Gene editing systems and their applications

Abstract

The present invention discloses a gRNA targeting VEGFA, a gene editing system, and applications thereof. The gRNA of the present invention comprises a guide sequence having at least 80% sequence identity to any one of SEQ ID NOS: 10 to 18 and 40 to 230. The gene editing system disclosed herein comprises the gRNA and its coding sequence, as well as an RNA-guided nuclease and its coding sequence, as disclosed herein.

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 2022113208591, filed on October 26, 2022, Chinese Patent Application No. 2023100880960, filed on January 18, 2023, and Chinese Patent Application No. 2023103961404, filed on April 12, 2023. This application cites the above Chinese patent applications in their entirety.

[0002] (Technical field) The present invention relates to the field of gene editing technology, specifically to a gene editing system and its application, more specifically to a gRNA and CRISPR system targeting VEGFA and its application. [Background technology]

[0003] The angiogenic signaling pathway involving vascular endothelial growth factor A (VEGFA) regulates various physiological processes and disease-related signaling pathways in living organisms, such as the angiogenesis process from embryonic development to various organs and tissues. When certain tissues or organs are exposed to hypoxia, intracellular VEGFA is activated by the hypoxia-inducible factor (HIF) transcription factor, resulting in the activation of VEGFA. Upregulation will be done. Upregulation The activated VEGFA binds to receptors, activating angiogenic signaling pathways and providing nutrients to the body. In various cancers and other diseases, tissues are hypoxic, resulting in significantly increased VEGFA expression and activation of angiogenic pathways. Research has shown that VEGFA-related angiogenic signaling pathways are elevated in various pathological processes, including cancer progression (e.g., lung cancer, breast cancer, liver cancer, glioblastoma), diabetic nephropathy, neovascular age-related macular degeneration, neovascular glaucoma, rheumatoid arthritis, psoriasis, and ovarian hyperstimulation syndrome, among other diseases in which VEGF expression is significantly elevated.

[0004] The already commercially available VEGF antibody aflibercept and the anti-VEGF overexpression drug RGX-314, currently undergoing clinical trials, are both used to treat diabetic macular edema (DME), macular edema after retinal vein occlusion (RVO), and diabetic retinopathy (DR), with relatively favorable results. Bayer's anti-VEGF drug aflibercept is already undergoing Phase III clinical trials for colorectal cancer, and Phase III clinical trials for non-small cell lung cancer are also underway. Genentech's anti-VEGF drug bevacizumab is used to treat colorectal cancer, lung cancer, breast cancer, glioma, kidney cancer, and other cancers. Clinical trials for the treatment of colorectal cancer and breast cancer are currently in Phase III.

[0005] Age-related macular degeneration (AMD) is the leading cause of blindness worldwide among people over the age of 50, resulting in irreversible visual impairment. AMD can be divided into dry (atrophic) and neovascular (wet) forms. Neovascular age-related macular degeneration (nAMD), accounting for 10–15% of cases, is one of the leading causes of vision loss. Clinical symptoms in nAMD patients include severe visual impairment, retinal hemorrhage and exudation, accompanied by the gradual or sudden development of choroidal neovascularization and macular discoid scarring. Neovascular age-related macular degeneration begins at age 50, with an incidence rate of 13% in people over 60. Current research suggests that the primary molecular mechanism underlying nAMD is overexpression of vascular endothelial growth factor (VEGFA) in retinal pigment epithelial (RPE) cells due to genetic or external stress stimuli, which activates a new angiogenic signaling pathway, leading to retinal hemorrhage and severe visual impairment. Therefore, intravitreal injection of VEGF antibody drugs can effectively suppress retinal and choroidal neovascularization and the deterioration of central vision loss.

[0006] Intravitreal injection of VEGF antibody drugs is currently the most effective treatment for nAMD and holds the largest market share. VEGF antibody drugs typically need to be injected intravitreously every 1–3 months.

[0007] The CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) system is currently the most widely used gene editing technology. However, in CRISPR-based disease treatment, obtaining a "Cas protein + gRNA" drug molecule with high editing efficiency and safety remains a significant challenge, and this remains a challenge that the field is working to resolve. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a guide RNA targeting VEGFA, a gene editing system and applications thereof. [Means for solving the problem]

[0009] A first aspect of the present disclosure provides a guide RNA (gRNA) for a gene editing system, comprising a guide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 10 to 18, 40 to 230.

[0010] In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0011] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 10-18, 40-230. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 10-18, 40-230. In some embodiments of the present disclosure, the guide sequence is selected from the sequences set forth in any one of SEQ ID NOs: 10-18, 40-230. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0012] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 40-43, 45-47, 52-60, 64, 65, 73, 216, 219, 220, 223, 227, 228, 229, and 230. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% sequence identity compared to the sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 40-43, 45-47, 52-60, 64, 65, 73, 216, 219, 220, 223, 227, 228, 229, and 230. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 40-43, 45-47, 52-60, 64, 65, 73, 216, 219, 220, 223, 227, 228, 229, and 230. In some embodiments of the present disclosure, the guide sequence is selected from the sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 40-43, 45-47, 52-60, 64, 65, 73, 216, 219, 220, 223, 227, 228, 229, and 230. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0013] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 47, 58, 59, 227, 228, and 230. In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 47, 58, 59, 227, 228, and 230. and at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% compared to the sequence set forth in any one of 230 orhas 100% sequence identity. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 47, 58, 59, 227, 228, and 230. In some embodiments of the present disclosure, the guide sequence is selected from the sequences set forth in any one of SEQ ID NOs: 12, 13, 15, 47, 58, 59, 227, 228, and 230. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0014] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 12. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 12. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 12. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0015] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 13. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 13. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 13. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 13. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0016] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 15. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 15. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0017] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity to the sequence set forth in SEQ ID NO: 40. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 40. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 40. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 40. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0018] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 41. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 41. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 41. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 41. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0019] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 42. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 42. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 42. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 42. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0020] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 43. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 43. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 43. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 43. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0021] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 45. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 45. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 45. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 45. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0022] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 46. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 46. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 46. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 46. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0023] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 47. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 47. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 47. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 47. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0024] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 52. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 52. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 52. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0025] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 53. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 53. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 53. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0026] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 54. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 54. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 54. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0027] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 55. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 55. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 55. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0028] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 56. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 56. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 56. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0029] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 57. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 57. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 57. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0030] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 58. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 58. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 58. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 58. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0031] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 59. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 59. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 59. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 59. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0032] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 60. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 60. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 60. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0033] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 64. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 64. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 64. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0034] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 65. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 65. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 65. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0035] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 73. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 73. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 73. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0036] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 216. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 216. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 216. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0037] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 219. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 219. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 219. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0038] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 220. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 220. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 220. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0039] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 223. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 223. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 223. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0040] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 227. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 227. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 227. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0041] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 228. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO: 228. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 228. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 228. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0042] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 229. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 229. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 229. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 229. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0043] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity compared to the sequence set forth in SEQ ID NO: 230. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 230. In some embodiments of the present disclosure, the guide sequence has 100% sequence identity compared to the sequence set forth in SEQ ID NO: 230. In some embodiments of the present disclosure, the guide sequence is the sequence set forth in SEQ ID NO: 230. In some embodiments, the guide sequence is designed to hybridize to a target RNA.

[0044] gRNA-1 through gRNA-10 target exon regions of VEGFA RNA that are conserved between humans and mice, gRNA-11 through gRNA-32 target exon regions of human VEGFA RNA, and gRNA-33 through gRNA-45 target splice sites in human VEGFA RNA. For example, gRNA-36 targets the intronic sequence adjacent to the 5' end of exon 2 of VEGFA pre-mRNA, and gRNA-35 targets the splice site at the 5' end of exon 2 of VEGFA pre-mRNA. gRNA-5-2, gRNA-5-3, and gRNA-5-5 target exons 1 and 2 of the VEGFA mature mRNA.

[0045] TIFF2025535495000056.tif244150TIFF2025535495000057.tif244150TIFF20255354950 00058.tif244150TIFF2025535495000059.tif244150TIFF2025535495000060.tif126150

[0046] In some embodiments of the present disclosure, the guide sequence is designed to hybridize to the target RNA with no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide mismatch.

[0047] In some embodiments of the present disclosure, the guide RNA is an RNA guide It can form a complex with a nuclease and induce sequence-specific binding of the complex to a target RNA.

[0048] In some embodiments of the present disclosure, the guide RNA is an RNA guide It can form a complex with a nuclease and induce the complex to bind to and cleave a target RNA.

[0049] In some embodiments of the present disclosure, RNA guide Nucleases are Cas proteins.

[0050] In some embodiments of the present disclosure, the guide RNA can form a CRISPR complex with a Cas protein and direct the sequence-specific binding of the CRISPR complex to a target RNA.

[0051] In some embodiments of the present disclosure, the guide RNA can form a CRISPR complex with a Cas protein and guide the CRISPR complex to bind to and cleave the target RNA.

[0052] In some embodiments of the present disclosure, RNA guide The nuclease is the Cas13 protein.

[0053] In some embodiments of the present disclosure, RNA guide The nuclease is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein.

[0054] In some embodiments of the present disclosure, RNA guide The nuclease is the Cas13d protein.

[0055] In some embodiments of the present disclosure, the Cas protein comprises an amino acid sequence having 50% or greater identity to the sequence set forth in any one of SEQ ID NOs: 1-3. In some embodiments of the present disclosure, the Cas protein comprises an amino acid sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.1% or greater, 99.2% or greater, 99.3% or greater, 99.4% or greater, 99.5% or greater, 99.6% or greater, 99.7% or greater, or 99.8% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0056] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0057] In some embodiments of the present disclosure, the Cas protein comprises a sequence set forth in any one of SEQ ID NOs: 1-3.

[0058] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or more identity to the sequence set forth in SEQ ID NO: 3. In some embodiments of the present disclosure, the Cas protein comprises a sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more identity to the sequence set forth in SEQ ID NO: 3.

[0059] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in SEQ ID NO:3.

[0060] In some embodiments of the present disclosure, the Cas protein comprises the sequence set forth in SEQ ID NO:3.

[0061] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or greater sequence identity to CasRx. In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 99.5% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0062] In some embodiments of the present disclosure, the Cas protein comprises 100% sequence identity to CasRx.

[0063] In some embodiments of the present disclosure, the Cas protein comprises the sequence of CasRx.

[0064] In some embodiments of the present disclosure, RNA guide The nuclease comprises a homologous or heterologous domain fused to a nuclease moiety.

[0065] In some embodiments of the present disclosure, RNA guide The nuclease comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0066] In some embodiments of the present disclosure, the Cas protein comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0067] In some embodiments of the present disclosure, the subcellular localization signal is a nuclear localization signal and outside transportation signal sequences.

[0068] In some embodiments of the present disclosure, the target RNA is VEGFA RNA. In some embodiments of the present disclosure, the target RNA is human VEGFA RNA. In some embodiments of the present disclosure, the target RNA is VEGFA pre-mRNA. In some embodiments of the present disclosure, the target RNA is mature VEGFA mRNA.

[0069] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence. In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence, and the direct repeat sequence interacts with an RNA-guided nuclease.

[0070] In some embodiments of the present disclosure, the complex reduces the level of a target RNA in an animal (eg, a human).

[0071] In some embodiments of the present disclosure, the complex reduces the level of a target RNA in a cell, such as the level of a target RNA in a cell that expresses the target RNA.

[0072] In some embodiments of the present disclosure, the complex reduces the level of target RNA in cells by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in target RNA level can be tested using conventional methods in the art, including but not limited to, the qPCR method described in the Examples, and untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as a negative control to calculate the target RNA knockdown level of the experimental group compared to the negative control.

[0073] In some embodiments of the present disclosure, when the complex binds to and cleaves the target RNA, the number of off-target genes is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection between the differentially expressed gene set determined by RNA sequencing and the set of off-target genes predicted by the program. As a non-limiting example, the program uses the EMBOSS-water program to predict the whole genome and whole cDNA sequences of the target species (Homo sapiens / Mus musculus), setting the parameters gap_extend=0.5 and gap_extend=10, using the forward and reverse strands of the gRNA guide sequence for comparison, filtering the prediction results, and obtaining predicted potential target genes (including on-target and off-target genes).

[0074] In some embodiments of the present disclosure, the conjugate reduces the level of a protein encoded by a target RNA in an animal (eg, a human).

[0075] In some embodiments of the present disclosure, the complex reduces the level of the protein encoded by the target RNA in cells.In some embodiments of the present disclosure, the target RNA encodes a protein called VEGFA protein.In some embodiments of the present disclosure, the complex reduces the level of VEGFA protein in cells.

[0076] In some embodiments of the present disclosure, the complex reduces intracellular VEGFA protein levels by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the level of the target RNA-encoded protein can be tested using conventional methods in the art, including, but not limited to, ELISA and Western blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as a negative control to calculate the knockdown level of the target RNA-encoded protein in the experimental group compared to the negative control.

[0077] A second aspect of the present disclosure provides an isolated nucleic acid encoding a guide RNA according to the present disclosure.

[0078] A third aspect of the present disclosure provides a vector comprising a polynucleotide sequence encoding a guide RNA according to the present disclosure and a regulatory sequence for regulating expression of the guide RNA.

[0079] In some embodiments of the present disclosure, the vector is an adeno-associated virus vector.

[0080] In some embodiments of the present disclosure, the regulatory sequence is a promoter and / or an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence and an enhancer sequence.

[0081] In some embodiments of the present disclosure, the regulatory sequence is selected from a native promoter, a ubiquitous promoter, a CAG promoter, a CBA promoter, a CBh promoter, a CMV promoter, and a tissue-specific promoter.

[0082] In some embodiments of the present disclosure, the regulatory sequence is a promoter, and the promoter is selected from RPE65, VDM2, Mlc1, GFAP, and ICAM-2 promoters.

[0083] In some embodiments of the present disclosure, the regulatory sequence is a U6 promoter.

[0084] In some embodiments of the present disclosure, the promoter is an eye-specific promoter.

[0085] In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0086] In some embodiments of the present disclosure, the regulatory sequence comprises an HRE enhancer element.

[0087] In some embodiments of the present disclosure, the regulatory sequence comprises a tandem NRS element and an HRE enhancer element.

[0088] A fourth aspect of the present disclosure provides a gene editing system comprising: (a) a guide RNA, or a polynucleotide sequence encoding the guide RNA, described in this disclosure; and (b) RNA guide Nuclease, or RNA guide a polynucleotide sequence encoding a nuclease; The guide RNA can form a complex with a nuclease and direct the complex to sequence-specific binding to the target RNA.

[0089] In some embodiments of the present disclosure, the guide RNA can form a complex with a nuclease and guide the complex to bind to and cleave the target RNA.

[0090] In some embodiments of the present disclosure, the guide sequence is designed to hybridize to the target RNA with no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide mismatch.

[0091] In some embodiments of the present disclosure, RNA guide The polynucleotide sequence encoding the nuclease is linked to a regulatory sequence, which in turn is an RNA guide Used to regulate the expression of nucleases.

[0092] In some embodiments of the present disclosure, the polynucleotide sequence encoding the guide RNA is linked to a regulatory sequence, and the regulatory sequence is used to regulate expression of the guide RNA.

[0093] In some embodiments of the present disclosure, expression of an RNA-guided nuclease is adjustment do adjustment The sequence controls the expression of the guide RNA. adjustment do adjustment The sequence may be the same or different.

[0094] In some embodiments of the present disclosure, the regulatory sequence is a promoter and / or an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence and an enhancer sequence.

[0095] In some embodiments of the present disclosure, the regulatory sequence is selected from a native promoter, a ubiquitous promoter, a CAG promoter, a CBA promoter, a CBh promoter, a CMV promoter, and a tissue-specific promoter.

[0096] In some embodiments of the present disclosure, the regulatory sequence is a promoter, and the promoter is selected from RPE65, VDM2, Mlc1, GFAP, and ICAM-2 promoters.

[0097] In some embodiments of the present disclosure, the regulatory sequence is a U6 promoter.

[0098] In some embodiments of the present disclosure, the promoter is an eye-specific promoter.

[0099] In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0100] In some embodiments of the present disclosure, the regulatory sequence comprises an HRE enhancer element (hypoxia response element). In some embodiments, the regulatory sequence comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine HRE enhancer elements in tandem. In some embodiments, the regulatory sequence comprises 2, 3, 4, 5, 6, 7, 8, or 9 HRE enhancer elements in tandem.

[0101] In some embodiments of the present disclosure, the regulatory sequence comprises an NRS element (neuron-restrictive silencer) from the human synapsin gene. In some embodiments, the regulatory sequence comprises an NRS element and an HRE enhancer element. In some embodiments, the regulatory sequence comprises a tandem NRS element and an HRE enhancer element. To enhance the regulatory effect, the tandem NRS element and the HRE enhancer element can be repeated multiple times. In some embodiments, the regulatory sequence comprises tandem NRS elements and HRE enhancer elements repeated at least two times, at least three times, at least four times, at least five times, or at least six times. In some embodiments, the regulatory sequence comprises a tandem NRS element and an HRE enhancer element repeated two, three, four, five, or six times.

[0102] In some embodiments of the present disclosure, the HRE enhancer element sequence is TGTCACGTCCTGCACGACGTA (SEQ ID NO: 233) or its reverse complement.

[0103] In some embodiments of the present disclosure, the NRS element sequence is TTCAGCGCGGACAGTGCC (SEQ ID NO: 234) or its reverse complement.

[0104] In some embodiments of the present disclosure, the regulatory sequence comprises the repeat TGTCACGTCCTGCACGACGTA (SEQ ID NO: 233) or its reverse complement.

[0105] In some embodiments of the present disclosure, the regulatory sequence comprises a tandem sequence of TTCAGCCCGCGGACAGTGCCTGTCACGTCCTGCACGACGTA (SEQ ID NO: 235) or its reverse complement.

[0106] In some embodiments of the present disclosure, the gene editing system is a CRISPR-Cas system.

[0107] In some embodiments of the present disclosure, RNA guide Nucleases are Cas proteins.

[0108] In some embodiments of the present disclosure, the guide RNA can form a CRISPR complex with a Cas protein and direct the sequence-specific binding of the CRISPR complex to a target RNA.

[0109] In some embodiments of the present disclosure, the guide RNA can form a CRISPR complex with a Cas protein and guide the CRISPR complex to bind to and cleave the target RNA.

[0110] In some embodiments of the present disclosure, RNA guide The nuclease is the Cas13 protein.

[0111] In some embodiments of the present disclosure, RNA guide The nuclease is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein.

[0112] In some embodiments of the present disclosure, RNA guide The nuclease is the Cas13d protein.

[0113] In some embodiments of the present disclosure, the Cas protein comprises an amino acid sequence having 50% or greater identity to the sequence set forth in any one of SEQ ID NOs: 1-3. In some embodiments of the present disclosure, the Cas protein comprises an amino acid sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.1% or greater, 99.2% or greater, 99.3% or greater, 99.4% or greater, 99.5% or greater, 99.6% or greater, 99.7% or greater, or 99.8% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0114] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0115] In some embodiments of the present disclosure, the Cas protein comprises a sequence set forth in any one of SEQ ID NOs: 1-3.

[0116] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or more identity to the sequence set forth in SEQ ID NO: 3. In some embodiments of the present disclosure, the Cas protein comprises a sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more identity to the sequence set forth in SEQ ID NO: 3.

[0117] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in SEQ ID NO:3.

[0118] In some embodiments of the present disclosure, the Cas protein comprises the sequence set forth in SEQ ID NO:3.

[0119] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or greater sequence identity to CasRx. In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 99.5% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0120] In some embodiments of the present disclosure, the Cas protein comprises 100% sequence identity to CasRx.

[0121] In some embodiments of the present disclosure, the Cas protein comprises the sequence of CasRx.

[0122] In some embodiments of the present disclosure, the gene editing system is a CRISPR-Cas system comprising: (a) a guide RNA, or a polynucleotide sequence encoding the guide RNA, described in this disclosure; and (b) a polynucleotide sequence encoding a Cas protein or a Cas13 protein; The guide RNA can form a complex with the Cas13 protein and direct the sequence-specific binding of the complex to the target RNA.

[0123] In some embodiments of the present disclosure, the gene editing system comprises: (a) a guide RNA, or a polynucleotide sequence encoding the guide RNA, described in this disclosure; and (b) a Cas13 protein or a polynucleotide sequence encoding a Cas13 protein; The guide RNA forms a complex with the Cas13 protein and guides the complex to bind and cleave the target RNA.

[0124] In some embodiments of the present disclosure, the polynucleotide sequence encoding the guide RNA is connected to a first regulatory sequence, and the first regulatory sequence is used to regulate the expression of the guide RNA. guide The polynucleotide sequence encoding the nuclease is connected to a second regulatory sequence, which is an RNA guide Used to regulate nuclease expression.

[0125] In some embodiments of the present disclosure, RNA guide The nuclease comprises a homologous or heterologous domain fused to a nuclease moiety.

[0126] In some embodiments of the present disclosure, RNA guide The nuclease comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0127] In some embodiments of the present disclosure, the Cas protein comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0128] In some embodiments of the present disclosure, the subcellular localization signal is a nuclear localization signal and outside transportation signal sequences.

[0129] The gene editing systems described herein include: (i) RNA guide (ii) as mRNA encoding the nuclease and guide RNA, (iii) as separate RNAs, either as part of a single vector or plasmid or split across multiple vectors or plasmids. guide as a nuclease and guide RNA, or (iv) RNA guide The RNP complex of nuclease and guide RNA can be introduced into cells (or cell-free systems) by a variety of non-limiting methods.

[0130] In some embodiments of the present disclosure, the target RNA is VEGFA RNA. In some embodiments of the present disclosure, the target RNA is VEGFA pre-mRNA. In some embodiments of the present disclosure, the target RNA is mature VEGFA mRNA.

[0131] In some embodiments of the present disclosure, the complex reduces the level of a target RNA in an animal (eg, a human).

[0132] In some embodiments of the present disclosure, the complex reduces the level of a target RNA in a cell, such as the level of a target RNA in a cell that expresses the target RNA.

[0133] In some embodiments of the present disclosure, the complex reduces the level of target RNA in a cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. That is, the editing efficiency of the gene editing system is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in target RNA levels can be tested using conventional methods in the art, including but not limited to the qPCR method described in the Examples, and untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls to calculate the target RNA knockdown level of the experimental group compared to the negative control.

[0134] In some embodiments of the present disclosure, when the complex binds to and cleaves the target RNA, the number of off-target genes is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection between the differentially expressed gene set determined by RNA sequencing and the set of off-target genes predicted by the program. As a non-limiting example, the program uses the EMBOSS-water program to predict the whole genome and whole cDNA sequences of the target species (Homo sapiens / Mus musculus), setting the parameters gap_extend=0.5 and gap_extend=10, using the forward and reverse strands of the gRNA guide sequence for comparison, filtering the prediction results, and obtaining predicted potential target genes (including on-target and off-target genes).

[0135] In some embodiments of the present disclosure, the conjugate reduces the level of a protein encoded by a target RNA in an animal (eg, a human).

[0136] In some embodiments of the present disclosure, the complex reduces the level of the protein encoded by the target RNA in cells.In some embodiments of the present disclosure, the target RNA encodes a protein called VEGFA protein.In some embodiments of the present disclosure, the complex reduces the level of VEGFA protein in cells.

[0137] In some embodiments of the present disclosure, the complex reduces intracellular VEGFA protein levels by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the level of the target RNA-encoded protein can be tested using conventional methods in the art, including, but not limited to, ELISA and Western blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as a negative control to calculate the knockdown level of the target RNA-encoded protein in the experimental group compared to the negative control.

[0138] A fifth aspect of the present disclosure provides a vector system comprising the gene editing system described herein, the vector system comprising a polynucleotide sequence encoding a guide RNA and a vector encoding a gene for expression of the guide RNA. adjustment The first adjustment RNA guide A polynucleotide sequence encoding a nuclease and an RNA guide Nuclease expression adjustment The second adjustment and an array.

[0139] In some embodiments of the present disclosure, the vector system comprises one or more vectors.

[0140] In some embodiments of the present disclosure, the vector system comprises only one vector.

[0141] In some embodiments of the present disclosure, the vector system comprises a plurality of vectors, each of which comprises a polynucleotide sequence encoding a guide RNA and a vector encoding the expression of the guide RNA. adjustment The first regulatory sequence is located on the first vector, and guidePolynucleotide sequences and RNA encoding nucleases guide Nuclease expression adjustment The corresponding second regulatory sequence is located on a second vector.

[0142] In some embodiments of the present disclosure, the first regulatory sequence and / or the second regulatory sequence are promoter and / or enhancer sequences.In some embodiments of the present disclosure, the first regulatory sequence or the second regulatory sequence is a promoter sequence.In some embodiments of the present disclosure, the first regulatory sequence and the second regulatory sequence are promoter and enhancer sequences.

[0143] A sixth aspect of the present disclosure provides an adeno-associated viral vector comprising a gene editing system described herein, wherein the adeno-associated viral vector encodes an RNA vector described herein. guide It contains DNA encoding a nuclease and a guide RNA.

[0144] A seventh aspect of the present disclosure provides a lipid nanoparticle comprising a gene editing system described herein, wherein the lipid nanoparticle comprises a guide RNA described herein and an RNA guide and mRNA encoding the nuclease.

[0145] An eighth aspect of the present disclosure provides a lentiviral vector comprising the gene editing system described herein, the lentiviral vector comprising a guide RNA described herein and an RNA guide and mRNA encoding a nuclease. Optionally, the lentiviral vector is mimicked with an envelope protein. Optionally, the RNA guide The mRNA encoding the nuclease is linked to an aptamer sequence.

[0146] A ninth aspect of the present disclosure provides a ribonucleoprotein complex comprising a gene editing system described herein, wherein the ribonucleoprotein complex comprises a guide RNA described herein and a nucleic acid sequence encoding the RNA. guide It is formed by nucleases.

[0147] A tenth aspect of the present disclosure provides a virus-like particle comprising the gene editing system described herein, wherein the virus-like particle comprises a guide RNA and an RNA guide Optionally, the ribonucleoprotein complex formed by the RNA guide The nuclease is fused to the gag protein.

[0148] An eleventh aspect of the present disclosure provides a eukaryotic cell comprising the gene editing system described herein. Optionally, the eukaryotic cell is a mammalian cell.

[0149] A twelfth aspect of the present disclosure provides a pharmaceutical composition comprising a gene editing system described in this disclosure, a vector system described in this disclosure, an adeno-associated viral vector described in this disclosure, a lipid nanoparticle described in this disclosure, a lentiviral vector described in this disclosure, a ribonucleoprotein complex described in this disclosure, a virus-like particle described in this disclosure, or a eukaryotic cell described in this disclosure.

[0150] In some embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0151] A thirteenth aspect of the present disclosure provides use of a guide RNA described in this disclosure, an isolated nucleic acid described in this disclosure, a vector described in this disclosure, a gene editing system described in this disclosure, a vector system described in this disclosure, an adeno-associated virus vector described in this disclosure, a lipid nanoparticle described in this disclosure, a lentiviral vector described in this disclosure, a ribonucleoprotein complex described in this disclosure, a eukaryotic cell described in this disclosure, a pharmaceutical composition described in this disclosure, and / or a virus-like particle described in this disclosure in the preparation of a medicament for carrying out any of the following, or any of the following schemes:

[0152] Cleavage or nicking of one or more target RNA molecules, activation or upregulation of one or more target RNA molecules, activation or inhibition of translation of one or more target RNA molecules, inactivation of one or more target RNA molecules, visualization, labeling or detection of one or more target RNA molecules, binding to one or more target RNA molecules, transport of one or more target RNA molecules, and masking of one or more target RNA molecules.

[0153] In some embodiments of the present disclosure, there is provided use of a guide RNA described in this disclosure, an isolated nucleic acid described in this disclosure, a vector described in this disclosure, a gene editing system described in this disclosure, a vector system described in this disclosure, an adeno-associated virus vector described in this disclosure, a lipid nanoparticle described in this disclosure, a lentiviral vector described in this disclosure, a ribonucleoprotein complex described in this disclosure, and a virus-like particle described in this disclosure in the preparation of a medicament for carrying out any of the following, or any of the following schemes:

[0154] One or more target Cleavage of RNA molecules and one or more target It binds to RNA molecules.

[0155] In some embodiments of the present disclosure, the target RNA is VEGFA RNA.

[0156] For example, as will be appreciated by those skilled in the art, guideNucleases can be fused or conjugated (e.g., via fusion proteins, linker peptides, etc.) to one or more homologous or heterologous domains. For example, Cas13 mutants that have completely or partially lost nuclease activity can be fused to homologous or heterologous domains. These homologous or heterologous domains can have a variety of activities, such as methylase activity, demethylase activity, deaminase activity, translational activation activity, translational repression activity, RNA cleavage activity, nucleic acid binding activity, base editing activity, or switching activity (e.g., photoinducible). Homologous or heterologous domains include, but are not limited to, localization signals (e.g., nuclear localization signal NLS, nuclear export signal NES), markers or detection markers (e.g., fluorescent dyes such as FITC or DAPI), targeting moieties, antigenic determinant tags (e.g., Hismyc, V5, FLAG, HA, VSV-G, Trx, etc.), deaminase or deamination domains (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD), methylases, demethylases, ssRNA cleavage activity domains, dsRNA cleavage activity domains, DNA or RNA ligases, or any combination thereof. For example, fusing a Cas13 protein with a deaminase and combining it with a gRNA to target the target RNA can achieve single-base editing of the target RNA molecule. For example, the homologous or heterologous domain can be a detectable label. When the CRISPR-CAS complex contacts or binds to the target nucleic acid, the complex containing the Cas13 nuclease cleaves or modifies the target nucleic acid, detecting the presence of the detectable label. The detectable label can be a fluorescent group, a chromogenic agent, a developing agent, or a radioisotope.

[0157] A fourteenth aspect of the present disclosure provides a method for diagnosing, treating, or preventing a disease or condition associated with a target RNA, the method comprising administering to a sample from or to a subject in need thereof an effective amount of a guide RNA described herein, an isolated nucleic acid described herein, a vector described herein, a gene editing system described herein, a vector system described herein, an adeno-associated virus vector described herein, a lipid nanoparticle described herein, a lentiviral vector described herein, a ribonucleoprotein complex described herein, a virus-like particle described herein, or a pharmaceutical composition described herein.

[0158] In some embodiments of the present disclosure, a disease or disorder associated with a target RNA refers to a disease or disorder caused by aberrant expression of a target RNA.

[0159] In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, age-related macular degeneration.

[0160] In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, dry age-related macular degeneration. In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, wet age-related macular degeneration.

[0161] In some embodiments of the present disclosure, the target RNA is VEGFA RNA.

[0162] A fifteenth aspect of the present disclosure provides the use of a guide RNA described in this disclosure, an isolated nucleic acid described in this disclosure, a vector described in this disclosure, a gene editing system described in this disclosure, a vector system described in this disclosure, an adeno-associated virus vector described in this disclosure, a lipid nanoparticle described in this disclosure, a lentiviral vector described in this disclosure, a ribonucleoprotein complex described in this disclosure, and a virus-like particle described in this disclosure in the manufacture of a medicament for diagnosing, treating, or preventing a disease or condition associated with a target RNA.

[0163] In some embodiments of the present disclosure, a disease or disorder associated with a target RNA refers to a disease or disorder caused by aberrant expression of a target RNA.

[0164] In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, age-related macular degeneration.

[0165] In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, dry age-related macular degeneration. In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, wet age-related macular degeneration.

[0166] In some embodiments of the present disclosure, the target RNA is VEGFA RNA.

[0167] The above-described preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present disclosure, provided that this is consistent with common knowledge in the art. [Brief explanation of the drawings]

[0168] [Figure 1] Figure 1 shows a schematic diagram of the editing efficiency results of different combinations of Cas13 and gRNA in cells. [Figure 2] Figure 2 shows a schematic diagram of the relative VEGFA RNA expression results. [Figure 3] Figure 3 is a schematic diagram showing the editing efficiency results of different tool combinations. [Figure 4A] Figure 4A is a schematic diagram showing a comparison of the editing efficiency at the RNA level between gRNA-5 and gRNA-6. [Figure 4B] Figure 4B is a schematic diagram showing a comparison of the editing efficiencies of gRNA-4 and gRNA-6 at the RNA level. [Figure 4C] FIG. 4C is a schematic diagram showing the results of comparing knockdown efficiency at the protein level. [Figure 5] FIG. 5 is a schematic diagram showing the results of editing efficiency in mice. [Figure 6] Figure 6 shows a schematic diagram of the editing efficiency results at the RNA level for several gRNAs, from gRNA-11 to gRNA-32. [Figure 7] Figure 7 is a schematic diagram showing the results of editing efficiency at the protein level from gRNA-11 to gRNA-32. [Figure 8] FIG. 8 is a schematic diagram showing the results of editing efficiency targeting regions near the splice sites of VEGFA RNA. [Figure 9A] Figures 9A and 9B show the editing efficiency of the flanking regions of the gRNA-30 target sequence. Figure 9A shows the specific locations of each target sequence and the corresponding editing efficiency, while Figure 9B shows the editing efficiency presented in the form of mean + standard deviation. [Figure 9B] Figures 9A and 9B show the editing efficiency of the flanking regions of the gRNA-30 target sequence. Figure 9A shows the specific locations of each target sequence and the corresponding editing efficiency, while Figure 9B shows the editing efficiency presented in the form of mean + standard deviation. [Figure 10] Figure 10 shows the read distribution of the targeted VEGFA genome coverage using IGV software after cell editing with C13-2-gRNA5 and CasRx-X-dual. This figure shows the results of three batches of experiments. [Figure 11] Figure 11 shows the design principles of gRNA-5-2 to gRNA-5-5. [Figure 12] Figure 12 shows the read distribution of the targeted VEGFA genome coverage displayed by the IGV software after editing cells with C13-2-gRNA. [Figure 13]Figure 13 shows that gRNA-4 and gRNA-5 significantly inhibited the development of new blood vessels. The white dotted circular selection boxes in the figure indicate the laser photocoagulation points, and the neovascularized areas were stained with IB4-488 7 days after modeling. There were 10 mice in each group. The AAV drug was delivered to both eyes of each mouse via subretinal injection. All eyes that successfully underwent 3 weeks of treatment were selected for laser modeling. Samples were collected, fixed, stained, and photographed 7 days after modeling, and the results were statistically analyzed. Bar = 200 μm. [Figure 14] Figure 14 shows that the CasRx of Example 12 also has higher editing efficiency when combined with the gRNAs disclosed herein. [Figure 15] Figure 15 shows the effects of inhibiting neovascularization in non-human primates (NHPs) called cynomolgus monkeys. The drug groups C13-2-gRNA-30 and C13-2-gRNA-5 significantly inhibited the formation of grade IV vitiligo, i.e., significantly inhibited the development of new blood vessels, with long-lasting effects. DETAILED DESCRIPTION OF THE INVENTION

[0169] The terms "guide sequence" and "targeting domain" are used interchangeably.

[0170] "guide array When referring to ", the "t" in the sequence is used synonymously with the "u".

[0171] When referring to an RNA sequence, "t" in the sequence is used interchangeably with "u".

[0172] When referring to "direct repeat sequences," "t" in the sequence is used interchangeably with "u." Definition section:

[0173] As used herein, the terms "gene editing system guide RNA," "guide RNA," "guide RNA," and "gRNA" are used interchangeably. The term guide RNA is used to refer to a molecule in a gene editing system that forms a complex with an RNA-guided nuclease and directs sequence-specific binding of the complex to a target sequence. A guide RNA is target When the RNA-guided nuclease is a Cas protein, particularly Cas13, the guide RNA typically consists of a direct repeat sequence connected to the guide sequence.

[0174] As used herein, the terms "guide sequence" and "target domain" are used interchangeably and refer to a contiguous nucleotide sequence within a gRNA that has partial or complete complementarity with a target sequence within a target RNA and can hybridize to the target sequence within the target RNA through base pairing promoted by an RNA-guided nuclease. Complete complementarity between the guide sequence and the target sequence described in the present invention is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a gene editing complex.

[0175] Suitable direct repeat (DR) sequences exist in the CRISPR locus structure of prokaryotes (such as bacteria and archaea) and can be obtained through experimental screening. These sequences can also be obtained through sequence modification or optimization, including, but not limited to, deletion, substitution, or addition of one, two, three, four, or more complementary base pairs in the complementary double-stranded region of the secondary structure of the DR sequence, and deletion, substitution, or addition of nucleotides in the loop of the stem-loop structure of the secondary structure of the DR sequence (e.g., an aptamer sequence can be inserted into the loop). Direct repeat sequences are typically several tens of nucleotides in size, with some of the fragments reverse-complementary to each other, meaning that secondary structures such as stem-loop structures (often called hairpin structures) form within the RNA molecule, while other fragments remain unstructured. guideWhen the nuclease is a Cas protein, the direct repeats are an invariant part of the gRNA molecule and contain strong secondary structure, facilitating the interaction between the Cas protein and the gRNA molecule.

[0176] The term "hybridization" or "hybridizing" refers to the process by which fully or partially complementary polynucleotide strands assemble under appropriate hybridization conditions to form a double-stranded structure or region that involves binding between nucleic acids through hydrogen bonds. As used herein, the term "hybridization" includes situations in which the double-stranded structure or region contains one or more bulges or mismatches. Hybridization and the strength of hybridization (i.e., the strength of the association between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the Tm of the hybrid formed. Hydrogen bonds are typically formed between adenine and thymine, adenine and uracil, or cytosine and guanine, although other non-classical base pairs can also form hydrogen bonds. Modified nucleotides may form hydrogen bonds in a non-classical manner that allows or facilitates hybridization.

[0177] As used herein, the term " target "RNA" refers to the specific sequence or its reverse complement that you wish to bind, target, or modify using a gene editing system. target A polynucleotide containing a sequence, which may be, for example, a complete mature mRNA molecule or a pre-mRNA molecule.

[0178] As used herein target The term sequence refers to a sequence that is complementary (fully or partially complementary) to the guide sequence of a gRNA molecule. targetThis refers to a short sequence within an RNA molecule. The gene editing complex specifically localizes to the target sequence via the guide sequence and performs its corresponding function at or near this location. The length of the target sequence is often several tens of nucleotides (nt), such as approximately 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, or 60 nt.

[0179] As used herein, the term "cleavage" or "cleavage" refers to breaking a covalent bond (eg, a covalent phosphodiester bond) within the ribosylphosphodiester backbone of a polynucleotide.

[0180] The ability of a guide RNA to direct sequence-specific binding of a gene editing complex to a target RNA can be assessed by any suitable assay. For example, components of a gene editing system sufficient to form a CRISPR complex, including a test guide RNA, can be provided to a host cell along with the corresponding target RNA molecule, such as by transfection of a vector encoding the components of the CRISPR complex, and then assessed for preferential cleavage within the target sequence. Similarly, cleavage of a target RNA sequence in a test tube can be assessed by providing a target RNA that is a component of a CRISPR complex containing the test guide RNA and a control guide RNA that is different from the test guide RNA, and comparing the ability of the test guide RNA to bind to the target RNA or the rate of cleavage of the target RNA between the test guide RNA and the control guide RNA. The ability of a guide RNA to direct the complex to cleave the target RNA can also be assessed by the assays described above.

[0181] The term RNA-guided nuclease refers to a polypeptide that binds in a sequence-specific manner to a particular target RNA sequence and is guided to the target RNA by a guide RNA that hybridizes to a target sequence on the target RNA in complex with the polypeptide. guide Cleavage of the target sequence by nucleases can result in strand breaks in RNA guide Nucleases can cleave target sequences upon binding, but RNA guideThe term nuclease includes nuclease-inactive RNA that can bind to but not cleave a target sequence. guide Nucleases are also included. RNA-guided nucleases disclosed herein include, but are not limited to, wild-type RNA-guided nucleases (e.g., C13-2, CasRx, etc.), mutants thereof (e.g., mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off-target effects, mutants with reduced side-cutting effects), and functional fragments or fusion proteins thereof.

[0182] As used herein, the term Cas protein refers to a CRISPR-associated (Cas) polypeptide or protein that, when complexed or functionally combined with one or more guide RNAs, is guided to a target sequence within a target RNA and can then bind to and cleave the target RNA. target Also included are nuclease-inactivated Cas proteins that can bind to but not cleave a sequence. Cas proteins disclosed herein include, but are not limited to, wild-type Cas proteins (e.g., C13-2, CasRx, etc.), mutants thereof (e.g., mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off-target effects, mutants with reduced side-cutting effects), and functional fragments or fusion proteins thereof.

[0183] As used herein, when referring to a nucleotide sequence / DNA / RNA that encodes a protein, RNA, or CRISPR complex, the coding sequence may be codon-optimized, for example, the coding sequence is codon-optimized for expression in a eukaryotic cell environment, the coding sequence is codon-optimized for expression in a mammalian cell environment, or the coding sequence is codon-optimized for expression in a human cell environment.

[0184] As used herein, the term "sequence identity" (identity or percent identity) refers to the sequence match between two polypeptides or two nucleic acids. If a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), the molecules are identical at that position. The "percent sequence identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, if six out of ten positions in two sequences are identical, the two sequences will have 60% sequence identity. Comparisons are typically performed by aligning two sequences to maximize sequence identity. Such alignments can be performed using publicly available, commercially available alignment algorithms and programs, such as ClustalΩ, MAFFT, Probcons, T-Coffee, Probalign, and BLAST, from which one of skill in the art can reasonably choose. Those skilled in the art can determine appropriate parameters for aligning sequences, including, for example, any algorithms needed to achieve better alignment or optimal comparison over the entire length of the sequences being compared, as well as any algorithms needed to achieve better alignment or optimal comparison over local regions of the sequences being compared. When the Cas13 proteins described in this disclosure are fused to other domains, the sequence identities of the Cas13 protein portions are compared.

[0185] As used herein, the term "regulatory sequence" includes promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly(U) sequences). Regulatory sequences include elements that direct continuous expression of a nucleotide sequence in many types of host cells and elements that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., neurons, lymphocytes). Regulatory sequences can also direct expression in a time-dependent manner, such as cell cycle-dependent or developmental stage-dependent, which may or may not be tissue- or cell-type-specific. The term regulatory sequence also includes enhancer elements, such as the WPRE, CMV enhancer, SV40 enhancer, and HRE enhancer element, as well as the intronic sequence between exons 2 and 3 of rabbit β-globin.

[0186] As used herein, the term "promoter" has its meaning generally recognized in the art.

[0187] As used herein, the term "enhancer" has its meaning generally recognized in the art.

[0188] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression desired, etc. The vector is introduced into a host cell to express the RNA described herein. guide Nucleases and / or guide RNAs can be produced.

[0189] As used herein, the term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant, pharmaceutical carrier, or other auxiliary substance administered with an active ingredient. The choice depends on the application and mode of administration. The excipient must not be incompatible with the active ingredient, e.g., it must not produce undesirable biological effects or interact in a deleterious manner with other components of the pharmaceutical composition. Pharmaceutical compositions can be prepared by methods known in the art of pharmaceutical preparation.

[0190] guide RNA

[0191] In some embodiments of the present disclosure, the guide RNA is an RNA guide It can form a complex with a nuclease (also called a gene editing complex) and induce sequence-specific binding of the complex to target RNA.

[0192] In some embodiments of the present disclosure, the guide RNA is an RNA guide It can form a complex with a nuclease and induce the complex to bind to and cleave a target RNA.

[0193] In some embodiments of the present disclosure, the complex reduces the level of a target RNA in an animal (eg, a human).

[0194] In some embodiments of the present disclosure, the complex reduces the level of a target RNA in a cell, such as the level of a target RNA in a cell that expresses the target RNA.

[0195] In some embodiments of the present disclosure, the complex reduces the level of target RNA in cells by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in target RNA level can be tested using conventional methods in the art, including but not limited to, the qPCR method described in the Examples, and untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as a negative control to calculate the target RNA knockdown level of the experimental group compared to the negative control.

[0196] In some embodiments of the present disclosure, when the complex binds to and cleaves the target RNA, the number of off-target genes is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection between the differentially expressed gene set determined by RNA sequencing and the set of off-target genes predicted by the program. As a non-limiting example, the prediction method of the program is to use the EMBOSS-water program to predict in the whole genome and whole cDNA sequences of the target species (Homo sapiens / Mus musculus), set the parameters gap_extend=0.5 and gap_extend=10, use the forward and reverse strands of the gRNA guide sequence for comparison, filter the prediction results, obtain predicted potential target genes (including on-target genes and off-target genes), and remove the off-target gene set after the on-target genes.

[0197] In some embodiments of the present disclosure, the conjugate reduces the level of a protein encoded by a target RNA in an animal (eg, a human).

[0198] In some embodiments of the present disclosure, the complex reduces the level of the protein encoded by the target RNA in cells.In some embodiments of the present disclosure, the target RNA encodes a protein called VEGFA protein.In some embodiments of the present disclosure, the complex reduces the level of VEGFA protein in cells.

[0199] In some embodiments of the present disclosure, the complex reduces intracellular VEGFA protein levels by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the level of the target RNA-encoded protein can be tested using conventional methods in the art, including, but not limited to, ELISA and Western blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as a negative control to calculate the knockdown level of the target RNA-encoded protein in the experimental group compared to the negative control.

[0200] In some embodiments of the present disclosure, RNA guide The nuclease is a Cas protein. guide The cleavage activity of the nuclease is completely or partially lost.

[0201] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein, and the guide RNA comprises a guide sequence and a direct repeat sequence.

[0202] In some embodiments of the present disclosure, the Cas protein is a Cas13 protein.

[0203] In some embodiments of the present disclosure, the Cas protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein. In some embodiments of the present disclosure, the Cas protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein in which the catalytic center RxxxxH motifs in both HEPN domains have mutations, completely or partially eliminating the cleavage activity of these Cas proteins. In some embodiments, the Cas protein is a CasRx protein. In some embodiments, the Cas protein is dCasRx in which both HEPN domains have mutations (R239A, H244A in HEPN-1 and R858A, H863A in HEPN-2).

[0204] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or greater identity to the sequence set forth in any one of SEQ ID NOs: 1-3. In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.1% or greater, 99.2% or greater, 99.3% or greater, 99.4% or greater, 99.5% or greater, 99.6% or greater, 99.7% or greater, or 99.8% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0205] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0206] In some embodiments of the present disclosure, the Cas protein comprises a sequence set forth in any one of SEQ ID NOs: 1-3.

[0207] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or more identity to the sequence set forth in SEQ ID NO: 3. In some embodiments of the present disclosure, the Cas protein comprises a sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more identity to the sequence set forth in SEQ ID NO: 3.

[0208] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in SEQ ID NO:3.

[0209] In some embodiments of the present disclosure, the Cas protein comprises the sequence set forth in SEQ ID NO:3.

[0210] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or more sequence identity to CasRx. In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0211] In some embodiments of the present disclosure, the Cas protein comprises 100% sequence identity to CasRx.

[0212] In some embodiments of the present disclosure, the Cas protein comprises the sequence of CasRx.

[0213] In some embodiments of the present disclosure, RNA guide The nuclease comprises a homologous or heterologous domain fused to a nuclease moiety.

[0214] In some embodiments of the present disclosure, RNA guide The nuclease comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0215] In some embodiments of the present disclosure, the guide sequence comprises 20-40, 20-35, 20-30, or 25-30 nucleotides.

[0216] In some embodiments of the present disclosure, the guide sequence hybridizes to the target RNA with no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch.

[0217] In some embodiments of the present disclosure, the guide sequence has 100% sequence identity with the target RNA, i.e., is perfectly complementary.

[0218] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence. In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence, and the direct repeat sequence interacts with an RNA-guided nuclease.

[0219] In some embodiments of the present disclosure, the guide sequence is located at the 3' or 5' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 3' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 5' end of the direct repeat sequence.

[0220] In some embodiments of the present disclosure, the direct repeat sequence comprises a sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% identical to a sequence set forth in any one of SEQ ID NOs: 19-21.

[0221] In some embodiments of the present disclosure, the direct repeat sequence comprises a sequence that is 100% identical to the sequence set forth in any one of SEQ ID NOs: 19-21.

[0222] In some embodiments of the present disclosure, the direct repeat sequence comprises the sequence set forth in any one of SEQ ID NOs: 19 to 21. In some embodiments of the present disclosure, the direct repeat sequence consists of the sequence set forth in any one of SEQ ID NOs: 19 to 21. In some embodiments of the present disclosure, the direct repeat sequence consists of the sequence set forth in SEQ ID NO: 21.

[0223] In some embodiments of the present disclosure, the direct repeat sequence comprises a sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence identity to the corresponding direct repeat sequence of a CasRx in an embodiment of the present disclosure.

[0224] In some embodiments of the present disclosure, the direct repeat sequence comprises a sequence having 100% sequence identity to the corresponding direct repeat sequence of a CasRx in an embodiment of the present disclosure.

[0225] In some embodiments of the present disclosure, the direct repeat sequences comprise direct repeat sequences corresponding to CasRx in embodiments of the present disclosure.

[0226] In some embodiments of the present disclosure, the guide RNA comprises an aptamer sequence.

[0227] In some embodiments of the present disclosure, the aptamer sequence is inserted into the loop of the stem-loop structure of the direct repeat secondary structure of the guide RNA.

[0228] In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides. Such modifications include, but are not limited to, 2'-O-methyl, 2'-O-methyl-3'-phosphorothioate, or 2'-O-methyl-3'-thioPACE modifications. In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides selected from deoxyribonucleotides and locked nucleic acids (LNA). In some embodiments, the guide RNA comprises at least one chemically modified nucleotide. In some embodiments, the guide RNA is a hybrid RNA-DNA guide, i.e., some RNA nucleotides in the guide RNA are replaced with DNA nucleotides. In some embodiments, the guide RNA is a hybrid RNA-LNA (locked nucleic acid) guide, i.e., some RNA nucleotides in the guide RNA are replaced with LNA nucleotides.

[0229] In some embodiments of the present disclosure, the target RNA is located in the nucleus and / or cytoplasm of a eukaryotic cell.

[0230] target RNA

[0231] The gene editing systems and compositions disclosed herein can be used to target one or more target RNA molecules, such as target RNA molecules present in a biological sample. In some embodiments, the target RNA is a pre-mRNA or mRNA (mature mRNA).

[0232] In some embodiments of the present disclosure, the target RNA is VEGFA RNA. In some embodiments of the present disclosure, the target RNA is human VEGFA RNA.

[0233] In some embodiments of the present disclosure, the target RNA is VEGFA RNA or a fragment thereof. Optionally, the VEGFA RNA is of human origin.

[0234] In some embodiments of the present disclosure, the target RNA is VEGFA mRNA or a fragment thereof. Optionally, the VEGFA mRNA is of human origin.

[0235] In some embodiments of the present disclosure, the target RNA is a VEGFA pre-mRNA or a fragment thereof. Optionally, the VEGFA pre-mRNA is of human origin.

[0236] In some embodiments of the present disclosure, the target RNA is located in the nucleus or cytoplasm of the cell.

[0237] In some embodiments of the present disclosure, the gene editing system described herein can be used to increase the expression level of VEGFA RNA, for example, an RNA fused to a translation activation domain. guide Nucleases are used to contact VEGFA RNA under the guidance of gRNA, increasing its translation level. In some embodiments, the gene editing system described in the present disclosure can reduce the expression level of VEGFA RNA. For example, RNA guide A nuclease is used to contact and cleave VEGFA RNA under the guidance of a gRNA. In some embodiments, the gene editing system described herein can be used to reduce the expression level of VEGFA RNA in cells.

[0238] RNA guide nuclease

[0239] In some embodiments of the present disclosure, RNA guide Nucleases are Cas proteins.

[0240] In some embodiments of the present disclosure, the RNA-guided nuclease is selected from a wild-type RNA-guided nuclease (including but not limited to, CasRx, C13-2, etc.), a mutant thereof (including but not limited to, a mutant that completely loses cleavage activity, a mutant that partially loses cleavage activity, a mutant that has increased cleavage activity, or a mutant that has reduced off-target / bypass effects), or a functional fragment thereof (e.g., a fragment that retains only the RNA-binding domain, a fragment that lacks the HEPN domain, etc.), or a fusion protein.

[0241] In some embodiments of the present disclosure, RNA guide The nuclease comprises a homologous or heterologous domain fused to a nuclease moiety.

[0242] In some embodiments of the present disclosure, RNA guide The nuclease comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0243] In some embodiments of the present disclosure, RNA guide The nuclease contains a subcellular localization signal.

[0244] In some embodiments of the present disclosure, RNA guide The nuclease contains a subcellular localization signal and a deaminase domain.

[0245] In some embodiments of the present disclosure, the subcellular localization signal is selected from a nuclear localization signal and a nuclear export signal.

[0246] In some embodiments of the present disclosure, the deaminase domain is selected from cytidine deaminase, adenosine deaminase.

[0247] In some embodiments of the present disclosure, the deaminase domain is selected from a PPR protein (pentatricopeptide repeat), an ADAR family protein, or an APOBEC family protein.

[0248] In some embodiments of the present disclosure, the translation activation domain is selected from the group consisting of domains of eIF4E and other translation initiation factors, yeast poly(A) binding protein, or GLD2.

[0249] In some embodiments of the present disclosure, the translation inhibitory domain is selected from a pumilio protein, a deadenylase (e.g., deadenylase CAF1), or an Argonaute protein.

[0250] In some embodiments of the present disclosure, the nuclease domain is selected from FokI, a PIN endonuclease domain, a NYN domain, an SMR domain from SOT1, and an RNase domain from Staphylococcal nuclease.

[0251] In some embodiments of the present disclosure, the methylase domain is selected from m6A methyltransferases.

[0252] In some embodiments of the present disclosure, the demethylation domain is selected from the RNA demethylase ALKBH5.

[0253] In some embodiments of the present disclosure, the regulatory splicing domain is selected from SRSF1, hnRNP A1, and RBM4.

[0254] In some embodiments of the present disclosure, RNA guide The nuclease is covalently linked to the fusion domain with or without a linker sequence. guideThe nuclease can be covalently linked to the fusion domain directly (no linker sequence) or via a linker sequence, typically consisting of 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5 amino acids.

[0255] Cas proteins

[0256] In some embodiments of the present disclosure, the Cas protein is a Cas13 protein.

[0257] In some embodiments of the present disclosure, the Cas protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein. guide The nuclease is the Cas13d protein.

[0258] In some embodiments of the present disclosure, the Cas protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein that has mutations in both HEPN domains, and the cleavage activity of these Cas proteins is completely or partially lost. In some embodiments, the Cas protein comprises the amino acid sequence of a CasRx protein. In some embodiments, the Cas protein is a CasRx protein. In some embodiments, the Cas protein is dCasRx, which has mutations in both HEPN domains (R239A, H244A in HEPN-1 and R858A, H863A in HEPN-2).

[0259] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or greater identity to the sequence set forth in any one of SEQ ID NOs: 1-3. In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.1% or greater, 99.2% or greater, 99.3% or greater, 99.4% or greater, 99.5% or greater, 99.6% or greater, 99.7% or greater, or 99.8% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0260] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0261] In some embodiments of the present disclosure, the Cas protein comprises a sequence set forth in any one of SEQ ID NOs: 1-3.

[0262] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or more identity to the sequence set forth in SEQ ID NO: 3. In some embodiments of the present disclosure, the Cas protein comprises a sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% or more identity to the sequence set forth in SEQ ID NO: 3.

[0263] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% identity to the sequence set forth in SEQ ID NO:3, wherein the Cas protein (a) The positions corresponding to residues R210 and H215 in the sequence shown in SEQ ID NO: 3 are mutated to other residues. (b) the positions corresponding to residues R785 and H790 in the sequence shown in SEQ ID NO:3 are mutated to other residues; (c) the positions corresponding to residues R210, H215, R785 and H790 in the sequence set forth in SEQ ID NO: 3 are mutated to other residues; or (d) The positions corresponding to residues R210, H215, R750A, H755A, R785 and H790 in the sequence set forth in SEQ ID NO: 3 are mutated to other residues.

[0264] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, or 99.8% identity to the sequence set forth in SEQ ID NO:3, wherein the Cas protein (a) The corresponding positions of residues R210 and H215 of the sequence shown in SEQ ID NO: 3 are mutated to A. (b) The positions corresponding to residues R785 and H790 of the sequence shown in SEQ ID NO:3 have been mutated to A. (c) the corresponding positions of residues R210, H215, R785 and H790 in the sequence set forth in SEQ ID NO: 3 are mutated to A; or (d) Residues R210, H215, R750A, H755A, R785 and H790 of SEQ ID NO: 3 are mutated to A.

[0265] In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 100% identical to the sequence set forth in SEQ ID NO:3.

[0266] In some embodiments of the present disclosure, the Cas protein comprises the sequence set forth in SEQ ID NO:3.

[0267] In some embodiments of the present disclosure, the Cas protein comprises a sequence having 50% or greater sequence identity to CasRx. In some embodiments of the present disclosure, the Cas protein comprises a sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 99.5% identical to the sequence set forth in any one of SEQ ID NOs: 1-3.

[0268] In some embodiments of the present disclosure, the Cas protein comprises 100% sequence identity to CasRx.

[0269] In some embodiments of the present disclosure, the Cas protein comprises the sequence of CasRx.

[0270] In some embodiments of the present disclosure, the Cas protein is a Cas13b protein. In some embodiments of the present disclosure, the Cas13b protein is Cas13X (also referred to as Cas13e) or Cas13Y (also referred to as Cas13f). In some embodiments of the present disclosure, the Cas13 protein has 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to any one of Cas13e.1 to Cas13e.8. In some embodiments of the present disclosure, the Cas13 protein has 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to any one of Cas13f.1 to Cas13f.7.

[0271] In some embodiments of the present disclosure, the Cas protein comprises a homologous or heterologous domain fused to a Cas protein portion.

[0272] In some embodiments of the present disclosure, the Cas protein comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0273] In some embodiments of the present disclosure, the Cas protein comprises a subcellular localization signal.

[0274] In some embodiments of the present disclosure, the Cas protein comprises a subcellular localization signal and a deaminase domain.

[0275] In some embodiments of the present disclosure, the subcellular localization signal is selected from a nuclear localization signal and a nuclear export signal.

[0276] In some embodiments of the present disclosure, the Cas protein is covalently linked to the fusion domain with or without a linker sequence. That is, the Cas protein can be covalently linked directly to the fusion domain (excluding the linker sequence) or covalently linked via a linker sequence. Typically, the linker sequence is comprised of 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5 amino acids.

[0277] guide RNA or RNA guide Nucleotide sequence encoding the nuclease

[0278] In some embodiments, RNA guide In some embodiments, the nucleotide sequence encoding the nuclease is a plasmid. guide The nucleotide sequence encoding the nuclease is part of the viral vector genome, such as the DNA genome of an AAV vector, flanked by ITRs. guide The nucleotide sequence encoding the nuclease is mRNA.

[0279] In some embodiments of the present disclosure, the nucleotide sequence encoding the guide RNA or RNA-guided nuclease is codon-optimized.

[0280] In some embodiments of the present disclosure, the nucleotide sequence encoding the guide RNA is DNA. In some embodiments, the DNA sequence is codon-optimized.

[0281] In some embodiments of the present disclosure, RNA guide The nucleotide sequence encoding the nuclease is DNA or mRNA. In some embodiments, the DNA sequence is codon-optimized. In some embodiments, the mRNA sequence is codon-optimized.

[0282] In some embodiments of the present disclosure, codon optimization is performed for expression in a desired cell type. In some embodiments, the protein is codon-optimized for expression in a eukaryotic cell environment. In some embodiments, the gene is codon-optimized for expression in a mammalian cell environment. In some embodiments, the gene is codon-optimized for expression in a human cell environment.

[0283] Generally, codon optimization refers to modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon in the original sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) with a codon more frequently used in the host cell's genes, while maintaining the original amino acid sequence. Computer programs or algorithms for codon optimization are also available. For example, tools or algorithms such as ExpOptimizer, Codon OptimWiz, NGTM Codon, Codon optimization, Synthetic Gene Designer, and DNAWorks can be used for codon optimization.

[0284] Vector or Vector System

[0285] Vectors can contain any type of nucleotide, including but not limited to DNA and RNA, can be single-stranded or double-stranded, can be derived in part from natural sources, and can contain natural, non-natural, or modified nucleotides. Suitable vectors include those designed for expression, such as plasmids and viruses.

[0286] In some embodiments, recombinant vectors optionally include regulatory sequences, such as transcriptional and translational initiation and termination codons, specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced.

[0287] In some embodiments, the recombinant vector optionally includes genetic vector elements (nucleic acids) such as a selectable marker region, lactose operon, CMV promoter, CAG promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, IRES sequence, WPRE element, ITR sequence, FLAG tag coding region, c-myc tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, ployA coding region, SV40 polyadenylation signal, SV40 origin of replication, Col E1 origin of replication, loxP site, or Cre recombinase coding region.

[0288] Regulatory sequences

[0289] In some embodiments of the present disclosure, the regulatory sequences include one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof.

[0290] In some embodiments of the present disclosure, the regulatory sequence comprises an HRE enhancer element (hypoxia response element). In some embodiments, the regulatory sequence comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine HRE enhancer elements in tandem. In some embodiments, the regulatory sequence comprises 2, 3, 4, 5, 6, 7, 8, or 9 HRE enhancer elements in tandem.

[0291] In some embodiments of the present disclosure, the regulatory sequence comprises an NRS element (neuron-restrictive silencer) from the human synapsin gene. In some embodiments, the regulatory sequence comprises an NRS element and an HRE enhancer element. In some embodiments, the regulatory sequence comprises a tandem NRS element and an HRE enhancer element. To enhance the regulatory effect, the tandem NRS element and the HRE enhancer element can be repeated multiple times. In some embodiments, the regulatory sequence comprises tandem NRS elements and HRE enhancer elements repeated at least two times, at least three times, at least four times, at least five times, or at least six times. In some embodiments, the regulatory sequence comprises a tandem NRS element and an HRE enhancer element repeated two, three, four, five, or six times.

[0292] In some embodiments of the present disclosure, the HRE enhancer element sequence is TGTCACGTCCTGCACGACGTA (SEQ ID NO: 233) or its reverse complement.

[0293] In some embodiments of the present disclosure, the NRS element sequence is TTCAGCGCGGACAGTGCC (SEQ ID NO: 234) or its reverse complement.

[0294] In some embodiments of the present disclosure, the regulatory sequence comprises the repeat TGTCACGTCCTGCACGACGTA (SEQ ID NO: 233) or its reverse complement.

[0295] In some embodiments of the present disclosure, the regulatory sequence comprises a tandem sequence of TTCAGCCCGCGGACAGTGCCTGTCACGTCCTGCACGACGTA (SEQ ID NO: 235) or its reverse complement.

[0296] promoter

[0297] In some embodiments of the present disclosure, the vector comprises a pol III promoter (e.g., U6 and H1 promoters), a pol II promoter (e.g., the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), an SV40 promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, or an EF1α promoter), or a pol III promoter and a pol II promoter.

[0298] In some embodiments of the present disclosure, the promoter is a constitutive promoter that is continuously active and is not controlled by external signals or molecules. Suitable constitutive promoters include, but are not limited to, CMV, RSV, SV40, EF1α, CAG, CBh, β-actin promoter, etc. In some embodiments, the promoter is an inducible promoter that is controlled by external signals or molecules (e.g., transcription factors).

[0299] In some embodiments of the present disclosure, the promoter is selected from RPE65, VDM2, Mlc1, GFAP, and ICAM-2 promoters.

[0300] In some embodiments of the present disclosure, the promoter is a tissue-specific promoter, which can be used to drive tissue-specific expression of the guide RNA or gene editing system described in this disclosure.

[0301] In some embodiments of the present disclosure, the promoter is an eye-specific promoter.

[0302] In some embodiments of the present disclosure, the eye-specific promoter is selected from a retinal separation protein promoter, a K12 promoter, a rhodopsin promoter, a rod-specific promoter, a cone-specific promoter, a rhodopsin kinase promoter, a GRK1 promoter, an interphotoreceptor retinoid-binding protein proximal (IRBP) promoter, and an opsin promoter (e.g., a red opsin promoter, a blue opsin promoter, etc.).

[0303] In some embodiments of the present disclosure, the promoter is a chicken β-actin (CB) promoter. The chicken β-actin promoter may be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., a chicken β-actin promoter) comprises an enhancer sequence, such as a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence may be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter comprises a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter comprises a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art will recognize that a short CMV enhancer can be used with a long CB promoter, and a long CMV enhancer can be used with a short CB promoter. In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0304] Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and its variants, and SPc5-12 synthetic promoter. Suitable immune cell-specific promoters include, but are not limited to, the B29 promoter (B cells), CD14 promoter (monocytes), CD43 promoter (leukocytes and platelets), CD68 promoter (macrophages), and SV40 / CD43 promoter (leukocytes and platelets). Suitable blood cell-specific promoters include, but are not limited to, the CD43 promoter (leukocytes and platelets), CD45 promoter (hematopoietic cells), INF-β (hematopoietic cells), WASP promoter (hematopoietic cells), SV40 / CD43 promoter (leukocytes and platelets), and SV40 / CD45 promoter (hematopoietic cells). Suitable pancreatic-specific promoters include, but are not limited to, the elastase-1 promoter. Suitable endothelial cell-specific promoters include, but are not limited to, the Fit-1 promoter and the ICAM-2 promoter. Suitable neuronal tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astrocytes), the SYN1 promoter (neurons), and the NSE / RU5' promoter (mature neurons). The neural tissue / cell-specific promoter may be the GFAP promoter or the SYN1 promoter. Suitable kidney-specific promoters include, but are not limited to, the NphsI promoter (podocytes). Suitable bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts). Suitable lung-specific promoters include, but are not limited to, the SP-B promoter (lung). Suitable liver-specific promoters include, but are not limited to, the SV40 / Alb promoter. Suitable cardiac-specific promoters include, but are not limited to, the α-MHC promoter.

[0305] Enhancer

[0306] In some embodiments of the present disclosure, the enhancer is selected from a WPRE, a CMV enhancer, an SV40 enhancer, an HRE enhancer element, and an intron sequence between exon 2 and exon 3 of rabbit beta globin.

[0307] In some embodiments of the present disclosure, enhancers are located upstream of promoter elements. However, enhancers can also be located downstream of or within the coding sequence controlled by the promoter and maintain their function. Thus, enhancers, or portions thereof, can be present in the RNA sequence transcribed from the coding sequence.

[0308] In some embodiments of the present disclosure, an enhancer can be located within 100, 200, 300, 400, 500, or more base pairs upstream or downstream of a coding sequence controlled by a promoter.

[0309] In some embodiments of the present disclosure, an enhancer increases expression of a coding sequence above that provided by a promoter.

[0310] Adeno-associated virus vector (AAV vector)

[0311] In some embodiments of the present disclosure, the AAV vector comprises a guide RNA flanked by ITRs and an RNA guide It contains a ssDNA genome that contains the coding sequence for the nuclease.

[0312] In some embodiments of the present disclosure, the guide RNA or gene editing system described herein is packaged into an AAV vector, for example, an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, AAV PHP.B2, AAV PHP.B3, AAV PHP.A, AAV PHP.eB, AAV PHP.eS, AAV2.7m8, AAV8.7m8, AAV ShH10, AAVrhlO, or AAVrh74 capsid.

[0313] In some embodiments of the present disclosure, the guide RNA or gene editing system described herein is packaged into an AAV2, AAV5, AAV6, AAV8, or AAV9 capsid.

[0314] In some embodiments of the present disclosure, the AAV vector described herein is selected from AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV2 / 12, AAV2 / 13, AAV2 / PHP.B, AAV2 / PHP.B2, AAV2 / PHP.B3, AAV2 / PHP.A, AAV2 / PHP.eB, AAV2 / PHP.eS, AAV2 / 2.7m8, AAV2 / 8.7m8, AAV2 / ShH10, AAV2 / rh10, and AAV2 / rh74.

[0315] In some embodiments of the present disclosure, the AAV vector described herein is selected from AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 8, and AAV2 / 9.

[0316] In some embodiments, the gene editing systems described herein are packaged into AAV vectors that comprise modified capsids with tissue tropism, such as modified ocular tissue-tropic capsids.

[0317] lipid nanoparticles

[0318] In some embodiments of the present disclosure, an RNA payload (RNA guide In addition to the mRNA encoding the nuclease and guide RNA, the lipid nanoparticle (LNP) further comprises four components: a cationic or ionizable lipid, cholesterol, a helper lipid, and a PEG lipid. In some embodiments, the cationic or ionizable lipid includes cKK-E12, C12-200, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, FTT5, Moderna SM-102, and Intellia LP01. In some embodiments, the PEG lipid includes PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159. In some embodiments, the helper lipid includes DSPC.

[0319] Lentiviral vectors

[0320] In some embodiments of the present disclosure, the lentiviral vector is mimicked with a homologous or heterologous envelope protein, such as VSV-G. In some embodiments, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

[0321] Aptamer / aptamer sequence

[0322] In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop of the guide polynucleotide. In some embodiments, the aptamer sequence is added to the end of the guide polynucleotide.

[0323] In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

[0324] Adaptor proteins

[0325] In some embodiments, the gene editing system further comprises a fusion protein comprising an adaptor protein and a fusion domain, or a nucleic acid encoding the fusion protein, wherein the adaptor protein is capable of binding to the aptamer sequence.

[0326] In some embodiments, the adapter protein comprises MS2 bacteriophage coat protein (MCP), PP7 bacteriophage coat protein (PCP), or Qβ bacteriophage coat protein (QCP). In some embodiments, the fusion domain comprises a cytosine deaminase domain, an adenosine deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, or an affinity or reporter tag or domain.

[0327] RNP complex (ribonucleoprotein complex)

[0328] In some embodiments of the present disclosure, RNP complexes (ribonucleoprotein complexes) can be delivered to eukaryotic, mammalian, or human cells by microinjection or electroporation. In some embodiments, the ribonucleoprotein complexes can be packaged into virus-like particles and delivered in vivo to mammalian or human subjects.

[0329] virus-like particles

[0330] In some embodiments of the present disclosure, the modified virus-like particles (VLPs) are pseudotyped with a homologous or heterologous envelope protein, such as VSV-G. guide The nuclease is fused to the gag protein (e.g., MLVgag) via a cleavable linker, and cleavage of the linker within the target cell separates the linker and RNA. guideIn some embodiments, the fusion protein comprises (e.g., from 5' to 3') a gag protein (e.g., MLVgag), one or more NESs, a cleavable linker, one or more NLSs, and an RNA. guide In some embodiments, the RNA guide The nuclease is fused to a first dimerization domain that can dimerize or heterodimerize with a second dimerization domain fused to a membrane protein, and the presence of a ligand promotes dimerization, resulting in the formation of an RNA fragment. guide The nuclease or fusion protein is concentrated into a VLP.

[0331] eukaryotic cell

[0332] In some embodiments of the present disclosure, the eukaryotic cells are mammalian or human cells. In some embodiments, the eukaryotic cells are primary eukaryotic cells, stem cells, tumor / cancer cells, circulating tumor cells (CTCs), blood cells (e.g., T cells, B cells, NK cells, Tregs, etc.), hematopoietic stem cells, specialized immune cells (e.g., tumor-infiltrating lymphocytes or tumor-suppressing lymphocytes, etc.), or stromal cells in the tumor microenvironment (e.g., cancer-associated fibroblasts, etc.). In some embodiments, the cells are neural cells (e.g., neurons, astrocytes, microglia, retinal ganglion cells, rod / cone cells, etc.) of the brain or central or peripheral nervous system.

[0333] Diseases or conditions associated with target RNA

[0334] In some embodiments of the present disclosure, the disease or disorder associated with target RNA refers to the disease or disorder caused by the abnormal expression of target RNA.In some embodiments of the present disclosure, the disease or disorder associated with target RNA refers to the disease or disorder caused by the abnormally high expression of target RNA.In some embodiments of the present disclosure, the disease or disorder associated with target RNA refers to the disease or disorder caused by the abnormally low expression of target RNA.

[0335] In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, age-related macular degeneration.

[0336] In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, dry age-related macular degeneration. In some embodiments of the present disclosure, the disease or condition associated with the target RNA includes, but is not limited to, wet age-related macular degeneration.

[0337] In some embodiments of the present disclosure, the target RNA is VEGFA RNA.

[0338] In some embodiments of the present disclosure, the target RNA is a mammalian VEGFA RNA.

[0339] In some embodiments of the present disclosure, the target RNA is a VEGFA RNA of a non-human primate.

[0340] In some embodiments of the present disclosure, the target RNA is human VEGFA RNA.

[0341] In some embodiments, the pharmaceutical composition is delivered to human subjects internally.The pharmaceutical composition can be delivered by any effective route, and a therapeutically effective amount of the pharmaceutical composition can be delivered to the subject in need thereof.Examples of administration routes include but are not limited to intravenous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, subcutaneous injection, intradermal injection, intraventricular injection, intravascular injection, intracerebellar injection, intraocular injection, subretinal injection, intravitreal injection, intracameral injection, intratympanic injection, intranasal administration and inhalation.

[0342] In some embodiments of the present disclosure, the delivery methods of the present disclosure can be used to deliver a therapeutically effective amount of the gene editing system or pharmaceutical composition described herein to a subject in need thereof to inhibit the expression or activity of VEGFA RNA or its encoded protein in ocular tissue cells and reduce the formation of new blood vessels, thereby diagnosing, preventing, and / or treating macular degenerative diseases, such as neovascular age-related macular degeneration.

[0343] The present invention will be further described below with reference to specific embodiments, but the scope of the present invention is not limited to these embodiments. In the following experimental examples, experimental methods without specific conditions are carried out according to conventional methods and conditions or selected according to the product instructions. [Example]

[0344] Experimental example 1: CRISPR gene editing reduces VEGFA gene transcription levels The restriction enzymes involved in this example were purchased from NEB. The small-scale plasmid DNA extraction kit, 2x Accurate Taq Master Mix, and genomic DNA extraction kit were purchased from Hunan Accurate Biotech Co., Ltd. (Accurate Biotech). OPTI-MEM, T4 DNA ligase, and Lipofectamine 2000 transfection reagent were purchased from Thermo Company. gRNA, PCR, and sequencing primers were synthesized using conventional methods.

[0345] 1. Obtaining Cas13 protein source and plasmid In this example, multiple Cas13 proteins are used to verify the effect of targeting VEGFA, including three Cas13 proteins recently discovered by the applicant: Cas13m.3 (not belonging to the Cas13d subtype), Cas13m.6 (not belonging to the Cas13d subtype), and C13-2 (also known as CasRfg.4, belonging to the Cas13d subtype), whose amino acid sequences are Cas13m.3 (SEQ ID NO: 1), Cas13m.6 (SEQ ID NO: 2), and C13-2 (SEQ ID NO: 3), respectively.

[0346] The Cas13m.3-BsaI plasmid (sequence shown in SEQ ID NO:4), Cas13m.6-BsaI plasmid (sequence shown in SEQ ID NO:5), and C13-2-BsaI plasmid (sequence shown in SEQ ID NO:6) were prepared by an experimental outsourcing service company and contained codon-optimized coding sequences for various Cas13 proteins (CMV-driven expression) and direct repeat sequences (U6-driven expression).

[0347] 2. Construction of recombinant plasmids

[0348] 1. gRNA design First, the RNA sequence after transcription of the VEGFA gene ( targetDetermine the gRNA guide sequence that will target the target RNA molecule. The gRNA guide sequence is the reverse complement of the target sequence on the target RNA molecule. Design gRNAs with guide sequences between 21 and 29 nt in length, as shown in Table 1 below. Use the Cas13m.3-BsaI (also known as Cas13m.3-V), Cas13m.6-BsaI (also known as Cas13m.6-V), and C13-2-BsaI (also known as C13-2-V) vectors as negative controls. (The Cas13m.3-BsaI and Cas13m.6-BsaI vectors contain the gRNA coding sequence, ggagaccacggcaggtctca (SEQ ID NO: 7), which is a disordered sequence and therefore theoretically will not target mammalian genomes. The C13-2-BsaI vector contains the gRNA coding sequence, ggagaccacggcaggtctca (SEQ ID NO: 7), which theoretically will not target mammalian genomes.)

[0349] [Table 1]

[0350] 2. Plasmid Construction All gRNA recombinant plasmids were annealed with gRNA forward and reverse primers, and the Cas13m.3-BsaI, Cas13m.6-BsaI, and C13-2-BsaI plasmids were digested with BsaI. Next, the positive and antisense strands of the DNA sequences corresponding to the gRNA guide sequences were annealed and ligated into the Cas13m.3-BsaI, Cas13m.6-BsaI, and C13-2-BsaI vectors, respectively, using T4 ligase. The directional repeat sequences of the gRNAs encoded by the Cas13m.3, Cas13m.6, and C13-2 vectors are as follows: 5'-GTTGTAGAAGCCGTTCATTCGGGACGGTATGACAAC-3' (SEQ ID NO: 19), 5'-GTTGTAGAAGCCTATCGTTAGGATAGGTATGACAAC-3' (SEQ ID NO: 20), 5'-GGAAGATAACTCTACAAACCTGTAGGGTTCTGAGAC-3' (SEQ ID NO: 21).

[0351] The sense and antisense strands of the DNA sequence corresponding to the gRNA guide sequence are synthesized by conventional methods. For the Cas13m.3-BsaI and Cas13m.6-BsaI vectors, cacc is added to the 5' end of the sense strand (if the 5' end of the guide sequence is not g, caccg is added), and caac is added to the 5' end of the antisense strand (if the 5' end of the guide sequence is not g, c is also added to the 3' end of the antisense strand). For the C13-2-BsaI vector, agac is added to the 5' end of the sense strand and aaaa is added to the 5' end of the antisense strand.

[0352] According to the following reaction scheme, 2 μl of the sense and antisense DNA sequences corresponding to the gRNA target sequences were mixed, 2 μl of NEB 10x CutterSmart buffer and 14 μl of H2O were added, and the mixture was incubated at 95°C for 5 minutes in a PCR machine. The mixture was then immediately removed and incubated on ice for 5 minutes to allow annealing and form double-stranded DNA with sticky ends.

[0353] Following the reaction system in Table 2, the digested plasmid, annealed primers, and DNA Ligation Kit Ver. 2.1 (TAKARA, 6022Q) were sequentially added and incubated at 16 °C for 1 hour in a PCR instrument to complete the ligation between the annealed product and the linearized backbone. This resulted in the production of plasmids targeting VEGFA RNA for Cas13m.3, Cas13m.6, and C13-2 (Cas13m.3-gRNA-n, Cas13m.6-gRNA-n, and C13-2-gRNA-n, where n corresponds to the number corresponding to the different guide sequences). These plasmids can then be used to express Cas13 protein and VEGFA RNA-targeting gRNAs (containing specific guide sequences and direct repeat sequences corresponding to the Cas13 protein).

[0354] [Table 2]

[0355] 3. Plasmid Transformation In a clean bench, the entire T4 ligation reaction product was quickly added to one tube (50 μl) of E. coli Stbl3 competent cells and incubated on ice for 30 minutes. The competent cells were heat-shocked in a 42°C water bath for 90 seconds and then returned to ice for 2 minutes. In a clean bench, 400 μl of antibiotic-free LB medium was added, and the bacterial solution was placed in a bacterial shaker and incubated at 37°C and 200 rpm for 45 minutes to recover. During the recovery period, the biochemical incubator was opened, and an LB agar plate containing an appropriate amount of ampicillin was placed inside the plate to dry. The bacterial solution was centrifuged at 12,000 rpm at room temperature for 1 minute, and most of the supernatant was aspirated, retaining approximately 50 μl to completely resuspend the pellet. A droplet of bacteria was pipetted onto the edge of the LB agar plate containing ampicillin and smeared onto the medium using the pipette tip. The plate was then inverted and placed in the biochemical incubator for 16 hours.

[0356] 4. Identification of Positive Clones In a clean bench, seven monoclonal clones were picked using a 1-10 μL pipette tip and placed in 50 μL of LB medium containing ampicillin. Mix the bacteria and LB medium several times by pipetting. Pipette 2 μL of the bacterial solution and add it to the colony PCR reaction solution. Mix well and briefly centrifuge the tube to collect the liquid at the bottom before running the PCR reaction. The remaining bacterial solution was placed in a biochemical incubator for further cultivation. After PCR amplification, agarose gel electrophoresis was performed, and clones with the correct electrophoretic band size, monoclonality, and normal brightness were selected as positive clones. A 10 μL aliquot of the bacterial solution was then sent for Sanger sequencing. The remaining bacterial solution was used for plasmid extraction. The procedure followed the instructions for the Accurate Plasmid Extraction Kit (Accurate Biotech, AG21001), and the resulting solution was finally eluted with 50 μL of Elution Buffer. Plasmid DNA concentration was measured using the Nanodrop double-stranded DNA assay protocol. One positive clone was selected for each plasmid, and 5–10 μl was provided for Sanger sequencing to screen for plasmids with completely correct sequences.

[0357] 3. Testing Editing Efficiency

[0358] 1. Detection of editing efficiency at the mRNA level HEK293T cells were transfected strictly according to LifeTech's Lipofectamine 2000 reagent protocol (Thermo Fisher 11668019). One day before transfection, 1 × 10 well-established 293T cells were transfected. 5The cells were seeded into a 24-well plate at 100 μl / well. On the day of transfection, add 500 ng of recombinant Cas13-targeting VEGFA RNA plasmid to 50 μl of OPTI-MEM (Thermo, 2120588) and 1 μl of Lipofactamine 2000 to 50 μl of OPTI-MEM culture medium. Mix well and let stand at room temperature for 5 minutes. Next, mix the diluted plasmid DNA with Lipofactamine 2000 and let stand at room temperature for 15 minutes. Next, add 100 μl of the plasmid DNA complex to each well of the 24-well plate and gently shake the plate to mix. (The negative control group was transfected with Cas13m.3-BsaI, Cas13m.6-BsaI, and C13-2-BsaI plasmids.) Incubate at 37°C for 72 hours.

[0359] Cells were harvested 72 hours post-transfection, and total RNA was extracted using the Accurate SteadyPure Universal RNA Extraction Kit (AG21017). mRNA concentrations were measured using Nanodrop. mRNA products were reverse-transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR (AG11728) reverse transcription kit, and the reverse transcription products were detected using the SYBR Green Premix Pro Taq HS qPCR Kit (Low Rox Plus) (AG11720) qPCR kit.

[0360] The primers used for qPCR are shown in Table 3 below. gVegfa-F1 / Vegfa-R1 are used to detect the editing efficiency of gRNA-5, and qVegfa-F2 / qVegfa-R2 are used to test the editing efficiency of all other gRNAs, including gRNA-4 and gRNA-7.

[0361] [Table 3]

[0362] The reaction system was prepared according to the instructions for the SYBR Green Premix Pro Taq HS qPCR Kit (Low Rox Plus) AG11720, and detection was performed using QuantStudio. TM 5 This was performed using a 96-well Real-Time PCR System.

[0363] 2.Calculation method In this experiment, the relative quantification method, i.e., 2- △△ Ct The change in target RNA was calculated using the method as follows: △ Ct = Ct(Vegfa)-Ct(GAPDH) ; △△ Ct = △ Ct (test group) - △ Ct (each negative control group); 2- △△ Ct = 2^(-△△ Ct).

[0364] This experiment was performed three times independently, and the relative RNA expression of Vegfa was calculated according to the method described above. The resulting data were obtained as the average of the three tests, as shown in Table 4 and Figure 1.

[0365] [Table 4]

[0366] NOTE: "**" indicates p < 0.01 compared to the corresponding negative control group, and "*" indicates p < 0.05 compared to the corresponding negative control group.

[0367] As can be seen from the table above, gRNA-2 through gRNA-10, designed by the inventors, can edit VEGFA RNA. Compared to gRNA-1, gRNA-2 through gRNA-10 have improved editing efficiency, with gRNA-4, gRNA-5, gRNA-6, and gRNA-7 showing the highest editing efficiency, followed by gRNA-9 and gRNA-10.

[0368] Experimental example 2: Gene editing using different combinations of Cas13 protein and gRNA-5

[0369] Construction of overexpression vectors

[0370] Using the method of Experimental Example 1, we constructed VEGFA RNA-targeting plasmids Cas13m.3-gRNA-5, Cas13m.6-gRNA-5, and C13-2-gRNA-5, which can express Cas13 protein and VEGFA RNA-targeting gRNA-5 (comprising the guide sequence shown in SEQ ID NO: 13 and the direct repeat sequence corresponding to the Cas13 protein). The Cas13m.3-BsaI, Cas13m.6-BsaI, and C13-2-BsaI vectors of Experimental Example 1 were used as negative controls.

[0371] Previous studies (CN112143701A, CN112430597A) have reported combinations of CasRx+gRNAs targeting VEGFA RNA (gRNAs containing different guide sequences or tandem combinations thereof: X-1, X-2, X-dual (i.e., X-1 + X-2), RJ-2, RJ-5, RJ-8, and RJ-5 + RJ-8 (i.e., RJ-5 + 8)). The guide sequences are listed in Table 5.

[0372] We ordered CasRx and its corresponding gRNA plasmids (purchased from Miaoling, pXR001:EF1a-CasRx-2A-EGFP and pXR004:CasRx pre-gRNA cloning backbone) and transformed them to obtain the pAAV-CasRx-gRNA plasmid (also called CasRx-V, SEQ ID NO: 28). Next, as in Experimental Example 1, the annealed products of forward and reverse primers corresponding to the guide sequences of the control gRNA of the prior art and the gRNA-5 disclosed in the present invention (primers with restriction enzyme sites, i.e., AAAC added to the 5' end of the sequence in Table 5 below to form the forward primer, and cttg added to the 5' end of the reverse complement of the sequence in Table 5 to form the reverse primer) were ligated into the BpiI restriction enzyme backbone of the pAAV-CasRx-gRNA plasmid, transformed into competent E. coli, cultured in medium containing the corresponding antibiotic, subjected to colony PCR, and positive clones were selected by electrophoresis. The plasmid was extracted and Sanger sequenced to obtain VEGFA-targeting CasRx. RNA plasmids that can be used to express CasRx and gRNA (such as X-1 gRNA and / or X-2 gRNA).

[0373] [Table 5]

[0374] The plasmids were transfected into HEK293T cells using the same method as in Experimental Example 1. The negative control group was transfected with the CasRx-V plasmid.

[0375] Total RNA was extracted 72 hours after transfection and reverse transcribed into cDNA. qPCR was used to detect editing efficiency at the mRNA level. The method was the same as in Example 1, and the primers used are listed in Table 6 below. This example experiment was repeated three times independently. The test results are shown in the figure. figureSee Tables 2 and 7–8 below. The results are the average of three tests. The editing efficiency of the different Cas13 protein and gRNA-5 combinations was higher than that of the control group, CasRx-X-dual (dual gRNA).

[0376] [Table 6]

[0377] [Table 7]

[0378] NOTE: "**" indicates p < 0.01 compared to the corresponding negative control group.

[0379] [Table 8]

[0380] Experimental example 3: Gene editing using different combinations of Cas13 protein and gRNA-4, gRNA-6, and gRNA-7 Using the method of Experimental Example 1, plasmids combining gRNA-4, gRNA-6, and gRNA-7 with different Cas13 proteins, Cas13m.6 and C13-2, respectively, were prepared (capable of expressing Cas13 and gRNA (containing specific guide sequences and direct repeat sequences corresponding to the Cas13 protein)). The CasRx-V plasmid and CasRx-X-2 plasmid (which can be used to express CasRx and X-2 gRNA) were prepared using the method of Experimental Example 2. The plasmids were introduced into 293T cells, and 72 hours after introduction, total cellular RNA was extracted and reverse-transcribed into cDNA. The qPCR detection method was the same as in Experimental Example 1. The primer sequences used in this example are listed in Table 9. The experiment was repeated three times independently. The test results are shown in Figure 1. 3 and Tables 10~11 Shown inData are the average of three experiments. gRNA-4 and gRNA-7, when combined with various Cas13 proteins, can significantly downregulate VEGFA RNA levels, with significantly improved editing efficiency compared to the control CasRx-X-2.

[0381] [Table 9]

[0382] [Table 10]

[0383] NOTE: "**" indicates p < 0.01 compared to the corresponding negative control group, and "*" indicates p < 0.05 compared to the corresponding negative control group.

[0384] [Table 11]

[0385] Experimental Example 4: Comparison of editing efficiency at the RNA and protein levels with existing technologies The Cas13-gRNA plasmid designed by the inventors and the conventional CasRx-gRNA plasmid were transfected in the same batch to compare editing efficiencies. The plasmids used in this experiment (to express Cas13 protein and gRNA (single or paired gRNAs)) were prepared using the methods described in Experiments 1 and 2. The plasmids were transfected into 293T cells, and 72 hours later, total cellular RNA was extracted and reverse-transcribed to obtain cDNA. The qPCR detection method was the same as in Experiment 1. The primers are listed in Table 12. Results from three independent biological replicates are shown in Figures 4A-4B and Tables 13-16, and the resulting data are the average of the three experiments. The primers used in Table 13 and Figure 4A were qGAPDHF1 / qGAPDHR1 and qVegfa-F1 / qVegfa-R1, and the P values ​​for the intergroup difference tests are listed in Table 14. The primers used in Table 15 and Figure 4B were qGAPDHF1 / qGAPDHR1 and qVegfa-F2 / qVegfa-R2, and the p-values ​​for the intergroup difference tests are shown in Table 16. The prior art combination of CasRx and dual gRNAs resulted in higher editing efficiency than the prior art combination of CasRx and a single gRNA. Compared to the prior art combinations of CasRx and a single gRNA and CasRx and dual gRNAs, the combinations of C13-2 and gRNA-4, gRNA-5, and gRNA-7 significantly improved VEGFA RNA editing efficiency.

[0386] Cell pellets were collected for total RNA extraction, while cell supernatants were collected for ELISA to detect changes in native VEGFA protein levels. We used the Elascience Human VEGF-A (Vascular Endothelial Growth Factor A) ELISA kit. Standards and process samples at various concentrations were prepared according to the kit's operating instructions. The final data were read at 450 nm using an ELISA reader (Thermo Fisher Multiskan FC). The corresponding equation between sample concentration and OD value was calculated based on the standard OD value. The experiment was repeated twice. The results are shown in Table 17 and Figure 4C. Compared to conventional techniques (e.g., CasRx-RJ-5, CasRx-RJ-8, and CasRx-RJ-5+8), VEGFA protein downregulation levels after editing with C13-2 in combination with gRNA-4, gRNA-5, and gRNA-7, respectively, were significantly increased.

[0387] [Table 12]

[0388] [Table 13]

[0389] NOTE: "**" indicates p < 0.01 compared to the corresponding negative control group, and "*" indicates p < 0.05 compared to the corresponding negative control group.

[0390] [Table 14]

[0391] [Table 15]

[0392] NOTE: "**" indicates p < 0.01 compared to the corresponding negative control group, and "*" indicates p < 0.05 compared to the corresponding negative control group.

[0393] [Table 16]

[0394] [Table 17]

[0395] NOTE: The VEGFA protein levels in the negative control C13-2-V and CasRx-V groups were set to 100%, and data for other groups were calculated based on their respective negative controls. "**" indicates p < 0.01 compared to the corresponding negative control group.

[0396] Experimental example 5: Editing in mice using AAV vectors The C13-2-gRNA-4 plasmid constructed in Experiment 1 was packaged into AAV8 WT virus (to express C13-2 and gRNA-4). The three plasmids were transfected into 293T cells to generate AAV, which was then purified by gradient centrifugation. Mice were anesthetized with an intraperitoneal injection of 2.5% tribromoethanol (100 μl–200 μl / 10 g), and Meiliduo's compound tropicamide eye drops were instilled to dilate the pupils. After dilating the pupils, Alcon's prilocaine hydrochloride eye drops were instilled to provide local anesthesia. After 1 minute of anesthesia, the eye drops were wiped off, and Yishukang's medical grade sodium hyaluronate gel was evenly applied to the mouse's eyes. Under an ophthalmic microscope, a 30G sharp needle was used to puncture the sclera close to the corneal limbus, and AAV8-C13-2-gRNA-4 was injected into the mouse eye through the subretinal space using a WPI 10 μl injection system (Nanofil-10 μl) containing the drug connected to a 35G flat needle. (The negative control group was injected with the WT serotype virus AAV8-C13-2-V, which was packaged with the aforementioned C13-2-BsaI plasmid and can express C13-2 and gRNA that do not target the mammalian genome.) Finally, tobramycin-dexamethasone ointment was applied to the eye and the eye was kept warm at 37°C. Once the mouse woke up, it was returned to its cage and continued to be cared for and observed. Four weeks after injection, the mouse was killed by cervical dislocation, the eyeball was peeled, and the retina was quickly and gently separated. The retinas were immediately immersed in Trizol, and RNA extraction was completed by strictly following the Trizol (Thermo Fisher) total RNA extraction procedure. cDNA was obtained by reverse transcription as in Experiment 1, and changes in mouse VEGFA expression and editing efficiency were detected by qPCR using mouse VEGFA-specific primers. The results are shown in Figure 5 and Table 19 below. Four weeks after AAV8-C13-2-gRNA-4 injection, VEGFA transcription levels were significantly reduced, and editing efficiency was high at 84.5%. The primers required for qPCR are listed in Table 18 below.

[0397] [Table 18]

[0398] [Table 19]

[0399] Experimental Example 6: Off-target detection According to the methods of Experimental Examples 1 and 2, a total of seven plasmids, C13-2-V, C13-2-gRNA-4, C13-2-gRNA-5, C13-2-gRNA-6, C13-2-gRNA-7, CasRx-V, and CasRx-X-dual, were constructed and transfected into 293T cells, respectively, with each plasmid transfected into three duplicate wells.

[0400] Seventy-two hours after transfection, total RNA from the cells was extracted and sent to a sequencing company. 1 μg of RNA was used for ribosomal RNA removal in all samples. cDNA was synthesized by RT and randomly fragmented. Next, the cDNA ends were repaired and A-tails were added. Libraries were amplified by PCR. After quality control testing, the libraries were sequenced on the BGI DNBSEQ platform with a read length of PE150. After quality control filtering, offline data were aligned to the human reference genome using STAR software, and gene expression was quantified using StringTie2. Next, significance analysis (compared to the respective negative control groups) was performed using DESeq2 software. A Padj (corrected P value) of ≤ 0.05 and an expression fold ratio of ≥ 2 (log2 (fold change) > 1) were used as significance criteria, and genes that met these criteria were identified as differentially expressed genes (DEGs).

[0401] The number of differentially expressed genes (DEGs) in each sample group is shown in Table 20 below. The number of differentially expressed genes in each C13-2 experimental group was significantly lower than that in the CasRx-X-dual group. Compared to the CasRx-X-dual group, editing using C13-2 in combination with each gRNA in the table resulted in less transcriptome disruption and was safer.

[0402] [Table 20]

[0403] We then intersected the differentially expressed genes with the predicted target gene set.

[0404] Off-target prediction method: The EMBOSS-water program was used to predict the entire genome and cDNA sequences of the target species (Homo sapiens) with the parameters gap_open=10.0; gap_extend=0.5. The forward and reverse strands of the gRNA guide sequences were used for comparison. Genes corresponding to transcripts with a minimum match of 18 bases and a mismatch + gap of 6 bases or less were selected as predicted target genes.

[0405] The crossover numbers in Table 21 below show that compared to CasRx-X-dual, the number of off-target genes for each combination of C13-2 and gRNA in the table is significantly reduced, indicating higher off-target safety.

[0406] [Table 21]

[0407] NOTE: * indicates that the target gene VEGFA is deleted.

[0408] From the above examples, we can see that gRNA-4, gRNA-5, and gRNA-7 outperform existing technologies in terms of editing efficiency and off-target safety, achieving unexpected technical benefits.

[0409] Experimental Example 7: Editing efficiency of several gRNAs at the RNA and protein levels The sense and antisense strands of DNA sequences corresponding to the guide sequences of gRNA-11 through gRNA-45 were synthesized using the method described in Example 1 (the sense strand had an agac added to the 5' end of the guide sequence, and the antisense strand had an aaaa added to the 5' end of the reverse complement of the guide sequence). The annealed product was ligated with the linearized product of the C13-2-BsaI plasmid using T4 ligase. After transformation of the plasmids and identification of positive clones, plasmid vectors targeting VEGFA for C13-2 and each gRNA were obtained. The plasmids were transfected into HEK293T cells, and 72 hours after transfection, total RNA was extracted and reverse transcribed into cDNA. Editing efficiency was then detected by qPCR.

[0410] The primers used for qPCR are shown in Table 22 below. qVegfa-F1 / qVegfa-R1 are used to detect the editing efficiency of gRNA-5, 17, 20, 21, 22, and 23. qVegfa-F2 / qVegfa-R2 are used to test the editing efficiency of gRNA-25, 26, 27, 28, 29, 30, 31, and 32.

[0411] [Table 22]

[0412] Relative quantification, i.e., 2- △△ Ct The change in target RNA was calculated using the method. The biological assay was independently repeated three times, and the resulting data were obtained as the average of the three tests. As shown in Table 23 and Figure 6.

[0413] [Table 23]

[0414] Seventy-two hours after introducing the above plasmids into the cells, we used the cell pellets to extract RNA and detect RNA levels by qPCR, while the cell supernatants were used to detect changes in protein levels. After observing relatively uniform cell growth and consistent cell density, we collected equal volumes of cell culture medium and centrifuged them at 14,000 rpm at 4°C for 15 minutes to remove dead cells and cell debris. Finally, we collected equal volumes of cell supernatants for ELISA detection. We strictly followed the instructions in the Elabscience Human VEGFA ELISA Kit (E-EL-H0111c) and set up two replicate wells for each sample. Different concentrations of the standards and treated samples were prepared, and the final data were read at 450 nm using an ELISA reader (Thermo Fisher Multiskan FC). A calibration curve was created based on the standard concentrations and OD values, and the corresponding formula was used to calculate the concentration of each sample corresponding to the OD value. The test results are shown in Table 24 and Figure 7. As can be seen in Table 24, compared to the negative control group C13-2-V, VEGFA protein levels were significantly reduced after editing with gRNA-30. VEGFA protein levels were also significantly reduced after editing with gRNA-5, 11, 12, 14, 18, 24, 26, 27, 28, and 29, consistent with the downward trend in RNA levels. After editing, VEGFA protein levels in the gRNA-30 group were significantly lower than those in the other groups (P<0.01). After editing, VEGFA protein levels in the gRNA-18 group were significantly lower than those in the gRNA-5 group (P<0.01). Therefore, unexpected technical effects were achieved.

[0415] [Table 24]

[0416] Experimental Example 8: Targeting the region near the VEGFA RNA splice site

[0417] The vector was constructed and edited in HEK293T cells using the method described in Example 1. The results are shown in Figure 8 and Table 25. As shown, gRNA-30 still has the highest editing efficiency. gRNA-35, gRNA-36, and gRNA-44, which target regions near splice sites, have relatively high editing efficiencies, but the editing efficiencies of the other gRNAs are low.

[0418] [Table 25]

[0419] Experimental Example 9: Targeted editing of the flanking regions of the gRNA-30 target sequence To test the editing efficiency, target We selected multiple gRNAs whose sequences flank gRNA-30.

[0420] Using the method described in Example 1, we synthesized the positive and antisense strands of DNA sequences corresponding to the guide sequences of gRNA-185 to gRNA-196. The annealed products were then ligated into the linearized C13-2-BsaI vector by enzymatic digestion with T4 ligase. After plasmid transformation and identification of positive clones, we obtained plasmid vectors capable of expressing C13-2 and each gRNA. The plasmids were then transfected into HEK293T cells. At 72 hours post-transfection, total RNA was extracted and reverse-transcribed into cDNA, and editing efficiency was measured by qPCR.

[0421] The following primers were used: qGAPDHF1:TCCAAAATCAAGTGGGGCGA (SEQ ID NO: 231) qGAPDHR1:TGATGACCCTTTTGGCTCCC (SEQ ID NO: 232) qVEGFA-F2: CAATGACGAGGGCCTGGAGT (SEQ ID NO: 26) qVEGFA-R2:TCTTTGGTCTGCATTCACAT (SEQ ID NO: 27)

[0422] The results are shown in Figure 9A, Figure 9B, and Table 26. gRNA-30, which targeted the VEGFA exon 4 region, had the highest editing efficiency, significantly higher than all other experimental groups, including gRNA-185 through gRNA-196 (P<0.01). gRNA-187, which differs by only one base from gRNA-30, also had a high editing efficiency, but it was significantly lower than gRNA-30 (P<0.01). gRNA-186, which differs by only one base from gRNA-30, only had an editing efficiency of 12%. The editing efficiencies of gRNA-190, gRNA-191, and gRNA-194 were above 70%, but significantly lower than gRNA-30 (P<0.01). Therefore, gRNA-30 achieved an unexpected technical effect.

[0423] [Table 26]

[0424] Experimental Example 10: Reduction of intron enrichment Whether the CRISPR-Cas13 system targeting VEGFA RNA exons generates new transcripts and thereby impacts the safety of in vivo treatment is a question worthy of further investigation. Therefore, we transfected HEK293T cells with a plasmid expressing the CRISPR-Cas13 system (Cas13 and gRNA) and performed RNA-seq to analyze changes at the transcriptome level.

[0425] Using the same methods as in Experiments 1 and 2, we constructed the plasmids, transfected them into HEK293T cells, extracted total RNA, and commissioned a gene sequencing company to construct ribosomal RNA-depleted RNA-seq libraries. The data was sequenced using a BGI DNBSEQ-T7 sequencer and analyzed using STAR and Kallisto. The RNA-seq sequencing data was mapped to the human reference genome using software, and then IGV software was used to display the read distribution within the genome for the target VEGFA.

[0426] As shown in Figure 10, after editing, the C13-2-gRNA-5 and CasRx-X-dual groups generated numerous intron-rich reads, resulting in long sequences (including multiple exon and intron sequences) that were suspected to be new transcripts through splicing. Although these intron reads do not generate new transcripts capable of encoding functional proteins, they may potentially regulate growth and development in vivo in the form of long non-coding RNAs, posing a potential safety risk.

[0427] Because gRNA-5 contains a 21-nt continuous nucleotide sequence complementary to the VEGFA pre-mRNA, we speculate that the C13-2 and gRNA-5 complex can bind to the pre-mRNA, triggering a new splicing mode of the pre-mRNA and leading to intron enrichment. To address this issue, we engineered multiple gRNAs near gRNA-5, with their guide sequences evenly distributed across the two exon sequences, in the hopes of reducing the C13-2-gRNA complex's binding to the pre-mRNA. Based on this, we obtained gRNA-5-2, gRNA-5-3, gRNA-5-5, and gRNA-5-4 as a control, as shown in Figure 11. The engineered gRNAs were combined with C13-2 for editing. The RNA-seq results are shown in Figure 12. gRNA-5-2, gRNA-5-3, and gRNA-5-5 did not result in enrichment of reads in introns. Conversely, gRNA-29 and gRNA-30 did not result in enrichment of reads within introns. gRNA-5-4 induced intron enrichment. The degree of intron enrichment with gRNA-18 was low. These results revealed that gRNA-30, gRNA-29, gRNA-5-2, gRNA-5-3, and gRNA-5-5 were safer than gRNA-5. The safety of the combination of gRNA-30, gRNA-29, gRNA-5-2, gRNA-5-3, gRNA-5-5, and C13-2 was superior to that of the conventional CasRx-X-dual technology. Therefore, gRNA-30, gRNA-29, gRNA-5-2, gRNA-5-3, gRNA-5-5, and gRNA-18 achieved unexpected technical effects.

[0428] Additionally, the method described in Experimental Example 1 was used to prepare the plasmids, transfect cells, and perform qPCR detection. The primer sequences used are listed in Table 27. The experiment was repeated three times independently. The results showed that the editing efficiency of the combinations of gRNA-5-2, gRNA-5-3, gRNA-5-4, gRNA-5-5, and C13-2 was greater than 50%.

[0429] [Table 27]

[0430] Experimental Example 11: Targeting VEGFA significantly inhibits angiogenesis in mice Using the two plasmids C13-2-gRNA-4 and C13-2-gRNA-5 constructed in Experiment 1, we outsourced the packaging of AAV8 wt virus. The three plasmids were transfected into 293T cells to generate AAV, which was purified by gradient centrifugation. The titer measured by qPCR was 1E13 vg / ml. Mice were intraperitoneally administered 200 μl / 20 g of 2.5% tribromoethanol anesthetic solution, followed by pupil dilation and local anesthesia. First, the edge of the cornea was punctured with a 30G sharp needle, and then 1 μl of AAV was injected into the subretinal space using the WPI Nanofil RPE kit. Successful administration was confirmed by the appearance of a retinal bubble under a microscope. Tobramycin antibiotics were then applied to the ocular surface, and the awake mice were placed in their cages. Laser modeling was performed three weeks after administration. Mice were anesthetized intraperitoneally with 380 μl / 20 g of 2.5% tribromoethanol anesthetic solution. After mydriasis and topical anesthesia, the fundus of each mouse was examined using a Lumenis Nova Spectra 532 nm laser therapy device. Laser photocoagulation was performed using 100 mW, 100 ms, and 100 μm lasers in four directions, evenly distributed around the optic disc, at a distance of 2–3 optic disc diameters from the optic disc. Mice exhibiting bleeding or cataracts were excluded. Seven days after modeling, samples were collected to observe the choroidal neovascularization area (CNV). The retinal pigment epithelium (RPE) / choroid / sclera complex was isolated and perfused with 4% paraformaldehyde (PFA). Neovascularization was stained with IB4-488 nm (Thermo Fisher I21411), and images were captured using a Lecia inverted fluorescence high-magnification microscope. The CNV regions were statistically analyzed using ImagJ 1.52v software. The results are shown in Figure 13. The CNV regions were significantly reduced after editing with C13-2-gRNA-4 and C13-2-gRNA-5, demonstrating that both can significantly inhibit the development of new blood vessels.

[0431] Experimental Example 12 Plasmids were constructed using the same method as in Example 2. The annealed products of forward and reverse primers corresponding to the guide sequences listed in Table 28, such as gRNA-4, gRNA-5, gRNA-5-3, gRNA-6, gRNA-7, gRNA-29, gRNA-30, gRNA-RJ-8, and gRNA-X-1, were selected and ligated to the BpiI restriction backbone of the pAAV-CasRx-gRNA plasmid (also known as CasRx-V, SEQ ID NO: 28). After transformation of competent E. coli, the plasmids were cultured in medium containing the corresponding antibiotics, colony PCR reactions were performed, positive clones were screened by electrophoresis, and the plasmids were verified by Sanger sequencing. CasRx targeting VEGFA RNA was obtained and used to express CasRx and gRNA (e.g., gRNA-5). Concurrently, for the publicly available tool Cas13X.1, the Cas13X.1-V vector (SEQ ID NO: 8) was prepared using conventional methods. The corresponding forward and reverse primers were synthesized according to the Cas13X.1-dual sequence (cacc was added to the 5' end of the sense strand, and cagc was added to the 5' end of the antisense strand). Cas13X.1-dual was constructed by connecting the primers to the backbone of the Cas13X.1-V vector cleaved with BsaI enzyme. Cas13X.1-V was used as a negative control. In this example, we also use the negative control C13-2-V, which corresponds to C13-2-gRNA-5 constructed in Example 1. The plasmid was transfected into 293T cells as in Example 4, and supernatants were collected 72 hours after transfection to detect VEGFA protein levels by ELISA. We used the Elascience Human VEGF-A (Vascular Endothelial Growth Factor A) ELISA kit. Various concentrations of standards and process samples were prepared in strict accordance with the kit's operating instructions.The final data were read at 450 nm using an ELISA reader (Thermo Fisher Multiskan FC). The corresponding equation between sample concentrations and OD values ​​was calculated based on the OD values ​​of the standards. The experiment was repeated twice.

[0432] [Table 28]

[0433] In this example, three combinations of Cas13 and different gRNAs were expressed in the same transfection batch. The results of this and the previous embodiments demonstrate that the gRNAs disclosed in this invention, when combined with C13-2 or CasRx, all exhibited high editing efficiency, significantly superior to the combination with the Cas13X.1-dual disclosed in the patent. As shown in Table 29 and Figure 14, C13-2-gRNA-5 performed best after plasmid transfection, achieving a target protein downregulation rate of up to 97.4%. CasRx-gRNA-30 and CasRx-gRNA-5-3 resulted in 85.4% and 82.2% target protein downregulation, respectively. CasRx-gRNA-5 and CasRx-gRNA-29 resulted in 75.7% and 71.8% target protein downregulation, respectively. CasRx-gRNA-4 and CasRx-gRNA-6 resulted in 61.0% and 69.7% target protein downregulation, respectively. The protein downregulation efficiency of these combinations was significantly superior to that of the CasRx-X-1, CasRx-RJ-8, and Cas13X.1 dual groups (P<0.01).

[0434] [Table 29]

[0435] NOTE: The VEGFA protein levels in the negative control CasRx-V, Cas13X.1-V, and C13-2-V groups were all set to 100%, and data for other groups were calculated based on their respective negative controls. "***" indicates p<0.001 compared to the corresponding negative control group.

[0436] Experimental Example 13 To compare the safety of the newly discovered high-editing gRNAs with published gRNAs, an experiment was designed using the VEGFA-targeting C13-2 plasmid constructed in the previous example. Using the same method as in Example 7, C13-2-RJ-2, C13-2-RJ-8, and C13-2-X-1 plasmids (expressing the corresponding gRNAs containing the RJ-2, RJ-8, and X-1 guide sequences, respectively) were constructed. A gRNA-luc targeting the luciferase gene (guide sequence: GAATGTAGCCATCCATCCTTGTCAA, SEQ ID NO: 237) was selected and constructed into C13-2-gRNA-luc as a control for RNA-seq using the same method. The plasmid transfection method for 293T cells was the same as in Example 1. Cell pellets were collected 72 hours post-transfection, and total RNA was extracted and sent to a third-party gene sequencing company for RNA library construction. RNA-seq data was obtained using a BGI DNBSEQ-T7 sequencer. Data were quality-controlled using fastqc and multiqc. Fastq was used to remove low-quality reads, and STAR software was used to remove rRNA sequence reads. Next, gene expression levels were quantified using StringTie2 software, and differential expression analysis was performed using DESeq2 software to analyze genes with a log2 fold change of >0.5, padj <0.05, and baseMean >2. Genes with a log2 fold change of >0.5 were considered differentially expressed. Using EMBOSS water software, transcripts with 21 or more bases aligned with the guide sequence, 6 or fewer mismatched bases, and 8 or more consecutive paired bases were considered as predicted gRNA-associated target transcripts, and the corresponding genes were considered as predicted target genes. Finally, the set of down-regulated differentially expressed genes was intersected with the set of predicted target genes to obtain off-target genes (VEGFA was excluded because it is an on-target gene).

[0437] The specific results are shown in Table 30. Compared to the control group, no gRNA-related differentially expressed genes were detected in gRNA-30 and gRNA-7, meaning no off-target genes were detected. However, gRNA-X-1, which is adjacent to the target sequence, has two off-target genes. In the case of the finished drug, this is not simply a difference in quantity, but a difference in quality.

[0438] Furthermore, the above example shows that the editing efficiency of gRNA-30 is significantly higher than that of gRNA-X-1. That is, when used in combination with the same Cas13 protein, the editing efficiency and safety of gRNA-30 are significantly higher than those of gRNA-X-1, which has adjacent target sequences, and it also has high druggability, achieving an unexpected technological effect.

[0439] gRNA-29 only had one gRNA-associated off-target gene, which offers certain advantages in terms of off-target safety.

[0440] The number of differentially expressed genes downregulated by editing with gRNA-29, gRNA-30, gRNA-7, gRNA-5-2, gRNA-5-3, and gRNA-5-5 was much lower than that by the conventional techniques gRNA-RJ-2, gRNA-RJ-8, and gRNA-X-1, indicating less perturbation to the transcriptome.

[0441] While gRNA-RJ-2 has dozens of off-target genes, gRNA-5-2, gRNA-5-3, and gRNA-5-5, which flank the target sequence, have far fewer off-target genes, making them safer in terms of off-target effects.

[0442] [Table 30]

[0443] Experimental Example 14: Editing efficiency of C13-2 mutants in combination with gRNA The inventors designed the C13-2 mutants shown in Table 31 and tested their editing efficiency in combination with gRNA-5.

[0444] Expression vectors for Cas13 (C13-2 and its variants) and gRNA-5 were constructed and transfected into 293T cells according to the instructions for Lipofectamine 2000 (Thermo). A 293T cell control group was set up without transfection of the plasmid.

[0445] qPCR detection of target RNA levels Forty-eight hours after transfection, RNA was extracted from the cells using the SteadyPure Universal RNA Extraction Kit, and the RNA concentration was measured using an ultramicrospectrophotometer. The RNA product was reverse-transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR Reverse Transcription Kit, and the reverse-transcribed product was detected using the SYBR Green Premix Pro Taq HS qPCR Kit (Low Rox Plus). Experiments were repeated three times, and the results were averaged.

[0446] The relative VEGFA RNA levels after editing of the C13-2 M01-M22 mutants were lower than 0.04 compared with the VEGFA RNA level of the C13-2-BsaI control group, indicating that the editing efficiency was higher than 96%.

[0447] [Table 31]

[0448] Example 15: Testing of C13-2 inactivation mutants in combination with gRNA The inventors designed the inactivating mutants shown in Table 32 and tested their editing efficiency in combination with gRNA-5.

[0449] An expression vector was constructed to express Cas13 and an inactivated variant of gRNA-5, and the vector was then introduced into the 293T cell line. Transfection was performed according to the instructions for Lipofectamine 2000 (Thermo). After 72 hours, RNA was extracted using the SteadyPure Universal RNA Extraction Kit. The RNA extracted from three batches of experiments was sent to a sequencing company for RNAseq sequencing, and the amount of VEGFA RNA detected is shown in Table 32 below.

[0450] [Table 32]

[0451] Experimental data showed that editing activity remained high after the introduction of the R750A+H755A mutation, but was weakened after the introduction of the R210A+H215A and / or R785A+H790A mutations. Editing activity was almost completely lost only after the introduction of the R210A+H215A, R750A+H755A, and R785A+H790A mutations.

[0452] Experimental Example 16 Based on the efficacy results in mice, C13-2-V, C13-2-gRNA-30, and C13-2-gRNA-5 were packaged into three types of AAV 2 / 8 using AAV capsid and helper plasmids, and the efficacy of these three types of AAV 2 / 8 in inhibiting laser-induced neovascularization in non-human primate (NHP) macaques was tested. Equal amounts of the three AAVs were injected subretinally into the eyes of NHP animals. The animals were first given general anesthesia, mydriasis, and ocular surface anesthesia, followed by subretinal injection. Finally, antibiotics were applied to the ocular surface to warm the eyes. After awakening, the animals were fed as usual. Three animals per group were treated. Four weeks after treatment, an Iridex laser treatment device was used to generate 6–10 photocoagulation points near the macula of the animals' eyes using the same parameters. Eyes with hemorrhage areas exceeding two photocoagulation points were excluded. Sodium fluorescein was injected intraperitoneally at different time points (weeks 6, 8, and 13) and fluorescence leakage was tracked and photographed. Finally, spot grading was assessed by three different technicians. The percentage of Grade IV spots is shown in Figure 15. The drug groups C13-2-gRNA-30 and C13-2-gRNA-5 significantly suppressed the occurrence of Grade IV spots at different time points, i.e., significantly inhibited the development of neovascularization, and this effect was sustained for a long period of time.

[0453] Based on the above-described embodiments, it can be seen that gRNAs with guide sequences of SEQ ID NOs: 12, 13, 15, 40-43, 45-47, 52-60, 64, 65, 73, 216, 219, 220, 223, 227, 228, 229, and 230 have high editing efficiency. The off-target risk of gRNAs with guide sequences of SEQ ID NOs: 58, 59, 227, 228, and 230 is significantly reduced, resulting in low off-target risk. gRNAs with guide sequences of SEQ ID NOs: 58, 59, 227, 228, and 230 essentially do not cause intron enrichment, resulting in low safety risks.

[0454] Although specific embodiments of the present disclosure have been described above, it will be understood by those skilled in the art that these are merely examples and that various changes or modifications can be made to these embodiments without departing from the principle and essence of the present disclosure. Accordingly, the scope of protection of the present disclosure is defined by the appended claims.

[0455] Some of the sequences used in this disclosure are:

[0456] Cas13m.3 (SEQ ID NO: 1):

[0457] Cas13m.6 (SEQ ID NO: 2):

[0458] C13-2 (SEQ ID NO: 3): MSKDKKTKAKRMGVKALLAHGEDKLTMTTFGKGNRSKIEFTEGYHGRALETPKHFGIRGFEVRRIDENVDLCGDLEEGKTIEALLVNPSEKVGEDYLKLKGTLEKRFFGRE FPHDNIRIQLIYNILDIYKILGMNVADILYALGNMQDTELDIDMFGQSLNNEDNLKECLKRMRPYMGYFGDIFKISPKGENIADREHNKKVLRCISVLRNATAHDKQDEYPW FKSSDIYETKIFKADMWKIIKDQYREKIKKVNKDFLSKNAVNMAILFDLLNARDVEQKKQITDEFYRFTIRKDGKNLGMNLVKIREIIIDRYASGLRDKKHDPHRQKINVI ADFLIFRALSQNQGIIDKTVSSLRLTKDEEEKDHVYQNAAELVWGMVSNCLTPYFNDPKNKYILKYKDAKTPGDFEDWITSKISEDDGEPFVKVLSFLCNFLEGKEINELLT AYIHKFECIQDFLNVISSLGENVQFQPRFALFNNASFAQNVAVQLRILASIGKMKPDLTEAKRPLYKAAIRMLCPPEKWEKYTSDEWLEKNMLLNSEDRKNDKKKKQVNPF RNFIAGNVIESRRFMYLVRYSKPKAVRAIMQNRSIVNYVLHRLPSEQVHRYASVFPENFADLEQEIDFLTKKLFEFSFEELLHEKDVILNNSRSHKPSLEIERLKAITGLYL SVAYIAIKNIVKANARYYIAFAVFERDKELVKAKDARIQTKIPETDFPDYFCLTQYYLDRDEEKKFPGDPRDKEAFFEHLRKTKRHFSKQWREWLNEKIADAKSSQATGLLL REARNDVEHLNVLRAIPDYIQDFRHGEKGETAMNSYFELYHYLMQRLMLKNTELDLSHWSGWIMRSGRPDRDLIQIAFVSLAYNLPRYRNLTKEHHFDDTVLQKIREKESLD

[0459] Cas13m.3-BsaI plasmid (SEQ ID NO: 4):

[0460] Cas13m.6-BsaI plasmid (SEQ ID NO: 5):

[0461]

[0462] Unordered sequence (SEQ ID NO: 7): ggagaccacggcaggtctca

[0463] Guide sequence of gRNA-1 (SEQ ID NO: 9): GGTACTCCTGGAAGATGTCCACCAGGGTCT

[0464] Guide sequence of gRNA-2 (SEQ ID NO: 10): CCACTGCGGCCCCCTCTCTTCCTTC

[0465] Guide sequence of gRNA-3 (SEQ ID NO: 11): GGCTGGAGCACTGTCTGCGCCACA

[0466] Guide sequence of gRNA-4 (SEQ ID NO: 12): AAGCTCATCTCTCCCTATGTGCTGGC

[0467] Guide sequence of gRNA-5 (SEQ ID NO: 13): TGGGTGCAGCCTGGGACCACTTGGCATGG

[0468] Guide sequence of gRNA-6 (SEQ ID NO: 14): TCTTTCTTTGGTCTGCATTCACAT

[0469] Guide sequence of gRNA-7 (SEQ ID NO: 15): GGCCTTGGTGAGGTTTGATCCGCAT

[0470] Guide sequence of gRNA-8 (SEQ ID NO: 16): ACAAACAAATGCTTTCTCCGCTCTGA

[0471] Guide sequence of gRNA-9 (SEQ ID NO: 17): GCAGGAACATTTACACGTCTGCGGATCT

[0472] Guide sequence of gRNA-10 (SEQ ID NO: 18): AGTACGTTCGTTTAACTCAAGCTGCC

[0473] Direct repeat sequence of the gRNA encoded by the Cas13m.3 vector (SEQ ID NO: 19): GTTGTAGAAGCCGTTCATTCGGGACGGTATGACAAC

[0474] Direct repeat sequence of the gRNA encoded by the Cas13m.6 vector (SEQ ID NO: 20): GTTGTAGAAGCCTATCGTTAGGATAGGTATGACAAC

[0475] Direct repeat sequence of gRNA encoded by C13-2 vector (SEQ ID NO: 21): GGAAGATAACTCTACAAACCTGTAGGGTTCTGAGAC

[0476] qGAPDHF1 primer (SEQ ID NO: 22): CCATGGGGAGGTGAAGGTC

[0477] qGAPDHR1 primer (SEQ ID NO: 23): GAAGGGGTCATTGATGGCAAC

[0478] qVegfa-F1 primer (SEQ ID NO: 24): ACCTCCACCATGCCAAGTGG

[0479] qVegfa-R1 primer (SEQ ID NO: 25): CAGGGTCTCGATTGGATGGC

[0480] qVegfa-F2 primer (SEQ ID NO: 26): CAATGACGAGGGCCTGGAGT

[0481] qVegfa-R2 primer (SEQ ID NO: 27): TCTTTGGTCTGCATTCACAT

[0482] pAAV-CasRx-gRNA Plasmid (SEQ ID NO: 28):

[0483] RJ-2 guide sequence (SEQ ID NO:29): GCAGCCTGGGACCACTTGGCATGGTGGAG

[0484] RJ-5 guide sequence (SEQ ID NO: 30): CCTGGAAGATGTCCACCAGGGTCTCGATTG

[0485] RJ-8 guide sequence (SEQ ID NO:31): ATGTTTGGACTCCTCAGTGGCACACACTCC

[0486] Tandem RJ-5 + RJ-8 (SEQ ID NO: 32): CCTGGAAGATGTCCACCAGGGTCTCGATTGcaagtaaacccctaccaactggtcggggtttgaaacATGTTGGACTCCTCCAGTGGGCACACACTCC

[0487] X-1 guide sequence (SEQ ID NO: 33): GTGCTGTAGGAAGCTCATCTCTCCTATGTG

[0488] X-2 guide sequence (SEQ ID NO: 34): GGTACTCCTGGAAGATGTCCACCAGGGTCT

[0489] X-Dual (Tandem X-1 + X-2) (SEQ ID NO: 35): GGTACTCCTGGAAGATGTCCACCAGGGTCTcaagtaaacccctaccaactggtcggggtttgaaacGTGCTGTAGGAAGCTCATCTCTCCTATGTG

[0490] qmVA-F primer (SEQ ID NO: 36): CTCAAGCCGTCCTGTGTGC

[0491] qmVA-R primer (SEQ ID NO: 37): GCTCATCTCTCCTATGTGCT

[0492] mGAPDH-F primer (SEQ ID NO: 38): GCTGAGTATGTCGTGGAGTCTA

[0493] mGAPDH-R primer (SEQ ID NO: 39): GTGGTTCACACCCATCACAA

[0494] The sequences from SEQ ID NO:40 to SEQ ID NO:235 are as described above.

[0495] Cas13X.1 dual-associated sequence (tandem X-1 + X-2 guide sequences used in Cas13X.1-associated vector construction) (SEQ ID NO: 236): GTGCTGTAGGAAGCTCATCTCTCCTATGTGGCTGGAGCAGCCCCGATTTGTGGGGTGATTACAGCGGTACTCCTGGAAGATGTCCACCAGGGTCT

[0496] Guide sequence of gRNA-luc targeting luciferase gene (SEQ ID NO: 237): GAATGTAGCCATCCATCCTTGTCAA

[0497] Cas13X.1-V vector (SEQ ID NO: 8):

Claims

1. A guide RNA for a gene editing system, comprising a guide sequence, wherein the guide sequence has at least 80% sequence identity compared to a sequence shown in any one of SEQ ID NOs: 10 to 18 and 40 to 230. Optionally, the guide sequence has at least 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 40-43, 45-47, 52-60, 64, 65, 73, 216, 219, 220, 223, 227, 228, 229, 230. Optionally, the guide sequence has at least 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 12, 13, 15, 47, 58, 59, 227, 228, and 230.

2. 2. The guide RNA of claim 1, wherein the guide sequence is designed to hybridize with a target RNA. Optionally, the guide sequence is designed to hybridize to the target RNA with a mismatch of up to six nucleotides.

3. The guide RNA according to claim 1, which is capable of forming a complex with an RNA-guided nuclease and inducing sequence-specific binding of the complex to a target RNA.

4. 2. The guide RNA of claim 1, which is capable of forming a complex with an RNA-guided nuclease and guiding the complex to bind to and cleave a target RNA.

5. 5. The guide RNA of claim 3 or 4, wherein the complex reduces the level of the target RNA in a cell.

6. 5. The guide RNA of claim 3 or 4, wherein the complex reduces the level of the target RNA in a cell by at least 5%.

7. The guide RNA according to claim 4, wherein the number of off-target genes that the complex cleaves when binding to and cleaving the target RNA is less than 40.

8. 8. The guide RNA of claim 7, wherein the number of off-target genes is determined by taking the intersection of a set of differentially expressed genes determined by RNA sequencing and a set of off-target genes predicted by the program.

9. 5. The guide RNA of claim 3 or 4, wherein the complex reduces the level of a protein encoded by the target RNA in a cell.

10. 5. The guide RNA of claim 3 or 4, wherein the complex reduces the level of the protein encoded by the target RNA in the cell by at least 5%.

11. The guide RNA according to any one of claims 3 to 10, wherein the nuclease induced by the RNA is a Cas protein. Optionally, the Cas protein is Cas13. Optionally, the Cas protein is Cas13a, Cas13b, Cas13c, or Cas13d.

12. 12. The guide RNA of claim 11, wherein the Cas protein is Cas13; Optionally, the Cas protein comprises an amino acid sequence having 50% or greater identity to a sequence set forth in any one of SEQ ID NOs: 1-3. Optionally, the Cas protein comprises an amino acid sequence having 50% or greater identity to the sequence set forth in SEQ ID NO:3, wherein the Cas protein (a) is mutated to other residues at positions corresponding to residues R210 and H215 of the sequence set forth in SEQ ID NO:3, (b) is mutated to other residues at positions corresponding to residues R785 and H790 of the sequence set forth in SEQ ID NO:3, (c) is mutated to other residues at positions corresponding to residues R210, H215, R785, and H790 of the sequence set forth in SEQ ID NO:3, or (d) is mutated to other residues at positions corresponding to residues R210, H215, R750A, H755A, R785, and H790 of the sequence set forth in SEQ ID NO:

3. Optionally, the Cas protein comprises a sequence having 50% or greater sequence identity to CasRx.

13. 13. The guide RNA of any one of claims 3 to 12, wherein the RNA-guided nuclease comprises a homologous or heterologous domain fused to a nuclease moiety. Optionally, the RNA-guided nuclease comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

14. 14. The guide RNA of any one of claims 1 to 13, wherein the guide sequence comprises 20 to 40 nucleotides.

15. 15. The guide RNA according to any one of claims 1 to 14, comprising a guide sequence and direct repeat sequences, wherein the direct repeat sequences interact with an RNA-guided nuclease.

16. 16. The guide RNA of claim 15, wherein the guide sequence is located at the 3' or 5' end of the direct repeat sequence.

17. The guide RNA according to any one of claims 15 to 16, wherein the direct repeat sequence comprises a sequence shown in any one of SEQ ID NOs: 19 to 21.

18. A guide RNA according to any one of claims 1 to 17, comprising an aptamer sequence.

19. 19. The guide RNA of claim 18, wherein the aptamer sequence is inserted into a loop in the secondary structure of the direct repeat sequence of the guide RNA.

20. 20. The guide RNA of any one of claims 1 to 19, wherein the guide RNA comprises modified nucleotides.

21. 21. The guide RNA of claim 20, wherein the modified nucleotides comprise 2'-O-methyl, 2'-O-methyl-3'-phosphorothioate, or 2'-O-methyl-3'-thio PACE modifications.

22. 21. The guide RNA of claim 20, wherein the modified nucleotides are selected from deoxyribonucleotides and locked nucleic acids.

23. A guide RNA according to any one of claims 2 to 22, wherein the target RNA is VEGFA RNA. Optionally, the target RNA is human VEGFA RNA. Optionally, the target RNA is VEGFA pre-mRNA or mature VEGFA mRNA.

24. A guide RNA according to any one of claims 2 to 23, wherein the target RNA is located in the nucleus or cytoplasm of a cell.

25. An isolated nucleic acid encoding a guide RNA according to any one of claims 1 to 24.

26. A vector comprising the nucleic acid of claim 25 and a control sequence for controlling the expression of a guide RNA.

27. 27. The vector of claim 26, wherein the vector is an adeno-associated virus vector.

28. 27. The vector of claim 26, wherein the regulatory sequence is a promoter or an enhancer.

29. 27. The vector of claim 26, wherein the regulatory sequence is a U6 promoter, an eye-specific promoter, or a CBh promoter.

30. A gene editing system comprising: (a) a guide RNA according to any one of claims 1 to 24, or a polynucleotide encoding said guide RNA; and (b) an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease; The guide RNA can form a complex with an RNA-guided nuclease and direct the complex to sequence-specific binding to the target RNA.

31. 31. The gene editing system of claim 30, wherein the guide RNA forms a complex with an RNA-guided nuclease and guides the complex to bind to and cleave the target RNA.

32. 31. The gene editing system of claim 30, wherein the RNA-guided nuclease is a Cas protein. Optionally, the Cas protein is a Cas13 protein. Optionally, the Cas protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein.

33. 33. The gene editing system of claim 32, wherein the Cas protein is Cas13; Optionally, the Cas protein comprises a sequence having 50% or greater identity to a sequence set forth in any one of SEQ ID NOs: 1-3. Optionally, the Cas protein comprises a sequence having 50% or greater identity to the sequence set forth in SEQ ID NO:3, wherein the Cas protein (a) is mutated to other residues at positions corresponding to residues R210 and H215 of the sequence set forth in SEQ ID NO:3, (b) is mutated to other residues at positions corresponding to residues R785 and H790 of the sequence set forth in SEQ ID NO:3, (c) is mutated to other residues at positions corresponding to residues R210, H215, R785, and H790 of the sequence set forth in SEQ ID NO:3, or (d) is mutated to other residues at positions corresponding to residues R210, H215, R750A, H755A, R785, and H790 of the sequence set forth in SEQ ID NO:

3. Optionally, the Cas protein comprises a sequence having 50% or greater sequence identity to CasRx.

34. 34. The gene editing system of any one of claims 30 to 33, wherein the RNA-guided nuclease comprises a homologous or heterologous domain fused to a nuclease moiety. Optionally, the RNA-guided nuclease comprises one or more of a subcellular localization signal, a deaminase domain, a translational activation domain, a translational repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag. Optionally, the subcellular localization signal is selected from a nuclear localization signal and a nuclear export signal sequence.

35. 31. The gene editing system of claim 30, wherein the polynucleotide sequence encoding the guide RNA is connected to a first regulatory sequence, and the first regulatory sequence is used to regulate expression of the guide RNA. The polynucleotide sequence encoding the RNA-guided nuclease is linked to a second regulatory sequence used to regulate expression of the RNA-guided nuclease.

36. A vector system comprising the gene editing system of any one of claims 30 to 35, the vector system comprising a polynucleotide sequence encoding a guide RNA and a first control sequence that controls expression of the guide RNA. It comprises a polynucleotide sequence that encodes an RNA-guided nuclease and a second regulatory sequence that controls expression of the RNA-guided nuclease.

37. 37. The vector system of claim 36, wherein the first regulatory sequence and / or the second regulatory sequence is a promoter or an enhancer.

38. An adeno-associated viral vector comprising the gene editing system of any one of claims 30 to 35, wherein the adeno-associated viral vector comprises DNA encoding an RNA-guided nuclease and a guide RNA.

39. A lipid nanoparticle comprising a gene editing system described in any one of claims 30 to 35, the lipid nanoparticle comprising a guide RNA and an mRNA encoding a nuclease guided by the RNA.

40. A lentiviral vector comprising the gene editing system of any one of claims 30 to 35, wherein the lentiviral vector comprises a guide RNA and an mRNA encoding an RNA-guided nuclease. Optionally, the lentiviral vector is mimicked with an envelope protein. Optionally, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

41. A ribonucleoprotein complex comprising the gene editing system of any one of claims 30 to 35, wherein the ribonucleoprotein complex is formed by a guide RNA and an RNA-guided nuclease.

42. 36. A virus-like particle comprising the gene editing system of any of claims 30 to 35, comprising a ribonucleoprotein complex formed by a guide RNA and an RNA-guided nuclease, optionally fused to a gag protein.

43. A eukaryotic cell comprising a gene editing system according to any one of claims 30 to 35.

44. A pharmaceutical composition comprising a gene editing system according to any one of claims 30 to 35, a vector system according to claim 36 or 37, an adeno-associated virus vector according to claim 38, a lipid nanoparticle according to claim 39, a lentiviral vector according to claim 40, a ribonucleoprotein complex according to claim 41, a virus-like particle according to claim 42, or a eukaryotic cell according to claim 43.

45. 45. The pharmaceutical composition of claim 44, comprising a pharmaceutically acceptable excipient.

46. Use of a guide RNA according to any one of claims 1 to 24, an isolated nucleic acid according to claim 25, a vector according to any one of claims 26 to 29, a gene editing system according to any one of claims 30 to 35, a vector system according to claim 36 or 37, an adeno-associated viral vector according to claim 38, a lipid nanoparticle according to claim 39, a lentiviral vector according to claim 40, a ribonucleoprotein complex according to claim 41, a virus-like particle according to claim 42, a eukaryotic cell according to claim 43 or a pharmaceutical composition according to claim 44 or 45 in the preparation of a medicament for carrying out any of the following, or any one of the following schemes: Cleavage or nicking one or more target RNA molecules, activation or upregulation of one or more target RNA molecules, activation or inhibition of translation of one or more target RNA molecules, inactivation of one or more target RNA molecules, visualization, labeling or detection of one or more target RNA molecules, binding one or more target RNA molecules, transporting one or more target RNA molecules, masking one or more target RNA molecules.

47. 47. The method of claim 46, wherein the target RNA is VEGFA RNA. Optionally, the target RNA is human VEGFA RNA.

48. 10. A method for diagnosing, treating or preventing a disease or condition associated with a target RNA, comprising administering to a sample from or to a subject in need thereof an effective amount of the guide RNA of any of claims 1 to 24, the isolated nucleic acid of claim 25, the vector of any of claims 26 to 29, the gene editing system of any of claims 30 to 35, the vector system of any of claims 36 to 37, the adeno-associated viral vector of claim 38, the lipid nanoparticle of claim 39, the lentiviral vector of claim 40, the ribonucleoprotein complex of claim 41, the virus-like particle of claim 42, or the pharmaceutical composition of claim 44 or 45.

49. 49. The method of claim 48, wherein the disease or condition associated with the target RNA is a disease or condition caused by aberrant expression of the target RNA.

50. 49. The method of claim 48, wherein the target RNA is VEGFA RNA. Optionally, the target RNA is human VEGFA RNA.

51. 41. A method of using a guide RNA according to any one of claims 1 to 24, an isolated nucleic acid according to claim 25, a vector according to any one of claims 26 to 29, a gene editing system according to any one of claims 30 to 35, a vector system according to any one of claims 36 to 37, an adeno-associated viral vector according to claim 38, a lipid nanoparticle according to claim 39, a lentiviral vector according to claim 40, a ribonucleoprotein complex according to claim 41, and a virus-like particle according to claim 42 in the manufacture of a medicament for diagnosing, treating, or preventing a disease or condition associated with a target RNA.

52. 52. The use of claim 51, wherein the disease or condition associated with the target RNA is a disease or condition caused by aberrant expression of the target RNA.

53. 52. The method of claim 51, wherein the target RNA is VEGFA RNA. Optionally, the target RNA is human VEGFA RNA.

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