction. Since ABE can achieve a directional replacement of A to G (A>G) in the genome, it can destroy the splicing junctions. After ABE gene editing, the common sequence GU...AG at the splicing junction is destroyed and becomes GC...AG or GU...GG, resulting in exon skipping or abnormal splicing of mRNA during the intromission process, thereby causing frameshift mutations and introducing premature stop codons, leading to mRNA degradation, and ultimately achieving the purpose of gene knockout.

[0011] There is currently no therapy on the market that targets AGT gene knockout, and there is a possibility of unpredictable toxicity after editing the AGT gene.

[0012] Citation or identification of any document in this disclosure is not an admission that such document is available as prior art to the present disclosure. Summary of the Invention

[0013] Based on this, in order to address the above problems, there is an urgent need to provide a gRNA targeting the AGT gene that can be used in gene editing therapy. It can achieve long-lasting and stable therapeutic effects through gene-level operations, reduce adverse reactions caused by long-term administration (such as drug resistance), and has the advantages of convenient treatment and relatively lower cost of long-term treatment.

[0014] The human AGT gene is located on chromosome 1. In order to obtain a gRNA that can effectively target and edit the AGT gene, the inventors investigated and experimentally verified the human AGT gene sequence and discovered a gRNA that can specifically target the AGT gene.

[0015] In one aspect, the present disclosure provides a gRNA that targets the splice junction region of the AGT gene.

[0016] In some embodiments, the splice junction region includes an intron 1 acceptor, an intron 2 donor / acceptor, and an intron 4 donor / acceptor.

[0017] In some embodiments, the gRNA targets the GU...AG splice junction in the splice junction region of the AGT gene.

[0018] In some embodiments, the gRNA guides the base editor to target the human AGT gene and change the AGT gene sequence, so that the expression of the AGT gene is downregulated by 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99%.

[0019] In some embodiments, the gRNA can guide the base editor to destroy the splicing junctions of the AGT gene, thereby reducing the expression level of the AGT gene.

[0020] In some embodiments, the targeting sequence of the gRNA is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar to the sequence shown in any one of SEQ ID NOs: 1-7, or is the reverse complementary sequence of a sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to the sequence shown in any one of SEQ ID NOs: 1-7.

[0021] In some embodiments, the targeting site of the gRNA is located in the splice junction region, and the target sequence targeted by the gRNA is based on the sequence shown in any one of SEQ ID NOs: 1-7, by deleting, adding or replacing some bases, such as differing from the sequence shown in any one of SEQ ID NOs: 1-7 by no more than 1, 2, 3, 4 or 5 nucleotides.

[0022] In some embodiments, the targeting sequence of the gRNA is as shown in any one of SEQ ID NOs: 1-7, or is the reverse complementary sequence of the sequence shown in any one of SEQ ID NOs: 1-7.

[0023] In some embodiments, the gRNA can be directly synthesized by chemical means, or prepared by other means, such as in vitro IVT.

[0024] In some embodiments, the gRNA includes a targeting sequence that targets a nucleic acid.

[0025] In some embodiments, the gRNA includes unmodified and modified gRNA.In some embodiments, the modified gRNA includes chemical modifications of bases.

[0026] In some embodiments, the chemical modification comprises methylation modification, methoxy modification, fluorination modification or thio modification.

[0027] In some embodiments, the base editor is a base editor fused to a nuclease. In some embodiments, the nuclease comprises a Cas protein.

[0028] In some embodiments, the nuclease comprises a type II Cas protein or a type V Cas protein.

[0029] In some embodiments, the nuclease is Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2c3, Cas12a, Cas12b, Cas12g, Cas12h, Cas12i, Cas13b, Cas13c, Cas13d, Cas14, or Argonaute (Ago).

[0030] In some embodiments, the base editor comprises an adenine base editor (ABE). In some embodiments, the base editor comprises ABE8e.

[0031] In yet another aspect, the present disclosure provides a nucleic acid encoding the gRNA of the present disclosure, or encoding a precursor of the gRNA of the present disclosure.

[0032] In another aspect, the present disclosure provides a vector comprising the gRNA of the present disclosure or the nucleic acid of the present disclosure.

[0033] In some embodiments, the vector further comprises a nucleic acid encoding a base editor.

[0034] In some embodiments, the vector comprises one or more vectors including:

[0035] a) a first regulatory element, which is operably linked to the gRNA,

[0036] b) a second regulatory element operably linked to the base editor;

[0037] Components (a) and (b) are located on the same or different carriers.

[0038] In some embodiments, the vector is a vector that targets and edits the AGT gene. In some embodiments, the vector comprises a plasmid or a viral vector.

[0039] In some embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0040] In another aspect, the present disclosure provides a gene editing system, comprising the gRNA of the present disclosure.

[0041] In some embodiments, the gene editing system further comprises a base editor.

[0042] In some embodiments, the base editor is a base editor fused to a nuclease.

[0043] In some embodiments, the nuclease comprises a Cas protein.

[0044] In some embodiments, the nuclease comprises a type II Cas protein or a type V Cas protein.

[0045] In some embodiments, the nuclease includes Cas9, CasX, CasY, Cpfl, C2cl, C2c2, C2c3, Casl2a, Casl2b, Casl2g, Casl2h, Casl2i, Casl3b, Casl3c, Casl3d, Casl4, and Argonaute (Ago).

[0046] In some embodiments, the base editor comprises an adenine base editor (ABE). In some embodiments, the base editor comprises ABE8e.

[0047] In some embodiments, the gene editing includes in vivo gene editing and in vitro gene editing.

[0048] In another aspect, the present disclosure provides a cell obtained by editing with the gene editing system described in the present disclosure.

[0049] In some embodiments, the cells are cells cultured in vitro.

[0050] In some embodiments, the cells include primary cells and passaged cells.

[0051] In some embodiments, the cells comprise mammalian cells.

[0052] In some embodiments, the cells comprise human cells.

[0053] In some embodiments, the cells include hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, fat cells, intestinal cells, cervical cells, and pancreatic cells.

[0054] In another aspect, the present disclosure provides a composition comprising a gRNA component and a protein component, wherein the gRNA comprises the gRNA of the present disclosure, and the protein component comprises a base editor.

[0055] In some embodiments, the gRNA component is selected from: the gRNA disclosed herein, or a nucleic acid encoding the aforementioned gRNA; the base editor is selected from: a base editor, or a nucleic acid encoding a base editor.

[0056] In some embodiments, the base editor is a base editor fused to a nuclease.

[0057] In some embodiments, the nuclease comprises a Cas protein.

[0058] In some embodiments, the nuclease comprises a type II Cas protein or a type V Cas protein.

[0059] In some embodiments, the nuclease includes Cas9, CasX, CasY, Cpfl, C2cl, C2c2, C2c3, Casl2a, Casl2b, Casl2g, Casl2h, Casl2i, Casl3b, Casl3c, Casl3d, Casl4, and Argonaute (Ago).

[0060] In some embodiments, the base editor comprises an adenosine base editor. In some embodiments, the base editor comprises ABE8e.

[0061] In some embodiments, the composition comprises a pharmaceutical composition. In some embodiments, the pharmaceutical composition is in liquid form.

[0062] In some embodiments, the dosage form of the pharmaceutical composition comprises an injection or an injection. In some embodiments, the dosage form of the pharmaceutical composition is an intravenous injection dosage form.

[0063] In yet another aspect, the present disclosure provides a composition comprising:

[0064] a system of the present disclosure or a cell of the present disclosure; and a pharmaceutically acceptable carrier.

[0065] In some embodiments, in the composition, the system or the cell accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the composition.

[0066] In another aspect, the present disclosure provides a delivery composition comprising an active ingredient and a delivery medium, wherein the active ingredient comprises the gRNA of the present disclosure, the vector of the present disclosure, or the system of the present disclosure.

[0067] In some embodiments, the delivery composition further comprises a base editor.

[0068] In some embodiments, the delivery vehicle comprises lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microbubbles, a gene gun, or a viral vector (e.g., a replication-defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).

[0069] In yet another aspect, the present disclosure provides a host cell comprising the gRNA of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the system of the present disclosure, the composition of the present disclosure, or the delivery composition of the present disclosure.

[0070] In some embodiments, the host cell comprises a mammalian cell. In some embodiments, the host cell comprises a human cell.

[0071] In some embodiments, the host cells include hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, fat cells, intestinal cells, cervical cells, and pancreatic cells.

[0072] In yet another aspect, the present disclosure provides a CRISPR complex comprising the gRNA of the present disclosure, a base editor, and a target nucleic acid bound to the gRNA.

[0073] In another aspect, the present disclosure provides a drug kit comprising: a first container, and an active ingredient or a drug containing the active ingredient, wherein the active ingredient or the drug is located in the first container, and the active ingredient comprises the disclosed gRNA, the disclosed nucleic acid, the disclosed vector, the disclosed system, the disclosed cell, the disclosed composition, or the disclosed delivery composition or the disclosed host cell or the disclosed CRISPR complex.

[0074] In some embodiments, the active ingredient or the drug is a monopreparation.

[0075] In some embodiments, the active ingredient or the drug is in the form of an injection or injection. In some embodiments, the active ingredient or the drug is in the form of an intravenous injection.

[0076] In some embodiments, the kit further contains instructions for administering the active ingredient or the drug to a subject, thereby (i) improving gene editing efficiency; and / or (ii) preventing and / or treating a disease.

[0077] In some embodiments, the condition or disease comprises an angiotensinogen-related disease.

[0078] In some embodiments, the angiotensinogen-related disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, Atherosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephroblastoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0079] In some embodiments, the angiotensinogen-related disease is selected from hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-related hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, primary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, stroke, diabetes, renal failure and systemic sclerosis.

[0080] In some embodiments, the angiotensinogen-related disease is pregnancy-related hypertension (eg, pregnancy-induced hypertension, preeclampsia, and eclampsia).

[0081] In some embodiments, the condition or disease is hypertension.

[0082] In some embodiments, the subject of administration is a cell.

[0083] In some embodiments, the subject of administration is a human cell.

[0084] In some embodiments, the administration targets include hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, fat cells, intestinal cells, cervical cells, and pancreatic cells.

[0085] In some embodiments, the subject of administration is a human or non-human mammal.

[0086] In some embodiments, the administration is contacting the active ingredient or the drug with human hepatocytes, or injecting the active ingredient or the drug into the human body.

[0087] In another aspect, the present disclosure provides a method for gene editing a cell, comprising contacting the cell with the gRNA of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the system of the present disclosure, the composition of the present disclosure, or the delivery composition of the present disclosure, or the host cell of the present disclosure, or the CRISPR complex of the present disclosure, or the drug kit of the present disclosure.

[0088] In some embodiments, the contacting is performed in vitro.

[0089] In some embodiments, the cells are cells cultured in vitro.

[0090] In some embodiments, the cells include primary cells and passaged cells.

[0091] In some embodiments, the cell comprises a mammalian cell. In some embodiments, the cell comprises a human cell.

[0092] In some embodiments, the cells include hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, fat cells, intestinal cells, cervical cells, and pancreatic cells.

[0093] In another aspect, the present disclosure provides a kit for gene editing, comprising the gRNA of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the system of the present disclosure, the composition of the present disclosure, or the delivery composition of the present disclosure, or the host cell of the present disclosure, or the CRISPR complex of the present disclosure, or the drug kit of the present disclosure.

[0094] In some embodiments, the kit further comprises a label or instructions.

[0095] In some embodiments, the label or the instructions contain instructions for administering the gRNA, nucleic acid, vector, system, composition, delivery composition, host cell, CRISPR complex, or kit of the present disclosure to the editing subject to perform gene editing.

[0096] In some embodiments, the editing subject comprises a cell.

[0097] In some embodiments, the cells include primary cells and passaged cells.

[0098] In some embodiments, the cells comprise mammalian cells.

[0099] In some embodiments, the cells comprise human cells.

[0100] In some embodiments, the cells include hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, fat cells, intestinal cells, cervical cells, and pancreatic cells.

[0101] In another aspect, the present disclosure provides the use of the gRNA of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the system of the present disclosure, the composition of the present disclosure, or the delivery composition of the present disclosure, or the host cell of the present disclosure, or the CRISPR complex of the present disclosure, or the drug kit of the present disclosure, or the kit of the present disclosure in gene editing, gene targeting, or gene cleavage.

[0102] In another aspect, the present disclosure provides uses of the gRNA of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the system of the present disclosure, the cell of the present disclosure, the composition of the present disclosure, or the delivery composition of the present disclosure, or the host cell of the present disclosure, or the CRISPR complex of the present disclosure, or the drug kit of the present disclosure, or the kit of the present disclosure, for preparing a medicament for preventing and / or treating a disease.

[0103] In some embodiments, the disease is a condition caused by a defect in a target sequence in a target locus, such as the AGT gene.

[0104] In some embodiments, the condition or disease comprises an angiotensinogen-related disease.

[0105] In some embodiments, the angiotensinogen-related disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, Atherosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephroblastoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0106] In some embodiments, the angiotensinogen-related disease is selected from hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-related hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, primary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, stroke, diabetes, renal failure and systemic sclerosis.

[0107] In some embodiments, the angiotensinogen-related disease is pregnancy-related hypertension (eg, pregnancy-induced hypertension, preeclampsia, and eclampsia).

[0108] In some embodiments, the condition or disease is hypertension.

[0109] In yet another aspect, the present disclosure provides a method of treating a disease, comprising administering to a subject an effective amount of the system, cell, composition, or delivery composition of the present disclosure, or the host cell, or the CRISPR complex, or the kit, or the reagent kit of the present disclosure.

[0110] In some embodiments, the administering comprises administering by injection.

[0111] In some embodiments, the subject is a human or non-human mammal.

[0112] In some embodiments, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0113] In some embodiments, the disease is a condition caused by a defect in a target sequence in a target locus, such as the AGT gene.

[0114] In some embodiments, the condition or disease comprises an angiotensinogen-related disease.

[0115] In some embodiments, the angiotensinogen-related disease is selected from hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, Atherosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephroblastoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

[0116] In some embodiments, the angiotensinogen-related disease is selected from hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-related hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, primary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, stroke, diabetes, renal failure and systemic sclerosis.

[0117] In some embodiments, the angiotensinogen-related disease is pregnancy-related hypertension (eg, pregnancy-induced hypertension, preeclampsia, and eclampsia).

[0118] In some embodiments, the condition or disease is hypertension.

[0119] It should be understood that within the scope of the present disclosure, the above-mentioned technical features of the present disclosure and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] FIG1 shows the positions of different gRNAs on the genome (the image from left to right (along the arrow direction) corresponds to the 5' end to the 3' end of the genome).

[0121] Figure 2 shows the deep sequencing results of base editing efficiency guided by different sgRNAs.

[0122] Figure 3 shows the changes in AGT mRNA levels caused by base editing guided by different sgRNAs. DETAILED DESCRIPTION

[0123] After extensive and in-depth research, the applicants of the present disclosure unexpectedly discovered for the first time that gRNA designed to target the splice junction region of the AGT gene (such as intron 1 acceptor, intron 2 donor / acceptor, and intron 4 donor / acceptor) can effectively inhibit the expression of the AGT gene. In addition, the applicants also discovered for the first time that the gRNA designed for the GU-AG splice site in the splice junction region of the present disclosure can mediate very high gene editing efficiency and has potential application prospects in the treatment of diseases.

[0124] the term

[0125] " splice site mutation " refers to changing or abolishing the correct splicing of the pre-mRNA sequence so that it produces a protein with an amino acid sequence different from the wild type. For example, one or more exons can be skipped during RNA splicing to produce a protein lacking the amino acids encoded by the skipped exons. Alternatively, the reading frame can be changed by incorrect splicing, or one or more introns can be retained, or other splicing donors or acceptors can be produced, or splicing can be initiated at other positions (e.g., within an intron), or other polyadenylation signals can be produced. Correct pre-mRNA splicing is a complex process that can be affected by a variety of mutations in the nucleotide sequence of the GTR encoding gene. In higher eukaryotes (such as plants), the major spliceosome contains an intron that contains GU at the 5' splice site (donor site) and AG at the 3' splice site (acceptor site). About 99% of splice sites in nuclear eukaryotic genes follow this GU-AG rule (or GT-AG rule; see Lewin, Genes VI, Oxford University Press 1998, pp. 885-920, ISBN 0198577788).

[0126] The term "in vitro transcription (IVT)" refers to a technique that uses linear DNA as a template to mimic the in vivo transcription process in an in vitro cell-free system containing RNA transcriptase and NTPs.

[0127] The term "cell transfection" refers to the technology of introducing exogenous molecules such as DNA and RNA into eukaryotic cells using transfection reagents.

[0128] The term "base editor", also referred to as "nucleobase editor (NBE)", refers to a reagent that binds to a polynucleotide and has nucleobase modification activity. In some embodiments, the base editor comprises a nucleobase modification polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain (e.g., Cas9, Cas12) bound to a guide polynucleotide (e.g., a guide RNA). In some embodiments, the domain with base editing activity is capable of deaminating bases within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating cytosine (C) or adenosine (A) within DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE). In some embodiments, the base editor is a nuclease-inactive Cas protein (e.g., dCas9, dCas12) fused to an adenosine deaminase.

[0129] The term "base editing activity" refers to an activity used to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting the target A·T to C·G. In another embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A, and adenosine or adenine deaminase activity, for example, converting A·T to G·C.

[0130] The term "gRNA", also known as guide RNA, crRNA or guide sequence, generally speaking, the guide RNA can comprise a scaffold sequence (e.g., a direct repeat sequence) and a guide sequence, or consists essentially of or consists of a scaffold sequence and a guide sequence. In some cases, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the CRISPR / Cas complex to specifically bind to the target sequence. When optimally aligned, the degree of complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% (fully complementary).

[0131] Gene editing system

[0132] The present disclosure provides a gene editing system, which includes the gRNA of the present disclosure, and the gRNA can target a target sequence.

[0133] In some embodiments, the gene editing system further comprises a base editor.

[0134] In some embodiments, the target sequence is the splice junction region of the AGT gene.

[0135] In some embodiments, the splice junction region includes an intron 1 acceptor, an intron 2 donor / acceptor, and an intron 4 donor / acceptor.

[0136] In some embodiments, the target sequence is a splice site in the splice junction region of the AGT gene.

[0137] The gene editing system disclosed herein can significantly improve gene editing efficiency.

[0138] If the Cas protein of the base editor selected is different, those skilled in the art can understand and select other possible conventional scaffold sequences and the targeting structure sequence or adjacent sequence in this embodiment, and achieve the editing of the target gene disclosed in this disclosure.

[0139] In the gRNA sequence disclosed herein, SEQ ID NOs: 1-7 are designed based on the sequence of the splicing site. By editing the targeted splicing site, splicing site mutations occur, affecting the transcription process of the target gene, thereby reducing the expression of the target gene.

[0140] In one embodiment, the sgRNA target sequence is selected from any one of AGT-gRNA1, AGT-gRNA2, AGT-gRNA3, AGT-gRNA4, AGT-gRNA5, AGT-gRNA6, and AGT-gRNA7. The present disclosure has experimentally confirmed that the above sequence has a good editing effect.

[0141] Pharmaceutical compositions and methods of administration

[0142] On the other hand, the present disclosure also provides a pharmaceutical composition comprising (a) a safe and effective amount of the gene editing system or cell of the present disclosure; and (b) a pharmaceutically acceptable carrier or excipient. The dosage of the gene editing system of the present disclosure is generally 10 μg to 100 mg per dose, optionally 100 to 1000 μg per dose.

[0143] In one embodiment, the concentration of the cells in the pharmaceutical composition is 1×10 3 -1×10 8 cells / kg body weight, preferably 1×10 5 -1×10 7 For the purposes of this disclosure, an effective dose is about 0.01 mg / kg to 50 mg / kg, preferably 0.05 mg / kg to 10 mg / kg of body weight of the gene editing system of the present disclosure. In addition, the gene editing system of the present disclosure can be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

[0144] The pharmaceutical compositions of the present disclosure can be administered in the form of cell therapeutic agents. For example, the gene editing system of the present disclosure can be used to modify cells, and then the modified cells are administered to subjects in need by intravenous injection. In certain embodiments, the gene editing system of the present disclosure can be used to modify cells in vivo or isolated. In certain embodiments, the cells can be hepatocytes, and the modification can include gene editing.

[0145] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and that are not unduly toxic upon administration. Such carriers are well known to those of ordinary skill in the art. A comprehensive discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Such carriers include, but are not limited to, saline, buffered solutions, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0146] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, etc. may also be present in these carriers.

[0147] Typically, therapeutic compositions can be formulated as injectables, such as liquid solutions or suspensions. They can also be formulated as solid forms suitable for dissolution or suspension in a liquid carrier prior to injection. Once formulated, the disclosed compositions can be administered by conventional routes, including, but not limited to, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated can be an animal, particularly a human.

[0148] When the pharmaceutical compositions of the present disclosure are used for actual treatment, various dosage forms of pharmaceutical compositions can be used depending on the intended use. Preferably, they are intravenous injections. These pharmaceutical compositions can be prepared by mixing, diluting, or dissolving according to conventional methods, and occasionally with the addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, and solubilizers. The preparation process can be carried out in a conventional manner depending on the dosage form.

[0149] The pharmaceutical composition of the present disclosure can also be administered in the form of a sustained-release agent. For example, the gene editing system of the present disclosure can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of sustained-release polymers, there can be exemplified ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinyl pyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc., preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

[0150] In one embodiment, the pharmaceutical composition may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The pharmaceutical composition of the present disclosure is preferably formulated for intravenous administration.

[0151] When the pharmaceutical composition of the present disclosure is used for actual treatment, the dosage of the gene editing system of the present disclosure as the active ingredient can be reasonably determined based on the weight, age, gender, and symptom severity of each patient to be treated.

[0152] The main advantages of the present disclosure include:

[0153] (1) For the first time, it was discovered that gRNAs designed to target the splice junction region of the AGT gene (intron 1 acceptor, intron 2 donor / acceptor, and intron 4 donor / acceptor) can effectively inhibit the expression of the AGT gene. The gRNAs designed for the GU-AG splice site in the splice junction region all had very good gene editing efficiency, and 5 of them had very high gene editing efficiency. This disclosure has potential application prospects in the treatment of diseases.

[0154] (2) The present disclosure provides a gRNA targeting the AGT gene, which can be used in CRISPR gene editing therapy. It can achieve long-lasting and stable therapeutic effects through genetic manipulation, reduce adverse reactions (such as drug resistance) caused by long-term administration, and has the advantages of convenient treatment and relatively lower cost of long-term treatment.

[0155] (3) The single-base editing method does not produce double-strand breaks and will not cause random insertion of large gene fragments. It has better therapeutic effects and is safe.

[0156] The following specific examples are provided to further illustrate some embodiments of the present disclosure. It should be understood that these examples are only used to illustrate some embodiments of the present disclosure and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally based on conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. Unless otherwise specified, the cell lines listed in the context of this disclosure are cultured according to the prior art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present disclosure. The terms used in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0157] Unless otherwise specified, the materials and reagents used in the examples are commercially available products.

[0158] Example 1 Chemical synthesis of sgRNA targeting the target sequence.

[0159] (1) The AGT gene sequence was obtained from the NCBI database (GeneBank, ID 8317). Based on the splice junction region of the human AGT gene, the corresponding sgRNA targeting sequence was designed to identify the GU-AG splice site that can disrupt the AGT gene. Seven sgRNA targeting sequences were designed. The locations of each sgRNA targeting sequence are shown in Figure 1. The specific design of the sgRNA targeting sequence is shown in Table 1.

[0160] Table 1 Various AGT-gRNA target sequences and their editing sites

[0161] The backbone sequences used for the sgRNAs in Table 1 above are as follows:

[0162] Each sgRNA sequence structure is a target sequence + backbone sequence (5'-3'). For example, the AGT-gRNA1 sequence is as follows:

[0163] The sgRNA sequence was commissioned to be chemically synthesized by GenScript Biotech Co., Ltd., and three bases at the 5' and 3' ends of the sgRNA were modified with thio and methoxy groups respectively.

[0164] Example 2 ABE8e mRNA Synthesis

[0165] ABE8e mRNA synthesis was performed using in vitro transcription (IVT). The specific method is as follows:

[0166] (1) ABE8e plasmid was purchased from Addgene (Plasmid, #138489) and then digested with BspQI restriction endonuclease (purchased from NEB). The resulting plasmid was linearized and used as a template for in vitro transcription. Using tweezers, a PCR tube was placed in a tube rack and labeled. The reaction components were then added in sequence using the appropriate pipette according to Table 2, and gently pipetted to mix thoroughly.

[0167] Table 2 ABE8e mRNA in vitro transcription (IVT) components

[0168] (2) After capping the PCR tube, place it in a PCR instrument and incubate at 37°C for 2 hours.

[0169] (3) After the incubation is complete, remove the PCR tube from the PCR instrument. Take another clean ice box and fill it halfway with ice. Take out DNaseI (purchased from NEB, M0303S) from the -20℃ refrigerator and transfer them together to the cell-free biosafety cabinet. First, use RNaseZap to remove the sample tube. TM Spray the tube with RNase Decontamination Solution (Invitrogen, AM9780) and wipe it clean with a clean paper towel before placing it in a PCR tube rack in a biosafety cabinet. Next, add 1 μl of DNase I (purchased from Takara Bio) to the reaction tube and gently stir to mix. The tube is then placed in a PCR instrument and incubated at 37°C for 15 minutes.

[0170] (4) After the reaction is completed, collect the sample in the biosafety cabinet. First, take a centrifuge column from the Rnaeasy mini kit (purchased from Qianen) and place it on the tube rack. Open the lid. Then take a clean 1.5ml centrifuge tube and place it on the tube rack. Use a 100μl pipette to aspirate the reaction solution into the tube. At the same time, add 350μl RLT Buffer solution and 250μl anhydrous ethanol to the tube. Use a 1ml pipette to gently mix and add to the previously prepared centrifuge column. After capping the tube, transfer it to the centrifuge and centrifuge at 12000rpm for 30 seconds.

[0171] (5) After centrifugation, transfer the sample column to a biosafety cabinet, aspirate the waste liquid with a 1ml pipette, then return the sample column to the waste liquid tube, open the lid, adjust the 1ml pipette range to 500μl, draw 500μl RPE Buffer into the sample column, cover the tube, and transfer to a centrifuge and centrifuge at 12000rpm for 30 seconds.

[0172] (6) After centrifugation, transfer the sample column to a biosafety cabinet, aspirate the waste liquid, and then return the sample column to the waste liquid tube. Open the lid, adjust the 1ml pipette range to 500μl, and draw 500μl RPE Buffer into the sample column. Cover the tube cap and transfer to a centrifuge and centrifuge at 12000rpm for 2min.

[0173] (7) After centrifugation, transfer the sample column to a biosafety cabinet, take out a clean waste tube, transfer the sample column to the clean waste tube, and centrifuge again at 12,000 rpm for 2 minutes.

[0174] (8) After centrifugation, transfer the sample column to a biosafety cabinet. Remove a clean 1.5ml EP tube, transfer the sample column to the clean EP tube, open the cap, and let it stand in a tube rack for 2 minutes. Adjust the 100μl pipette scale to 50μl, draw 50μl of enzyme-free water and add it to the middle of the sample column membrane. Then, cover the tube, let it stand in the tube rack for 1 minute, and transfer it to a centrifuge and centrifuge at 12000rpm for 1 minute.

[0175] (9) After centrifugation, transfer the sample column to the biosafety cabinet and use the 50 μl pipette to draw another 50 μl of enzyme-free water and add it to the middle position of the sample column membrane. After covering the tube cap, let it stand on the tube rack for 2 minutes and then centrifuge it at 12,000 rpm for 1 minute.

[0176] (10) After centrifugation, transfer the sample column to a biosafety cabinet. Take out a clean PCR tube and EP tube, place them in a tube rack, collect all the eluate in the newly prepared 1.5ml EP tube, then use a 10μl pipette to draw 3μl of sample into the PCR tube for subsequent concentration determination. Label the sample name and date. Place both tubes on ice.

[0177] (11) The concentration was marked in the tube containing the mRNA sample, sealed with sealing film, and frozen at -80°C. The obtained ABE8e mRNA concentration was 1542 ng / μl after measurement by Nanodrop.

[0178] Example 3 Detection of base editing efficiency at AGT target gene sites

[0179] 1. Cell culture

[0180] The Human Protein Atlas (https: / / www.proteinatlas.org / ), a human genome expression information website, shows that the AGT gene is efficiently expressed in the cell line HepG2 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-510), so this cell line was selected as the base editing target.

[0181] 1.1 Preparation before the experiment

[0182] (1) Before use, TM I Reduced Serum Medium (Gibco, 31985070) was placed at room temperature for equilibration for 30 min.

[0183] (2) After the biosafety cabinet operating table is wiped and disinfected with 75% alcohol cotton, turn on the ultraviolet light for 30 minutes.

[0184] 1.2 Preparation of culture medium

[0185] (1) Culture medium preparation

[0186] 50 ml of FBS and 5 ml of double antibody (Gibco, #15140122) were added to 500 ml of DMEM high glucose medium (purchased from Gibco).

[0187] (2) Filter the prepared culture medium through a filter, mark the bottle and store it in a refrigerator at 4°C.

[0188] 1.3 Operation

[0189] (1) After the wind speed of the biosafety cabinet stabilizes, wipe the operating table with alcohol gauze and put in the required consumables and reagents (items entering and leaving the operating table must be wiped and disinfected with alcohol gauze);

[0190] (2) Take out the cell culture dish from the incubator and observe the cell growth under an inverted microscope. When the cell density reaches 80-90%, the cells can be passaged.

[0191] (3) Remove the culture dish and discard the supernatant. Add 3 ml of DPBS to a 100 mm culture dish, cover the bottom of the dish, and then discard it;

[0192] (4) Add 1 ml of 0.25% trypsin-EDTA per 100 mm culture dish and place in a 37°C incubator for 5 min. Gently shake the dish to detach the cells.

[0193] (5) Add 3 ml of complete culture medium, collect the cells into a centrifuge tube, pipette to mix, and record the total volume;

[0194] (6) Take 10 μl of cell suspension, add 10 μl of AO / PI (purchased from Beijing Biolab Technology Co., Ltd., #HR0462), pipette evenly, transfer to a cell counting plate, count using a cell counter, and calculate the total cell number;

[0195] (7) Centrifuge at 400 g for 5 min at room temperature;

[0196] (8) After centrifugation, discard the supernatant and add an appropriate amount of culture medium based on the total number of cells to ensure a cell concentration of 20 million / ml. Resuspend the cells by repeated pipetting and inoculate 1 ml of cells into a new 100 mm culture dish.

[0197] (9) Mix the culture dishes crosswise and place them in the incubator, and record the time;

[0198] (10) Take out the sealed reagents and consumables in the biosafety cabinet, wipe the disinfection table with alcohol, and turn on the ultraviolet disinfection lamp.

[0199] 2. Cell transfection

[0200] (1) HepG2 cells were passaged when the cell confluence reached 80-90%, counted, and seeded into 48-well plates at 40,000 cells / well. Cell density was observed after 24 hours, and mRNA transfection was performed when the cell confluence reached 70%.

[0201] (2) Dosage: Calculate the amount of RNA per well of a 48-well plate and transfect with Lipofectamine MessengerMax. Three RNA gradients are available: 100 ng, 300 ng, and 500 ng. The weight ratio of ABE mRNA to sgRNA is 1:1. For example, for a 500 ng RNA transfection, the total amount is 500 ng RNA (250 ng sgRNA + 250 ng ABE8e mRNA) and 1.5 μl Lipofectamine MessengerMax (Invitrogen, LMRNA003).

[0202] Prepare two 1.5ml EP tubes, add 20μl Opti-MEM medium to each tube, add 250ng sgRNA and 250ng ABE8e to one tube, and add 1.5μl Lipofectamine MessengerMax to the other tube. Mix them separately and let them stand at room temperature for 10 minutes.

[0203] (3) Mix the reagents in the two EP tubes and let them stand at room temperature for 5 minutes; then add the mRNA-transfection reagent complex to the cells for transfection, and the editing time is 72 hours.

[0204] 3. Genome Extraction

[0205] Genomic DNA of HepG2 cells was extracted using a blood / cell / tissue genomic DNA extraction kit (Tiangen, DP304).

[0206] (1) Resuspend the cells in 200 μl Buffer GA.

[0207] (2) Add 20 μl Proteinase K and mix well

[0208] (3) Add 200 μl of Buffer GB, mix thoroughly by inversion, and incubate at 70°C for 10 min until the solution becomes clear. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0209] (4) Add 200 μl of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0210] (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 back into the collection tube.

[0211] (6) Add 500 μl of Buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and return the adsorption column CB3 to the collection tube.

[0212] (7) Add 600 μl of Buffer PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and return the adsorption column CB3 to the collection tube.

[0213] (8) Repeat step (7).

[0214] (9) Centrifuge at 12000 rpm for 2 min and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0215] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μl of enzyme-free water to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution into the centrifuge tube.

[0216] (11)Use NanoDrop TM DNA concentration was measured using a Lite spectrophotometer (Thermo Scientific).

[0217] 4. Determination of base editing efficiency

[0218] (1) The target sequence site detection primers for each target gene are shown in Table 3. All primers were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0219] Table 3 Target sequence site detection primers

[0220] (2) PCR reaction system and reaction.

[0221] The upstream and downstream primers used in the detection are shown in Table 3. The PCR reactions all used the high-fidelity enzyme KOD-Plus-Neo (purchased from TOYOBO, product number KOD-401) to amplify the target fragments.

[0222] The amplification system was as follows: 10× PCR Buffer for KOD-Plus-Neo 5 μl, dNTP 5 μl, Mg 2+

[0223] 3μl, KOD-Plus-Neo 1μl, 1μl each of upstream and downstream primers for target sequence site detection, 50ng DNA Template, and ddH2O to make up to 50μL.

[0224] The PCR reaction program was as follows: initial denaturation: 95°C for 5 min, 35 cycles (95°C for 10 s; 60°C for 10 s; 68°C for 10 s), extension: 68°C for 5 min, and storage temperature: 4°C.

[0225] The obtained PCR products were commissioned to Sangon Biotech (Shanghai) Co., Ltd. for deep sequencing.

[0226] The sequencing results are shown in Figure 2, which show that:

[0227] When the transfection amount = 100 ng, the base editing efficiency mediated by AGT-gRNA1 was 0.5%, the base editing efficiency mediated by AGT-gRNA2 was 35.0%, the base editing efficiency mediated by AGT-gRNA3 was 11.5%, the base editing efficiency mediated by AGT-gRNA4 was 10.0%, the base editing efficiency mediated by AGT-gRNA5 was 26.0%, the base editing efficiency mediated by AGT-gRNA6 was 0.0%, and the base editing efficiency mediated by AGT-gRNA7 was 6.5%.

[0228] When the transfection amount = 300 ng, the base editing efficiency mediated by AGT-gRNA1 was 1.5%, the base editing efficiency mediated by AGT-gRNA2 was 61.5%, the base editing efficiency mediated by AGT-gRNA3 was 27.5%, the base editing efficiency mediated by AGT-gRNA4 was 6.5%, the base editing efficiency mediated by AGT-gRNA5 was 43.5%, the base editing efficiency mediated by AGT-gRNA6 was 5.0%, and the base editing efficiency mediated by AGT-gRNA7 was 3.0%.

[0229] When the transfection amount = 500 ng, the base editing efficiency mediated by AGT-gRNA1 was 0.0%, the base editing efficiency mediated by AGT-gRNA2 was 66.5%, the base editing efficiency mediated by AGT-gRNA3 was 32.5%, the base editing efficiency mediated by AGT-gRNA4 was 6.0%, the base editing efficiency mediated by AGT-gRNA5 was 53.0%, the base editing efficiency mediated by AGT-gRNA6 was 6.0%, and the base editing efficiency mediated by AGT-gRNA7 was 4.5%.

[0230] From this, it can be seen that the base editing efficiency mediated by gRNA2 and gRNA5 is higher, especially the base editing efficiency mediated by gRNA2 is the highest, while the editing efficiency of gRNA1 and gRNA6 is low and they play almost no mediating role.

[0231] Example 4 Detection of mRNA transcription levels after AGT target gene editing

[0232] 1. Use Trizol method to extract total RNA from HepG2 cells with RNA transfection amount = 300ng.

[0233] (1) Collect cells by centrifugation, add 1 ml of Trizol, and mix repeatedly.

[0234] (2) Place the homogenized sample at room temperature (15-30°C) for 5 minutes to completely separate the nucleic acid-protein complex.

[0235] (3) Add 0.2 ml of chloroform per 1 ml of Trizol, shake vigorously for 15 seconds, and let it stand at room temperature for 10 minutes.

[0236] (4) Centrifuge at 12,000 × g for 15 minutes at 4°C. The sample will separate into three layers: a red organic phase at the bottom, a colorless aqueous phase at the top, and an intermediate layer. The RNA is primarily in the aqueous phase, which accounts for approximately 60% of the volume of the Trizol reagent used. Transfer the upper aqueous phase to a new EP tube.

[0237] (5) Add isopropanol (about 500 μL) in an equal volume to the upper aqueous phase (about 500 μL) to precipitate the RNA in the aqueous phase with isopropanol. After adding isopropanol, invert and mix thoroughly. Let it stand at room temperature for 10 minutes.

[0238] (6) Centrifuge at 12,000 × g for 10 minutes at 4°C. No RNA precipitate is visible before centrifugation, but a gelatinous precipitate appears on the sides and bottom of the tube after centrifugation. Remove the supernatant.

[0239] (7) Wash the RNA pellet with 1 mL of 75% ethanol, centrifuge at 12,000 × g for 5 minutes at 4°C, and discard the supernatant.

[0240] (8) Leave the RNA precipitate to dry at room temperature for about 5-10 minutes. Over-drying will greatly reduce the solubility of RNA.

[0241] (9) Add 50 μL of enzyme-free water and mix well. Measure the concentration using Nanodrop and store at -80°C until ready for use.

[0242] 2. cDNA synthesis and real-time PCR (qPCR)

[0243] (1) The reverse transcription system (10 μl) is as follows:

[0244] ⅡSuperMix plus 5μL, total RNA 500ng, the balance is RNase-free H2O.

[0245] (2) The RT-PCR procedure is as follows:

[0246] 25℃5min, 42℃30min, 85℃5min.

[0247] The collected cDNA products were stored at -20°C for later use.

[0248] 3. Real-time PCR reaction

[0249] (1) Take 10 μl of the obtained cDNA, add 40 μl of DNase / RNase-free water for dilution (i.e., dilute the cDNA 5 times) and use it as a template for subsequent real-time qPCR.

[0250] The real-time PCR reaction system (20 μl) is as follows:

[0251] qPCR SYBR Green Master Mix 10 μl, upstream and downstream primers 0.5 μl each, Real-time qPCR template 1 μl, and the balance is sterile ultrapure water.

[0252] Human AGT qPCR Primer Pair was provided by Shanghai Biotechnology Co., Ltd., catalog number QH01141S.

[0253] The real-time PCR reaction procedure is as follows:

[0254] Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing / extension at 60°C for 30 s, for a total of 40 cycles. Melting curves were drawn using the instrument default settings. -ΔΔCTMethods: mRNA expression levels were analyzed. The untreated group was selected as the control group (control), the NT group was selected as the negative control group 1 (i.e., non-targeting group), and the AAVS1 group was selected as the negative control group 2 (i.e., AAVS1 site editing group).

[0255] The mRNA expression levels are shown in Figure 3. Compared with the control group, AGT-gRNA1-mediated editing reduced AGT mRNA expression by 45.4%, AGT-gRNA2-mediated editing knocked down AGT mRNA expression by 76.8%, AGT-gRNA3-mediated editing knocked down AGT mRNA expression by 61.2%, AGT-gRNA4-mediated editing knocked down AGT mRNA expression by 36.6%, AGT-gRNA5-mediated editing knocked down AGT mRNA expression by 57.1%, AGT-gRNA6-mediated editing reduced AGT mRNA expression by 55.4%, and AGT-gRNA7-mediated editing reduced AGT mRNA expression by 35.0%.

[0256] The above results show that the use of AGT-gRNA2 in the present disclosure can significantly knock down the expression of AGT mRNA, and the use of other AGT-gRNAs also knocked down the expression of AGT mRNA to a certain extent, such as AGT-gRNA3, AGT-gRNA5 and AGT-gRNA6. It is worth noting that although the editing efficiency of the targeted AGT gene mediated by AGT-gRNA1 is very low, it still knocked down the expression of AGT mRNA by nearly 50%, which shows that the use of AGT-gRNA1 still has application prospects.

[0257] In summary, the use of ABE (adenosine base editor, such as ABE8e) and the gRNA disclosed herein can effectively inhibit the expression of the AGT gene and has potential therapeutic application prospects.

[0258] All documents mentioned in this disclosure are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above disclosure, those skilled in the art may make various changes or modifications to the disclosure, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A gRNA, characterized in that, The gRNA targets the splicing junction region of the AGT gene; Optionally, the splicing junction region includes intron 1 acceptor, intron 2 donor / acceptor, and intron 4 donor / acceptor; Optionally, the gRNA targets the GU…AG splicing junction in the splicing junction region of the AGT gene; Optionally, the gRNA guides the base editor to target the human AGT gene and change the AGT gene sequence, resulting in a 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% downregulation of AGT gene expression; Optionally, the gRNA can guide the base editor to disrupt the splicing junctions of the AGT gene, thereby reducing the expression level of the AGT gene; Optionally, the targeting sequence of the gRNA has a similarity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to the sequence shown in any one of SEQ ID NO: 1-7, or is the reverse complementary sequence of a sequence with a similarity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to the sequence shown in any one of SEQ ID NO: 1-7; Optionally, the targeting site of the gRNA is located in the splicing junction region, and the target sequence targeted by the gRNA is based on the sequence shown in any one of SEQ ID NO: 1-7, with some bases deleted, added, or substituted, such as differing from the sequence shown in any one of SEQ ID NO: 1-7 by no more than 1, 2, 3, 4, or 5 nucleotides, etc.; Optionally, the targeting sequence of the gRNA is as shown in any one of SEQ ID NO: 1-7, or is the reverse complementary sequence of the sequence shown in any one of SEQ ID NO: 1-7; Optionally, the gRNA can be directly synthesized by chemical means or prepared by other means, such as by in vitro IVT means; Optionally, the gRNA includes a targeting sequence for targeting nucleic acids; Optionally, the gRNA includes unmodified and modified gRNAs; Optionally, the modified gRNA includes chemical modifications of bases; Optionally, the chemical modifications include methylation modification, methoxy modification, fluorination modification, or thiolation modification; Optionally, the base editor is a base editor fused with a nuclease; Optionally, the nuclease includes a Cas protein; Optionally, the nuclease includes a type II Cas protein or a type V Cas protein; Optionally, the nuclease is Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2c3, Cas12a, Cas12b, Cas12g, Cas12h, Casl12i, Cas13b, Cas13c, Cas13d, Cas14, Argonaute (Ago); Optionally, the base editor includes an adenine base editor (ABE); Optionally, the base editor includes ABE8e.

2. A nucleic acid, characterized in that, The nucleic acid encodes the gRNA of claim 1, or a precursor encoding the gRNA of claim 1.

3. A carrier, characterized in that, The vector contains the gRNA of claim 1 or the nucleic acid of claim 2; Optionally, the vector further includes a nucleic acid encoding a base editor; Optionally, the vector includes one or more vectors, including: a) a first regulatory element, which is operably linked to the gRNA, b) a second regulatory element, which is operably linked to the base editor; wherein components (a) and (b) are on the same or different vectors; Optionally, the vector is a vector targeting the editing of the AGT gene; Optionally, the vector includes a plasmid or a viral vector; Optionally, the viral vector is selected from the group consisting of: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, SV40, poxvirus, or a combination thereof.

4. A gene editing system, characterized in that, The gene editing system includes the gRNA of claim 1; Optionally, the gene editing system further includes a base editor; Optionally, the base editor is a base editor fused with a nuclease; Optionally, the nuclease includes a Cas protein; Optionally, the nuclease includes a type II Cas protein or a type V Cas protein; Optionally, the nuclease includes Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2c3, Cas12a, Cas12b, Cas12g, Cas12h, Cas12i, Cas13b, Cas13c, Cas13d, Cas14, Argonaute (Ago); Optionally, the base editor includes an adenine base editor (ABE); Optionally, the base editor includes ABE8e; Optionally, the gene editing includes in vivo gene editing and in vitro gene editing.

5. A cell, characterized in that, The cell is obtained after being edited by the gene editing system of claim 4; Optionally, the cell is a cell cultured in vitro; Optionally, the cells include primary cells and passaged cells; Optionally, the cells include mammalian cells; Optionally, the cells include human cells; Optionally, the cells include liver cells, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, fat cells, intestinal cells, cervical cells, pancreatic cells.

6. A composition, characterized in that, It includes a gRNA component and a protein component, the gRNA includes the gRNA of claim 1, and the protein component contains a base editor; Optionally, the gRNA component is selected from the group consisting of: the gRNA of claim 1, or a nucleic acid encoding the aforementioned gRNA; the base editor is selected from the group consisting of: a base editor, or a nucleic acid encoding a base editor; Optionally, the base editor is a nuclease-fused base editor; Optionally, the nuclease includes a Cas protein; Optionally, the nuclease includes a type II Cas protein or a type V Cas protein; Optionally, the nuclease includes Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2c3, Cas12a, Cas12b, Cas12g, Cas12h, Cas12i, Cas13b, Cas13c, Cas13d, Cas14, Argonaute (Ago); Optionally, the base editor includes an adenosine base editor; Optionally, the base editor includes ABE8e; Optionally, the composition includes a pharmaceutical composition; Optionally, the pharmaceutical composition is liquid; Optionally, the dosage form of the pharmaceutical composition includes an injection or a syringe; Optionally, the dosage form of the pharmaceutical composition is an intravenous injection dosage form.

7. A composition, characterized in that, Comprising: The system according to claim 4 or the cell according to claim 5; And A pharmaceutically acceptable carrier; Optionally, in the composition, the system or the cell accounts for 1-99 wt% of the total weight of the composition, preferably 10-90 wt%, more preferably 30-70 wt%.

8. A delivery composition, characterized in that, Containing an active ingredient and a delivery medium, the active ingredient contains the gRNA according to claim 1, the carrier according to claim 3, or the gene editing system according to claim 4; Optionally, the delivery composition further includes a base editor; Optionally, the delivery medium includes lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microbubbles, gene guns, or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

9. A host cell, characterized in that, The host cell contains the gRNA according to claim 1, the nucleic acid according to claim 2, the carrier according to claim 3, the gene editing system according to claim 4, the composition according to claim 6, or the delivery composition according to claim 8; Optionally, the host cell includes mammalian cells; Optionally, the host cell includes human cells; Optionally, the host cell includes liver cells, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, adipocytes, intestinal cells, cervical cells, pancreatic cells.

10. A CRISPR complex, characterized in that, The complex includes the gRNA according to claim 1, a base editor, and a target nucleic acid bound to the gRNA.

11. A medicine box, characterized in that, Comprising: A first container, and an active ingredient or a drug containing the active ingredient, wherein the active ingredient or the drug is located in the first container, and the active ingredient comprises the gRNA according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, the gene editing system according to claim 4, the cell according to claim 5, the composition according to any one of claims 6 or 7, the delivery composition according to claim 8, the host cell according to claim 9, or the CRISPR complex according to claim 10; Optionally, the active ingredient or the drug is a single-agent formulation; Optionally, the dosage form of the active ingredient or the drug is an injection dosage form or a syringe; Optionally, the dosage form of the active ingredient or the drug is an intravenous injection dosage form; Optionally, the kit further contains an instruction manual, which records instructions for administering the active ingredient or the drug to a subject to be administered, so as to (i) improve gene editing efficiency; and / or (ii) prevent and / or treat diseases; Optionally, the disorder or disease includes angiotensinogen-related diseases; Optionally, the angiotensinogen-related diseases are selected from hypertension, borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other states of glucocorticoid excess (including chronic steroid therapy), pheochromocytoma, reninoma, secondary hyperaldosteronism and other states of mineralocorticoid excess, sleep apnea, thyroid / parathyroid diseases, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, nephropathy, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR) and fetal growth restriction; Optionally, the angiotensinogen-related diseases are selected from hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-related hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other states of glucocorticoid excess (including chronic steroid therapy), pheochromocytoma, primary hyperaldosteronism and other states of mineralocorticoid excess, sleep apnea, thyroid / parathyroid diseases, heart failure, myocardial infarction, stroke, diabetes, renal failure and systemic sclerosis; Optionally, the angiotensinogen-related disease is pregnancy-related hypertension (e.g., pregnancy-induced hypertension, preeclampsia and eclampsia); Optionally, the disorder or disease is hypertension; Optionally, the subject of administration is a cell; Optionally, the subject of administration is a human cell; Optionally, the subject of administration includes hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, adipocytes, intestinal cells, cervical cells, pancreatic cells; Optionally, the subject of administration is a human or non-human mammal; Optionally, the administration is to contact the active ingredient or the drug with human hepatocytes, or to inject the active ingredient or the drug into the human body by injection.

12. A method for gene editing of cells, characterized in that, Including contacting the cell with the gRNA according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, the gene editing system according to claim 4, the composition according to claim 6 or 7, the delivery composition according to claim 8, the host cell according to claim 9, the CRISPR complex according to claim 10 or the kit according to claim 11; Optionally, the contacting is carried out in vitro; Optionally, the cell is a cell cultured in vitro; Optionally, the cell includes primary cells and passaged cells; Optionally, the cell includes mammalian cells; Optionally, the cell includes human cells; Optionally, the cell includes hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, adipocytes, intestinal cells, cervical cells, pancreatic cells.

13. A kit for gene editing, characterized in that, The kit includes the gRNA according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, the gene editing system according to claim 4, the composition according to claim 6 or 7, the delivery composition according to claim 8, the host cell according to claim 9, the CRISPR complex according to claim 10 or the kit according to claim 11; Optionally, the kit further includes a label or an instruction manual; Optionally, the label or the instruction manual records the instruction for gene editing by administering the gRNA according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, the system according to claim 4, the composition according to claim 6 or 7, or the delivery composition according to claim 8 or the host cell according to claim 9 or the CRISPR complex according to claim 10 or the kit according to claim 11 to the editing subject; Optionally, the editing subject includes a cell; Optionally, the cell includes primary cells and passaged cells; Optionally, the cell includes mammalian cells; Optionally, the cell includes human cells; Optionally, the cells include hepatocytes, basal ganglia cells, brain cells, cardiomyocytes, nerve cells, gallbladder cells, kidney cells, skeletal muscle cells, choroid plexus cells, retinal cells, pituitary cells, prostate cells, adrenal cells, thyroid cells, adipocytes, intestinal cells, cervical cells, pancreatic cells.

14. Use of the gRNA according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, the gene editing system according to claim 4, the composition according to claim 6 or 7, the delivery composition according to claim 8, the host cell according to claim 9, the CRISPR complex according to claim 10, the kit according to claim 11 or the reagent kit according to claim 13 in gene editing, gene targeting or gene cleavage.

15. Use of the gRNA according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, the system according to claim 4, the cell according to claim 5, the composition according to claim 6 or 7, the delivery composition according to claim 8, the host cell according to claim 9, the CRISPR complex according to claim 10, the kit according to claim 11 or the reagent kit according to claim 13, characterized in that For the preparation of a drug for the prevention and / or treatment of a disease; Optionally, the disease is a disorder caused by a defect in a target sequence in a target locus (such as the AGT gene); Optionally, the disorder or disease includes angiotensinogen-related diseases; Optionally, the angiotensinogen-related diseases are selected from hypertension, borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other states of glucocorticoid excess (including chronic steroid therapy), pheochromocytoma, reninoma, secondary aldosteronism and other states of mineralocorticoid excess, sleep apnea, thyroid / parathyroid diseases, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR) and fetal growth restriction; Optionally, the angiotensinogen-related diseases are selected from hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-related hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other states of glucocorticoid excess (including chronic steroid therapy), pheochromocytoma, primary aldosteronism and other states of mineralocorticoid excess, sleep apnea, thyroid / parathyroid diseases, heart failure, myocardial infarction, stroke, diabetes, renal failure and systemic sclerosis; Optionally, the angiotensinogen-related disease is pregnancy-related hypertension (for example, pregnancy-induced hypertension, preeclampsia and eclampsia); Optionally, the disorder or disease is hypertension.

16. A method for treating a disease, characterized in that, Comprising administering to a subject an effective amount of the gene editing system according to claim 4, the cell according to claim 5, the composition according to claim 6 or 7, the delivery composition according to claim 8, the host cell according to claim 9, the CRISPR complex according to claim 10, the kit according to claim 11, or the reagent kit according to claim 13; Optionally, the administration comprises injection administration; Optionally, the subject is a human or non-human mammal; Optionally, the non-human mammal comprises rodents and primates, preferably mice, rats, rabbits, monkeys; Optionally, the disease is a disorder caused by a defect in a target sequence at a target locus (such as the AGT gene); Optionally, the disorder or disease comprises angiotensinogen-related diseases; Optionally, the angiotensinogen-related diseases are selected from hypertension, borderline hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other states of glucocorticoid excess (including chronic steroid therapy), pheochromocytoma, reninoma, secondary hyperaldosteronism and other states of mineralocorticoid excess, sleep apnea, thyroid / parathyroid diseases, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR) and fetal growth restriction; Optionally, the angiotensinogen-related diseases are selected from hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-related hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other states of glucocorticoid excess (including chronic steroid therapy), pheochromocytoma, primary hyperaldosteronism and other states of mineralocorticoid excess, sleep apnea, thyroid / parathyroid diseases, heart failure, myocardial infarction, stroke, diabetes, renal failure and systemic sclerosis; Optionally, the angiotensinogen-related disease is pregnancy-related hypertension (e.g., pregnancy-induced hypertension, preeclampsia and eclampsia); Optionally, the disorder or disease is hypertension.

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