RNA editing method and reagent

Abstract

The present invention relates to an RNA editing method and a reagent. The method uses a type III CRISPR complex and introduces a bulge loop structure of at least one base onto gRNA to edit a target RNA molecule. The method can bypass the characteristics of a 6-nucleotide-based cleavage rule, thereby enabling cleavage and ligation of RNA bases that are not multiples of 6 nucleotides.

Description

Methods and reagents for RNA editing

[0001] This application claims priority to Chinese Patent Application No. 202411582077.4, filed on November 7, 2024, entitled "Method and Reagent for RNA Editing", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of nucleic acid editing technology, and more particularly to methods and reagents for RNA editing. Background Technology

[0003] Precise nucleic acid editing technologies, whether at the DNA or RNA level, are of great value to biological research and therapy. CRISPR RNA-guided endonucleases have enabled programmable DNA cutting and have driven the development of new therapies. First-generation CRISPR genome editing used the Cas9 nuclease to create double-strand DNA breaks, which were then edited at the break sites through cellular repair. While DNA editing has the potential to cure genetic diseases, it can lead to unintended changes in the genome and activate cellular stress responses, resulting in toxicity. In contrast, RNA editing technologies can regulate biological functions by altering the gene expression program of cells without introducing hereditary mutations into the DNA.

[0004] CRISPR-mediated selective DNA binding or cleavage has revolutionized DNA editing, including base editing, leader editing, and manipulation of large DNA fragments. However, progress in RNA editing tools has lagged behind DNA editing. Cas13, a type VI CRISPR RNA-guided endonuclease, has been used to knock down cellular transcripts, but its target recognition also activates non-sequence-specific nuclease activity, degrading non-target RNA. A nuclease-inactivating mutant of Cas13 (dCas13) has been used to induce exon skipping, trans-splicing, or specific base editing for conversions from adenosine to inosine or cytosine to uracil. However, similar descriptions exist for programmable RNA deletion introduced by DNA editors.

[0005] To meet the demand for versatile and easily manipulated RNA, Nemudraia developed a technique for sequence-specific RNA editing in living cells. This technique utilizes a type III CRISPR system to programmably cut target RNA. Unlike Cas13, which has non-sequence-specific nuclease activity, the type III CRISPR system cuts target RNA only in 6-nt intervals. Nemudraia used this precise cutting activity to remove 6-nt interval fragments from human transcripts and demonstrated that the resulting RNA fragments could be repaired.

[0006] Currently, the type III CRISPR system relies on a 6-nt spacer for RNA splicing. When applied to the CDS region, it can only generate in-frame deletions, thus it cannot repair frameshift mutations and is mainly suitable for repairing nonsense mutations. However, alternative tools such as ADAR base editing, engineered tRNA, and programmable RNA pseudouracillation can achieve similar nonsense mutation repair at the RNA level. This fixed 6-nt spacer limits its ability to precisely regulate genetic information; it cannot perform non-6-nt spacer splicing to adjust open reading frames or repair specific types of genetic mutations, thus limiting the widespread application of this tool. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method and reagents for RNA editing based on the CRISPR type III system, which bypasses the 6-nucleotide-based cutting rule and aims to cut sequences that are not multiples of 6 nucleotides.

[0008] The RNA editing method provided by this invention includes: editing target RNA molecules using a type III CRISPR system and gRNA;

[0009] The gRNA targets the target RNA and introduces a raised loop of at least one base at different positions.

[0010] The method provided in this invention is a selective RNA cleavage and intramolecular splicing technique, abbreviated as SCISSOR. This method is based on the principle of calculating the base length in gRNA according to the 5' rule using a type III CRISPR system, and then cleaving the target RNA at 6-nt intervals in the opposite position. Through engineered gRNA design, bases are added or deleted to create raised loops in the target-binding region (the raised loops are located on either the target RNA side or the gRNA side), thereby allowing for cleavage of lengths other than 6-nt multiples on the RNA molecule. This provides greater flexibility and allows for precise control of RNA molecule editing. This is particularly important for applications requiring fine-tuning of open reading frames or repair of specific genetic mutations.

[0011] In this invention, the sequence of the gRNA is inversely complementary to that of the target RNA, and its total length is 20–100 nt. Experiments show that the editing efficiency increases with the increase of gRNA length, and the optimal editing efficiency is obtained for gRNAs of 50-nt and 56-nt length.

[0012] Preferably, the length is 30–60 nt, more preferably 38–56 nt. For example, in embodiments of the present invention, the length of the gRNA is 38 nt, 44 nt, 50 nt, or 56 nt.

[0013] In this invention, the bases added or deleted by the gRNA relative to the target RNA are located within the cleavage window of the type III CRISPR complex.

[0014] In this invention, the gRNA is formed by adding or deleting one or more bases in a sequence that is inversely complementary to the target RNA.

[0015] Preferably, the bases added or deleted by the gRNA relative to the target RNA are located between the 3rd and 4th bases within the cleavage window.

[0016] As a feasible example, the deleted or added bases are located 1–25 nt from the 5' end, preferably 10–25 nt from the 5' end; more preferably 10–20 nt from the 5' end; and even more preferably 10–15 nt, 12–17 nt, 15–20 nt, or 17–22 nt from the 5' end. For example, in gRNA, the deleted or added bases are located 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt from the 5' end of the gRNA relative to the target RNA.

[0017] In other cases, the deleted or added bases are located 1–25 nt from the 3' end, preferably 10–25 nt from the 3' end; more preferably 10–20 nt from the 3' end; and even more preferably 10–15 nt, or 12–17 nt, 15–20 nt, or 17–22 nt from the 3' end. For example, in gRNA, the deleted or added bases are located 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt from the 3' end of the gRNA relative to the target RNA.

[0018] In this invention, the bases added or deleted by the gRNA relative to the target RNA are located within the cleavage window of a type III CRISPR system, such as the Csm complex or the Cas7-11 system.

[0019] The Csm complex has a cleavage window of 6 bases, starting from the 5' end of the gRNA. This invention validates the position of the raised loop; RNA cleavage can be achieved regardless of its location within the cleavage window, but the editing efficiency is highest when the raised loop is located in the middle of the cleavage window. Preferably, the bases added or deleted by the gRNA relative to the target RNA are located between the 3rd and 4th bases within the cleavage window.

[0020] In this invention, to achieve cleavage of target RNA at lengths other than 6-nt multiples, the gRNA is augmented or deleting 3n+1 or 3n+2 bases relative to the target RNA, where n is an integer ≥ 0. For example, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, n is 0, 1, or 2. In embodiments of this invention, the gRNA is augmented or deleted by 1nt, 2nt, 4nt, 5nt, 7nt, 8nt, or 10nt relative to the target RNA.

[0021] In this invention, as another feasible approach, RNA is flexibly edited, with the gRNA adding or deleting 3 to 60 bases relative to the target RNA. By designing the gRNA and introducing 3-nt to 30-nt raised loops, the target RNA can also be knocked down with good editing efficiency. In the embodiments of this invention, the gRNA adds or deletes 3 to 30 bases relative to the target RNA; preferably, the gRNA adds or deletes 3nt, 6nt, 9nt, 18nt, 24nt, or 30nt relative to the target RNA.

[0022] This invention validated whether the protrusion loop is located on the gRNA side or the target RNA side. Preferably, the protrusion loop is located on the target RNA side, which is more beneficial for improving editing efficiency. Therefore, the gRNA is missing at least one base relative to the target RNA. Furthermore, a 1-nt deficiency is more effective than a 2-nt deficiency.

[0023] This invention also validated the presence of mismatched gRNAs. In the embodiments, the mismatches occurred on the gRNA in portions other than deleted and / or added bases. The results showed that even with partial base mismatches, good editing results could still be achieved. However, there was a negative correlation between the number of mismatches and the knockout efficiency. Preferably, the matching rate between the portion of the gRNA other than deleted and / or added bases and the target RNA was not less than 30%. In specific embodiments, in a 38nt gRNA, the number of mismatched bases did not exceed 6, preferably not exceeding 5, 4, 3, 2, or 1.

[0024] The Csm complex of the type III CRISPR system includes: Csm1 subunit, Csm2 subunit, Csm3 subunit, Csm4 subunit and Csm5 subunit; preferably, the amino acid sequence of the Csm is shown in any one of SEQ ID NO:104 to 108.

[0025] The type III CRISPR Cas7-11 system comprises a single Cas7-11 protein. Preferably, the amino acid sequence of the Cas7-11 system is shown in SEQ ID NO:109.

[0026] This invention does not limit the proteins in the type III CRISPR system; any protein with similar function can be used to implement the scheme of this invention.

[0027] In this invention, the type III CRISPR system includes a Cas7 protein, a Cas10 protein, a Csm protein complex, or a Cmr protein complex. For example, the Csm protein complex includes at least one of the Csm1, Csm2, Csm3, Csm4, Csm5, and Csm6 subunits, and the Cmr protein complex includes at least one of the Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 subunits.

[0028] As a feasible example, the type III CRISPR system is a Csm complex, which includes: Csm1 subunit, Csm2 subunit, Csm3 subunit, Csm4 subunit and Csm5 subunit; preferably, the amino acid sequence of the Csm is shown in any one of SEQ ID NO:104 to 108;

[0029] In another example, the type III CRISPR Cas7-11 system comprises a single Cas7-11 protein; preferably, the amino acid sequence of the Cas7-11 system is shown in SEQ ID NO:109.

[0030] Specifically, the RNA editing method of the present invention includes: transfecting cells containing target RNA with the gRNA and the Csm complex in the type III CRISPR system or the Cas7-11 system.

[0031] The RNA editing method provided by this invention has wide applications in RNA editing of organisms. Preferably, the cell is a eukaryotic cell. In this invention, the cell is an animal cell, plant cell, and / or microbial cell. For example, it is a cell, embryo, and / or tissue derived from an animal. Or, for example, it is a cell, tissue, seed, or germ derived from a plant. Or, for example, it is a cell or spore derived from a eukaryotic microorganism. In other embodiments, the organism is a prokaryote, specifically a cell of a prokaryotic microorganism. In the embodiments of this invention, human cells were used as the test subject for efficacy verification; however, in fact, the method of this invention can achieve good RNA editing effects on a variety of biological cells.

[0032] As a feasible example, the animals are humans, primates, rodents, even-toed ungulates, birds, felines, and / or canines. The plants are algae, mosses, ferns, angiosperms, and / or gymnosperms. Examples include cyanobacteria, green algae, spirulina, rice, soybeans, wheat, oats, corn, sorghum, apples, pears, peaches, cabbage, radishes, carrots, tobacco, or Arabidopsis thaliana. The microorganisms are yeast, Escherichia coli, Bacillus thuringiensis, Lactobacillus, or Bifidobacterium.

[0033] In this invention, the type of target RNA is not limited. The targeted RNA is selected from at least one of pre-messenger RNA (premRNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA), small nuclear RNA (snRNA), micronuclear interfering RNA (miRNA), and Piwi-interacting RNA (piRNA). In embodiments of this invention, the target RNA is the RNA of a reporter gene and / or disease-related RNA. According to the method of this invention, any RNA can be edited. As a feasible example, the target RNA molecule is the encoding gene of the GFP reporter gene, the gene encoding Nano-Luc luciferase, and / or endogenous transcripts PARK7, HEXA, RNF43, TCERG1, and CKB. In specific embodiments of this invention, the gRNA has the nucleic acid sequence shown in any one of SEQ ID NO: 1 to 103.

[0034] The present invention also provides gRNA as described in the previously described RNA editing method.

[0035] Furthermore, the present invention also provides a composition comprising the gRNA and the protein complex of the type III CRISPR system as described above.

[0036] Furthermore, the present invention also provides a plasmid vector comprising the gRNA as described above; or comprising the gRNA as described above and a protein complex of the CRISPR type III system.

[0037] The plasmid vector described in this invention may include only gRNA, or it may include a protein complex of gRNA and a type III CRISPR system; this invention does not limit the inclusion of gRNA. Preferably, the plasmid vector described in this invention includes both gRNA and a Csm complex or a Cas7-11 system. More preferably, the Csm complex backbone vector is pDAC627 (Addgene, Plasmid #195240).

[0038] The present invention also provides a host that is transformed or transfected with the plasmid vector as described above.

[0039] The host described in this invention is used for the preservation or transfection of the plasmid vector as described above. This invention does not limit the use of the host. For example, the host can be Escherichia coli, yeast, 293T cells, etc.

[0040] Furthermore, the present invention also provides an RNA editing reagent comprising at least one of the following:

[0041] i) gRNA as described above;

[0042] ii) The composition as described above;

[0043] iii) Plasmid vectors as described above;

[0044] iv) Host cells as described above.

[0045] In this embodiment of the invention, the RNA editing reagent further includes reagents, adjuvants, or excipients required for transfection and / or transformation;

[0046] The reagents include electroporation transfection reagents, ionotropic transfection reagents, and / or liposome transfection reagents; for example, the electroporation transfection reagents include trypsin, PBS buffer, sodium chloride, potassium chloride, lactate, acetic acid, Tween, poloxamer, and Span; the ionotropic transfection reagents include calcium-phosphorus transfection reagents; and the liposome transfection reagents include... 3000, Lipofectamine RNAiMAX, Lipo8000, etc.

[0047] The adjuvant includes at least one of the following: cytokines (e.g., interferons such as IFN-α, IFN-β and IFN-γ, lymphokines such as IL-2, IL-3, IL-4, IL-5 and IL-10, etc.), monokines such as IL-1, IL-6, IL-8 and IL-12, and other cytokines such as TNF and GM-CSF), co-stimulatory molecules (e.g., B7-1 and B7-2), emulsifying adjuvants (Titer MaxGold(TMG)), Freund's adjuvants, saponins, and monophosphate acyl lipids.

[0048] The excipients include polyethylene glycol and / or polylysine.

[0049] Furthermore, the present invention also provides at least one of the following applications in the preparation of vaccines for the prevention and / or treatment of RNA-related diseases:

[0050] i) gRNA as described above;

[0051] ii) The composition as described above;

[0052] iii) Plasmid vectors as described above;

[0053] iv) Host cells as described above;

[0054] v) RNA editing reagents as described above.

[0055] In this invention, the RNA-related diseases include tumors, nervous system diseases, immune system diseases, metabolic diseases and / or infectious diseases.

[0056] For example, the tumor includes malignant and benign tumors, selected from at least one of breast cancer, lung cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, bladder cancer, and melanoma; the neurological disease includes at least one of Parkinson's disease, Alzheimer's disease, stroke, multiple sclerosis, meningitis, spinal cord injury, and brain tumor; the immune system disease includes at least one of rheumatoid arthritis, systemic lupus erythematosus, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, scleroderma, and systemic sclerosis; the metabolic disease includes at least one of diabetes, obesity, hypertension, hyperlipidemia, hyperthyroidism, hypothyroidism, and osteoporosis; and the infectious disease includes at least one of AIDS, hepatitis B, hepatitis C, tuberculosis, malaria, dengue fever, cholera, influenza, and pneumonia.

[0057] Furthermore, the present invention also provides a medicament for the prevention and / or treatment of RNA-related diseases, comprising at least one of the following:

[0058] i) gRNA as described above;

[0059] ii) The composition as described above;

[0060] iii) Plasmid vectors as described above;

[0061] iv) Host cells as described above;

[0062] v) RNA editing reagents as described above.

[0063] Furthermore, the present invention also provides a method for preventing and / or treating RNA-related diseases, comprising administering the drug as described above to a subject.

[0064] The method provided by this invention uses a type III CRISPR system and a raised loop structure that introduces at least one base onto gRNA to edit target RNA molecules. This method can bypass the characteristics of the 6-nucleotide-based cleavage rule, thereby achieving RNA base cleavage and ligation that are not multiples of 6 nt. Attached Figure Description

[0065] Figure 1 shows a schematic diagram of the design principle of SCISSOR. SCISSOR utilizes a type III CRISPR system, such as the Csm complex and engineered gRNA, to flexibly excise RNA, wherein:

[0066] The top diagram illustrates a Csm-mediated RNA cleavage model. The gRNA associated with the Csm complex can recognize target RNA with complementary sequences, thus forming a gRNA-target RNA duplex. The Csm3 subunit cleaves the target RNA at 6-nt intervals. In the duplex structure shown in the diagram, the gRNA strand is on top and the target RNA strand is on the bottom.

[0067] The diagram shows that adding a 1-nt protrusion loop to the target RNA or gRNA can cause the Csm3 subunit to cleave the target RNA at 7-nt or 5-nt.

[0068] Figure 2 illustrates the construction of the SCISSOR reporting system, where:

[0069] (A) Top: Schematic diagram of three types of stop-GFP reporter genes; Bottom: Definition of editing modes;

[0070] (B) Cell imaging using fluorescence microscopy 48 hours after transfection; no GFP signal was observed for the three types of stop-GFP reporter genes;

[0071] (C) The positive control reporter genes produce strong GFP fluorescence, which are generated by introducing 6-nt, 7-nt and 8-nt deletions into the window containing the stop codon in the corresponding stop GFP reporter genes, respectively;

[0072] (D) shows a schematic diagram of the SCISSOR method for repairing frameshifted GFP;

[0073] (E) shows a suggested method for introducing a 1-nt raised loop into the target RNA (lower strand) or gRNA (upper strand) to produce a 7-nt or 5-nt cleavage in the target RNA; gRNAs with raised loops opposite positions 1 to 6 of the second window are shown as examples; TL, gRNA with a raised loop introduced on the target side;

[0074] Figure 3 shows the validation of SCISSOR on the reporting system, where:

[0075] (A) Shows the relative GFP RNA expression levels in HEK293T cells co-transfected with a GFP reporter gene and a Csm complex expressing gRNA with a 1-nt target protrusion loop; qPCR signals were normalized to the puromycin gene expressed by the GFP reporter plasmid, and the non-target gRNA control was set to 1; error bars represent the mean standard deviation of three biological replicates; NTC, negative control co-transfected with a GFP reporter gene and a Csm complex expressing non-target gRNA; Note that due to sequence configuration, in window 2, TL1 = TL2, TL5 = TL6; in window 3, TL2 = TL3;

[0076] (B) shows the editing efficiency of the sample in (A), quantified by targeted RNA-seq; the error bars represent the average standard deviation of three biological replicates;

[0077] (C) shows the target deletion patterns of engineered gRNAs with introduced 1-nt target protrusion loops; the deletion frequency of each pattern is quantified as the average of three biological replicates; diagonal stripes indicate deletions including the target window; Del, deletion; p, location;

[0078] (D) is the same as (A), but the engineered gRNA introduces a 1-nt gRNA protrusion loop;

[0079] (E) is the same as (B), but the engineered gRNA introduces a 1-nt gRNA protrusion loop;

[0080] (F) is the same as (C), but the engineered gRNA introduces a 1-nt gRNA protrusion loop;

[0081] (G) A recommended approach to introduce a 2-nt protrusion loop into the target to generate 8-nt cleavage in the target RNA;

[0082] (H) is the same as (A), but engineered gRNAs introduce a 2-nt target protrusion loop;

[0083] (I) is the same as (B), but engineered gRNAs introduce a 2-nt target protrusion loop;

[0084] (J) is the same as (C), but engineered gRNAs introduce a 2-nt target protrusion loop;

[0085] (K) Relative GFP RNA expression levels in HEK293T cells co-transfected with a GFP reporter gene and a Csm complex expressing gRNA with a 2-nt target protrusion loop; error bars represent the mean standard deviation of three biological replicates; note that in window 2, TL2 = TL3 due to sequence composition.

[0086] Figure 4 shows the introduction of raised loops of different lengths into gRNA, where:

[0087] (AB) shows the cutting pattern of introducing protruding rings of different lengths into the target;

[0088] (C) shows the relative GFP RNA expression level in HEK293T cells co-transfected with a GFP reporter gene and a Csm complex expressing gRNA with target loops of different lengths; error bars represent the mean standard deviation of three biological replicates.

[0089] (D) shows the editing efficiency of the sample in (C), quantified by targeted RNA-seq; the error bars represent the average standard deviation of three biological replicates;

[0090] (E) Deep sequencing results showing the introduction of protrusion loops of different lengths into the target using engineered gRNAs;

[0091] (F) shows that gRNA with a large protruding loop restored protein expression of the stop GFP reporter gene;

[0092] Figure 5 shows the effect of extending gRNA, where:

[0093] (A) shows the design of gRNAs with extended length;

[0094] (B) shows the relative GFP RNA expression level in HEK293T cells co-transfected with a GFP reporter gene and a Csm complex expressing extended-length gRNA; the error bars represent the mean standard deviation of three biological replicates.

[0095] (C) shows the editing efficiency of the sample in (B), quantified by targeted RNA-seq; the error bars represent the average standard deviation of three biological replicates;

[0096] Figure 6 shows the effect of incorporating a 5' mismatch into gRNA, where:

[0097] (A) shows the design that incorporates a 5' mismatch between gRNA and target mRNA;

[0098] (B) shows the relative GFP RNA expression level in HEK293T cells co-transfected with a GFP reporter gene and a Csm complex expressing gRNAs; error bars represent the mean standard deviation of three biological replicates.

[0099] (C) shows the editing efficiency of the sample in (B), quantified by targeted RNA-seq; the error bars represent the average standard deviation of three biological replicates;

[0100] (D) shows the deep sequencing results of engineered gRNAs with 5' mismatch introduced in different windows, with each row representing a biological replicate;

[0101] Figure 7 illustrates that the Cas7-11 system with engineered gRNAs can perform flexible cleavage, wherein:

[0102] (A) Schematic diagram of GFP-stop-Luc-fs1 and GFP-stop-Luc-fs2 reporter genes; GFP is used to confirm transfection efficiency;

[0103] (B) Relative reporter gene expression levels in HEK293T cells co-transfected with reporter genes and gRNAs expressing Cas7-11; error bars represent the mean standard deviation of three biological replicates;

[0104] (C) shows the editing efficiency of the sample in (B), quantified by targeted RNA-seq; the error bars represent the average standard deviation of three biological replicates;

[0105] (D) shows representative examples of RNA editing results for selected gRNAs; for each gRNA, the top three most common RNA editing results are shown; dashed lines indicate the location of RNA breaks; deletion frequencies are quantified as the average standard deviation of three biological replicates;

[0106] Figure 8 illustrates SCISSOR-mediated flexible cutting of endogenous targets, where:

[0107] (A) Shows gRNAs designed to introduce flexible excision of PARK7 transcripts; target protrusion loops are introduced at different positions in window 2;

[0108] (B) Shows the expression level of PARK7 RNA in HEK293T cells transfected with the Csm complex expressing gRNAs; error bars represent the mean standard deviation of three biological replicates; note that due to sequence composition, P1-3 = P1-4, P1-5 = P1-6.

[0109] (C) shows PARK7 editing efficiency, quantified by targeted RNA-seq; the error bars represent the average standard deviation of three biological replicates.

[0110] (D) Shows the targeting deletion patterns of engineered gRNAs. The deletion frequency of each pattern is the average of three biological replicates;

[0111] Figure 9 shows SCISSOR-mediated flexible cutting of endogenous target CKB, where:

[0112] (A) Five gRNAs were designed to introduce flexible excision of the CKB transcript. A raised loop was introduced in the middle of window 3.

[0113] (B) Expression level of CKB RNA in HEK293T cells transfected with the Csm complex expressing gRNAs. Error bars represent the mean standard deviation of three biological replicates.

[0114] (C) CKB editing efficiency, quantified by targeted RNA-seq, with error bars representing the mean standard deviation of three biological replicates;

[0115] (D) Targeted deletion patterns of engineered gRNAs, with the deletion frequency of each pattern being the average of three biological replicates;

[0116] Figure 10 illustrates the repair of pathogenic frameshift mutations mediated by SCISSOR, where:

[0117] (A) Above: Schematic diagram of the HEXA4bp insertion mutation and the introduction of the premature stop codon caused by this mutation;

[0118] The image below shows the design of gRNAs to repair frameshift mutations. We introduced a raised loop in the middle of window 2 to design 5 gRNAs.

[0119] (B) RNA expression level of HEXA reporter gene in HEK293T cells, co-transfection of HEXA reporter gene and Csm complex expressing 1-nt target protrusion loop gRNA in HEK293T cells, error bars represent the average standard deviation of three biological replicates;

[0120] (C) HEXA editing efficiency, quantified by targeted RNA-seq, with error bars representing the average standard deviation of three biological replicates;

[0121] (D) Targeted deletion patterns of engineered gRNAs, with the deletion frequency of each pattern being the average of three biological replicates;

[0122] (E) Western blot shows the HEXA reporter protein obtained by SCISSOR editing using gRNA H1, with NT, non-targeting gRNA, and GAPDH as internal reference gene. The HEXA-HA fusion protein was detected using HA-tag antibody.

[0123] (F) Above: Schematic diagram of generating HEXA-mutant HEK293T cells using the CRISPR / Cas9 system;

[0124] The image below shows that the Sanger results indicate a successful 4-bp insertion in the mutant cells.

[0125] RNA expression level of (G)HEXA endogenous transcript in 1278ins4 mutant HEK293T cells transfected with a Csm complex expressing gRNA H1. Error bars represent the mean standard deviation of three biological replicates.

[0126] Editing efficiency of (H)HEXA endogenous transcripts, with error bars representing the average standard deviation of three biological replicates;

[0127] (I) is the same as (D), but it targets HEXA endogenous transcripts;

[0128] Figure 11 shows that SCISSOR can generate immunogenic frameshift multiple epitopes, wherein:

[0129] Above: Schematic diagram of reporter plasmids for detecting SCISSOR-edited transcripts at the protein level using HA-tag;

[0130] The image below shows that for each gene, we introduced a raised loop in the middle of window 2 to design 5 gRNAs.

[0131] (B) RNA expression levels of TCERG1 and RNF43 reporter plasmids in HEK293T cells; error bars represent the mean standard deviation of three biological replicates.

[0132] (C) The editing efficiency of TCERG1 and RNF43 report plasmids was quantified by targeted RNA-seq, and the error bars represent the average standard deviation of three biological replicates;

[0133] (D) Deletion patterns of engineered gRNAs targeting the TCERG1 reporter plasmid, with the deletion frequency of each pattern quantified as the average of three biological replicates.

[0134] (E) is the same as (D), but it is for the RNF43 reporter plasmid;

[0135] (F) Western blot shows the expression of frameshift multiepitopes from TCERG1 and RNF43 transcripts. PTC is the positive control; NT is the non-targeting gRNA; FS is the frameshift HA. The positive control group was transfected with 0.9 μg and 0.2 μg of reporter plasmid, respectively, and the experimental group was transfected with 1.8 μg of reporter plasmid, respectively. Vinculin is an internal control protein. The HA-tag antibody was used to detect the frameshift multiepitope-HA fusion protein.

[0136] (G) RNA expression levels of endogenous TCERG1 and RNF43 transcripts in HEK293T cells transfected with the Csm complex expressing gRNAs T2 and R3. Error bars represent the mean standard deviation of three biological replicates.

[0137] Editing efficiency of (H)TCERG1 and RNF43 endogenous transcripts, with error bars representing the mean standard deviation of three biological replicates;

[0138] (I) is the same as (D), but targets endogenous transcripts of TCERG1 and RNF43;

[0139] (J) is the same as (G), but it is specific to the U2OS cell line;

[0140] (K) is the same as (H), but it is specific to the U2OS cell line;

[0141] (L) is the same as (I), but it is for the U2OS cell line. Detailed Implementation

[0142] This invention provides methods and reagents for RNA editing. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0143] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0144] In this invention, "and / or" is used to describe the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0145] In this invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0146] In this invention, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions "consisting of...".

[0147] In this invention, "RNA" stands for ribonucleic acid, a long-chain molecule formed by the condensation of ribonucleotides via phosphodiester bonds. In this invention, the main bases of RNA are A (adenine), G (guanine), C (cytosine), and U (uracil). Generally, there are three main types of RNA molecules: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). In addition, there are some special RNAs, such as pre-messenger RNA, long non-coding RNA, small nuclear RNA, micro-interfering RNA, and Piwi-interacting RNA.

[0148] In this invention, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) refers to short palindromic repeat sequences that are regularly spaced in clusters. CRISPR is a natural immune system, and the CRISPR-Cas system can be used for genome editing. By guiding RNA to complementarily pair with target DNA or RNA sequences, it enables precise editing of specific sites on the genome.

[0149] In this invention, SCISSOR (Selective Cleavage and Intramolecular Stitches of RNA) refers to the selective cleavage and intramolecular splicing of RNA.

[0150] In this invention, CDS refers to the coding region of a gene, also known as the coding sequence, which is the part of the DNA or RNA of a gene that encodes a protein.

[0151] In this invention, "inDel" refers to Insertions and Deletions, which involve inserting one or more DNA bases into the genome or deleting one or more DNA bases from the genome.

[0152] In this invention, gRNA refers to guide RNA (gRNA) or single guide RNA (sgRNA), which is a short sequence of RNA that can act as a guide for Cas9 endonuclease or other Cas proteins that can cleave double-stranded DNA, thereby enabling gene editing.

[0153] In this invention, frameshift mutation refers to a gene mutation caused by the insertion or deletion (indel) of a number of nucleotides that is not a multiple of three. Because gene codons consist of three nucleotides during expression, such insertions or deletions alter the reading frame, causing the mRNA to be translated into a completely different protein after this mutation. Frameshift mutations occur further upstream in the open reading frame and have a greater impact on the protein.

[0154] In this invention, nonsense mutation refers to a mutation in which a specific codon for an amino acid in a gene is converted into a stop codon for polypeptide chain synthesis due to base substitution or frameshift mutation, resulting in the interruption of polypeptide chain synthesis and the production of a non-functional protein product.

[0155] In this invention, an open reading frame (ORF) refers to a sequence within a given reading frame that does not contain a stop codon. This sequence is a portion of the genome of an organism that may serve as a protein-coding sequence.

[0156] In this invention, the cleavage window refers to a cleavage window that starts from the 5' end of the gRNA of the Csm complex and consists of 6 bases.

[0157] In this invention, "treatment" refers to surgical or therapeutic treatment aimed at preventing or mitigating (reducing) undesirable physiological changes or lesions, such as cancer and tumors, in the treated subject. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Subjects requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as mitigation, reduction, weakening, mitigation, and remission are used, they also include elimination, disappearance, and non-occurrence.

[0158] In this invention, "medicine" means a formulation which exists in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the pharmaceutical composition.

[0159] In this invention, the "medication" can preferably be administered by injection, enteral administration, or local administration in any of the following ways. For example, the injection includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and / or intracranial injection. The enteral administration mainly includes oral and / or rectal administration. The local administration includes, but is not limited to, spray inhalation, nasal administration, buccal administration, and vaginal administration.

[0160] In this invention, "subject" refers to an organism receiving treatment for a specific disease or symptom as described in this invention. Exemplarily, "subject" includes mammals receiving treatment for a disease or symptom, including bovines, equines, sheep, suidae, canines, felines, rodents, and primates, with preferred mammals being humans, cats, dogs, or pigs.

[0161] In this invention, "effective amount" refers to the amount of a therapeutic agent, when administered alone or in combination with another therapeutic agent to cells, tissues, or other subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective amount" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating related medical conditions, or increasing the rate at which these conditions are treated, cured, prevented, or alleviated. When an active ingredient is administered alone to an individual, the therapeutically effective dose refers solely to that ingredient. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0162] In this invention, the drug can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferably, the dosage form of the drug depends on the intended route of administration and therapeutic use. The pharmaceutical compositions of this invention should be sterile and stable under the conditions of production and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the gRNA of this invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Furthermore, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0163] In this invention, "cancer" and "tumor" are not mutually exclusive. "Cancer" refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. "Tumor" or "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. More specifically, the tumor or tumor refers to a tumor or tumor associated with RNA modification.

[0164] In this invention, the design and modification of gRNA based on CRISPR-Csm involves introducing deletions of 1nt, 2nt, or multiple base lengths at specific positions on the gRNA to achieve non-6nt cleavage and ligation on the mRNA. This novel type of RNA editing has broad application prospects, such as treating diseases caused by certain frameshift mutations or using frameshift mutations for tumor treatment. Furthermore, introducing longer base-pairing sequences at one or both ends of the gRNA enhances binding to the target site, thereby increasing the efficiency of cleavage and ligation. Even further, introducing mismatched bases at the target cleavage site reduces or eliminates cleavage at that location, further improving the purity of the product. The design principle and mechanism of the SCISSOR technology in this invention are as follows: Structural analysis shows that the type III CRISPR complex calculates the base length in the gRNA according to the 5' rule, and then cleaves the target RNA at 6-nt intervals at the opposite positions. Therefore, we believe that by designing gRNA to create raised loops in the gRNA-target binding region, it may be possible to enable the type III CRISPR system to cleave target RNA in cells at lengths not exceeding 6-nt (Figure 1).

[0165] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0166] The present invention will be further illustrated below with reference to the embodiments:

[0167] Example

[0168] I. Experimental Procedure

[0169] 1.1. Cell Culture

[0170] HEK293T and U2OS cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection (CBTCCCAS). Their identity was verified using short tandem repeat (STR) analysis via CBTCCCAS, which involved simultaneously amplifying 17 STR markers along with amelogenin to confirm cell identity. Both cell lines were maintained in DMEM (Gibco) with 10% FBS (HyClone) added. Mycoplasma contamination was checked via CBTCCCAS, and routine mycoplasma testing was performed using PCR on conditioned medium.

[0171] 1.2. Construction of Type III CRISPR composite plasmids and their reporter plasmids

[0172] The gRNA fragment was synthesized by Suzhou Hesheng Biotechnology Co., Ltd., and subcloned into either the Csm plasmid (from pDAC627 (Addgene, Plasmid #195240)) or the Cas 7-11 gRNA plasmid (backbone plasmid pC0043). To generate various GFP reporter plasmids, GFP was amplified from the pCDNA3.1 EGFP vector using PCR. Additional elements, such as stop codons, frameshift mutations, Luciferase, and HA-tags, were integrated into the GFP gene using overlap PCR.

[0173] To generate reporter plasmids HEXA-HA (wild-type) and HEXA-frameshift-HA (1278ins4), the wild-type gene fragment was synthesized by Suzhou Synthetic Biotechnology Co., Ltd. The 1278ins4 mutation was introduced using overlap PCR while retaining introns 11-13.

[0174] 1.3. Plasmid transfection for qPCR and targeted RNA-seq experiments

[0175] HEK293T cells were seeded one day prior to the experiment and transfected when 60%-80% confluence was reached. Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. For experiments editing the GFP reporter gene, 700 ng of plasmid DNA encoding the Csm complex and gRNA or Cas7-11 and gRNA was co-transfected with 300 ng of plasmid DNA encoding the GFP reporter gene into 12-well plates. Cells were harvested 48 hours after transfection. For experiments editing the HEXA and frameshift multi-epitope host reporter genes, 700 ng of plasmid DNA encoding the Csm complex and gRNA was co-transfected with 300 ng of plasmid DNA encoding the target gene into 12-well plates. Cells were harvested 48 hours after transfection. For experiments editing endogenous expression genes, 500 ng of plasmid DNA encoding the Csm complex and gRNA was transfected into 24-well plates. Twenty-four hours after transfection, cells were isolated and seeded in 12-well plates in medium supplemented with puromycin (1 g / mL). Cells were harvested 72 hours after puromycin resistance selection.

[0176] 1.4. qPCR

[0177] Total RNA was extracted from cells using TRIzol reagent and the Direct-zol RNA kit (Zymo Research). RNA was treated with DNase I and reverse transcribed using HiScript II Q RTSuper Mix (Vazyme) with random primers. Each cDNA sample underwent three PCRs using ChamQ SYBR qPCR Master Mix (Vazyme). For GFP reporter gene quantification, the primer pair amplifying the aminoglycoside phosphotransferase in the reporter gene plasmid was used as an internal control. The same internal control was used for HEXA and frameshift multiepitope reporter gene quantification. For endogenous gene quantification, the housekeeping gene GAPDH was used as an internal control.

[0178] 1.5. Targeted RNA-seq

[0179] In the first round of PCR, the target sequence was amplified using STARmix Taq DNA polymerase (GenStar) according to the following program: 94°C for 3 minutes; 25 cycles of 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 20 seconds; and heating at 72°C for 1 minute. Next, the PCR product was purified using DNA Clean Beads (Vazyme) and dissolved in 10 μl of water. Using sequencing adapters, 1 μl of the purified product was amplified for 5 cycles using the same program as above. Finally, the PCR product was recovered using the Zymoclean Gel DNA Recovery Kit (Zymo Research) and sequenced.

[0180] 1.6. Western blot

[0181] HEK293T cells were seeded in 6-well plates the day before the experiment. In the experiment of editing the HEXA reporter gene, the experimental group was transfected with 1.2 μg of plasmid DNA encoding the mutant HEXA reporter gene and 0.8 μg of plasmid DNA encoding the Csm complex and gRNA, respectively. The negative control group was transfected with 1.2 μg of plasmid DNA encoding the mutant HEXA reporter gene and 0.8 μg of plasmid DNA encoding the Csm complex and non-target gRNA, respectively. The positive control group was transfected with 0.2 μg of plasmid DNA encoding the wild-type HEXA reporter gene and 1.8 μg of plasmid DNA encoding the Csm complex and non-target gRNA, respectively. For the experiments of editing the TCERG1 and RNF43 reporter genes, the positive control group was transfected with 0.9 μg and 0.2 μg, respectively, and the experimental group was transfected with 1.8 μg of reporter gene, respectively.

[0182] All transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. After 48 hours, cells were washed with frozen PBS (Thermo Fisher Scientific) and lysed with RIPA buffer (Beyotime). Total protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Cell lysates were mixed with 5x loading buffer (Beyotime) and heated at 100°C for 10 min. Protein samples were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% defatted dry milk (Bio-Rad) at room temperature for 1 hour and incubated overnight at 4°C with polyclonal anti-HA antibody (Proteintech, cat#510642-AP) as the primary antibody. IRDye 680RD (LI-COR, cat#92568072) and 800CW (LI-COR, cat#925-32210) were conjugated with anti-mouse antibodies as secondary antibodies, and the proteins were visualized using an Odyssey CLx Imager (LI-COR).

[0183] 1.7. Generation of mutant cell lines

[0184] HEXA mutant cell lines were generated by CRISPR / Cas9 knock-in using an ssDNA template. The sgRNA targeting the HEXA gene was cloned into lentiCRISPR v2 (Addgene plasmid #52961), and the ssDNA template was synthesized by Synbio Technologies, Suzhou. Cells were transfected into 12-well plates with 500 ng of sgRNA plasmid and 500 ng of ssDNA. After 48 hours, cells were selected with puromycin, and surviving cells were selected for integration using Sanger sequencing.

[0185] 1.8. Sequencing Data Analysis

[0186] First, use Cutadapt (version 3.7) to trim low-quality bases (quality score <20) and sequencing adapter sequences. Use STAR (version 2.7.10a) to build a reference genome. Map reads to the genome using STAR with the following parameters: "--runThreadN 30 --outSAMprimaryFlag AllBestScore --alignEndsType EndToEnd --scoreDelOpen0 --scoreDelBase0".

[0187] The aligned BAM files were sorted using Samtools (version 1.6), and the edit level was analyzed using a custom Python script. In short, the deletion type of each read was identified by examining the CIGAR values ​​located at the reference target gene. The edit level was calculated by determining the proportion of reads that repaired or introduced frameshift mutations at the target site relative to the total number of reads.

[0188] Based on the above steps, the following experiment was conducted:

[0189] 2.1 Construction of reporter plasmids encoding GFP fused with an N-terminal HA tag. We introduced a stop codon and a 2-nt or 1-nt deletion before the start codon of GFP, resulting in 1-nt and 2-nt frameshift GFP variants (stop-GFP-fs1 and stop-GFP-fs2) (Figure 2, A). After transfecting these stop-GFP-fs1 and stop-GFP-fs2 plasmids into cells, we did not observe a GFP signal (Figure 2, B). Conversely, plasmids with the same sequence but without the stop codon and deletion produced strong GFP fluorescence (Figure 2, C). Next, we designed gRNAs to remove the stop codon and correct the frameshift mutation (Figure 2, D). We designed gRNAs with a 1-nt protrusion loop introduced in one of the windows to achieve a 7-nt or 5-nt deletion in the target RNA. We selected a 32-nt gRNA that showed the best results and designed it as a raised loop corresponding to positions 1 through 6 of the selected window (E in Figure 2). The sequences of the gRNA are shown in Table 1, denoted as window2_1nt_TL1~6 and window3_1nt_TL1~6. The results showed that setting the raised loop between positions 3 and 4 resulted in the best editing effect.

[0190] 2.2 An integrated plasmid expressing the Csm complex and gRNA was transfected into HEK293T cells along with a reporter plasmid, and cells were collected 48 hours after transfection. We found that engineered gRNAs with a 1-nt raised loop on the target side resulted in a reduction of target RNA by more than 90% (Fig. 3A). The cutting window was excised in 17–45% of transcripts, depending on the location of the raised loop (Fig. 3B). Most importantly, all deletions across the cutting window resulted in the removal of exactly 7 nt within the window (Fig. 3C). For gRNAs with a raised loop on the gRNA side, which edit the stop-GFP-fs2 reporter gene, the target window was excised in 2%–35% of transcripts (Fig. 3DE). Furthermore, all deletions across the target window resulted in the removal of 5 nt within the window (Fig. 3F). We also explored the possibility of rescuing the 2-nt frameshift by introducing a 2-nt target-side raised loop in a second or third window (Fig. 3G). This strategy successfully knocked out the target RNA (Fig. 3H). Similarly, all deletions across the target window resulted in the removal of 8-nt within the window (IJ in Figure 3), which restored the protein expression of stopGFP-fs2 (K in Figure 3). The sequences of the gRNAs with protrusion loops on the target genes are shown in Table 1, denoted as window2_2nt_TL1~6 and window3_2nt_TL1~6. The sequences of the gRNAs with protrusion loops on the gRNAs are also shown in Table 1, denoted as window2_2nt_GL1~6 and window3_2nt_GL1~6.

[0191] 2.3 We designed gRNAs targeting the GFP reporter gene and introduced raised loops ranging from 3-nt to 30-nt (3-, 6-, 9-, 18-, 24-, and 30-nt, AB in Figure 4) opposite position 4 of the third window. To ensure adequate binding of the gRNA sequences upstream and downstream of the deletion region, a 38-nt gRNA was used. We found that the target RNA was significantly knocked down in all cases (C in Figure 4) and exhibited high editing efficiency (DE in Figure 4). GFP signals were detected in all groups after editing (F in Figure 4). The gRNA sequences are shown in Table 1, denoted as window3_(3 / 6 / 9 / 18 / 24 / / 30)nt_TL.

[0192] 2.4 Design of the target side protrusion loop. First, we attempted to increase the gRNA length. We extended the gRNA length in 6-nt increments, ranging from 1 to 3 additional units (Figure 5A). Notably, both gRNAs showed a significant increase in editing efficiency with increasing length, reaching peak values ​​of 73% and 87% at 12-nt and 18-nt extensions, respectively (Figure 5BC). The gRNA sequences are shown in Table 1, denoted as w3d2-3+(0 / 6 / 12 / 18) or w3d1-2+(0 / 6 / 12 / 18).

[0193] 2.5 A series of gRNAs incorporating 5' mismatches were designed (Figure 6A), and their deletion patterns were examined. We found a negative correlation between the number of mismatches and the knockout efficiency (Figure 6B). Although the introduction of 5-position mismatches effectively suppressed the cleavage of the corresponding window, it also reduced the editing efficiency (Figure 6CD). The sequences of the gRNAs are shown in Table 1, denoted as 1-(0 / 1 / 2 / 3)-PS or 2-(0 / 1 / 2 / 3)-PS.

[0194] 2.6 Cas7-11 with engineered gRNAs allows for flexible cleavage: Plasmids expressing GFP and Nano-Luc luciferase were developed and linked to the viral 2A peptide. We introduced a stop codon and a 2-nt or 1-nt deletion before the start codon of Nano-Luc to generate 1-nt and 2-nt frameshift Nano-Luc variants (GFP-stop-Luc-fs1 and GFP-stop-Luc-fs2) (Figure 7A). Next, we engineered gRNAs to remove the stop codon and correct the frameshift. The plasmid expressing Cas7-11 and the gRNAs were co-transfected into HEK293T cells, and cells were collected 48 hours post-transfection. We observed that all engineered gRNAs resulted in successful RNA knockout (Figure 7B). Deep sequencing of the target RNA amplicon confirmed that most of the samples contained RNA deletions that corrected the frameshift mutation (Figure 7CD). The sequences of the gRNAs are shown in Table 1, denoted as Luc-TG(1 / 2 / 3 / 4 / 5)-1nt, Luc-TG(1 / 2 / 3 / 4 / 5)-2nt, or Luc-GL(1 / 2 / 3 / 4 / 5)-1nt.

[0195] 2.7 SCISSOR-mediated flexible cleavage of endogenous targets: We tested targeting the endogenous transcript PARK7 (Figure 8A). qPCR showed that different gRNAs caused varying levels of knockdown of the PARK7 transcript (Figure 8B). Deep sequencing confirmed the presence of 7-nt and 8-nt deletions (Figure 8CD). The editing efficiency of gRNAs with 1-nt loops was 8.3%, and that with 2-nt loops was 1.4%. The sequences of the gRNAs are shown in Table 1, denoted as P1-(1–5) or P2-(1–5).

[0196] 2.8 SCISSOR was applied to the endogenous transcript CKB (Figure 9, A). The raised loop was located at position 4 of the CKB gRNA, and qPCR showed that different gRNAs caused different levels of knockdown of the CKB transcript (Figure 9, B). The results showed that SCISSOR successfully achieved 7-nt and 8-nt deletions in the CKB transcript, with the best-performing gRNAs achieving editing efficiencies of 4.7% and 4%, respectively (Figure 9, CD).

[0197] 2.9 Protein expression can be restored by removing pathogenic frameshift mutations in RNA using SCISSOR:

[0198] To determine the therapeutic potential of SCISSOR in repairing frameshift mutations, clinically relevant frameshift mutations in the HEXA gene were targeted. This mutation, a 4 bp insertion (TATC, 1278ins4) in exon 11 of the HEXA gene, is the most common mutation causing Tay-Sachs disease (TSD), accounting for approximately 80% of TSD cases. This mutation leads to the production of premature stop codons downstream, and the mutated transcript is rapidly degraded via nonsense-mediated messenger RNA (mRNA) degradation (NMD) pathway.

[0199] First, the HEXA-1278ins4 reporter plasmid was constructed, encoding a small HEXA gene fused to a C-terminal HA tag (Figure 10, A). Only the repaired transcript can express the C-terminal HA-tag, facilitating detection of the repaired transcript at the protein level. Five gRNAs with 1nt target protrusion loops were designed (Figure 10, A). The reporter gene and the Csm complex expressing the gRNA were co-transfected into HEK293T cells. Among them, gRNA H1 showed the highest editing efficiency, reaching 53% (Figure 10, BD). Co-transfection of the reporter gene and the Csm complex expressing gRNA H1 successfully restored the C-terminus of the HEXA protein (Figure 10, E).

[0200] To further evaluate the ability of SCISSOR to correct this frameshift mutation in endogenous HEXA transcripts, a HEK293T cell line containing a 1278ins4 insertion was constructed using the CRISPR / Cas9 system (F in Figure 10). The efficiency of editing the mutant HEXA endogenous transcript using the Csm complex expressing gRNA H1 reached 56% (GI in Figure 10).

[0201] 2.10 SCISSOR can generate immunogenic frameshift polyepipants (fs-poly-epitopes).

[0202] Two frameshift multiepitopes, which have been shown to be highly immunogenic in microsatellite instability tumors, were selected for testing. These two frameshift multiepitopes originated from frameshift mutations in the TCERG1 (CA>C, position 2861) and RNF43 (GT>T, position 1,975) genes, respectively.

[0203] First, reporter plasmids for TCERG1 and RNF43 were generated. Each plasmid expressed GFP and a flanking region around the TCERG1 or RNF43 frameshift mutation site, and a HA-tag was attached to the protein terminus (Figure 11, A). Only transcripts with defined frameshifts could generate frameshift epitopes and C-terminal HA-tags, facilitating the detection of frameshift epitopes at the protein level. Five gRNAs with 1-nt target protrusion loops were designed for each of the two genes (Figure 11, A). The reporter genes and Csm complexes expressing the gRNAs were co-transfected into HEK293T cells. The best-performing gRNAs achieved editing efficiencies of 32% and 9%, respectively (Figure 11, BC), and successfully generated frameshifts (Figure 11, DE). Consistent with this, Western blot analysis confirmed that the edited transcripts generated frameshift epitopes (Figure 11, F).

[0204] Finally, SCISSOR was applied to the endogenously expressed TCERG1 and RNF43 transcripts in HEK293T cells, achieving editing efficiencies of 30% and 19%, respectively, using the previously identified best-performing gRNAs (GI in Figure 11). We also applied SCISSOR to the endogenously expressed TCERG1 and RNF43 transcripts in U2OS cells, achieving editing efficiencies of 7% and 6%, respectively, using the previously identified best-performing gRNAs (JL in Figure 11).

[0205] Table 1 SCISSOR gRNA Design

[0206] Table 2. CSM protein amino acid sequence

[0207] Table 3 Amino acid sequence of Cas7-11 protein

[0208] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. RNA editing methods, including: Editing target RNA molecules using the type III CRISPR system and gRNA; The gRNA targets the target RNA and has at least one base added or deleted relative to the target RNA.

2. The RNA editing method according to claim 1, characterized in that, The gRNA has a length of 20–100 nt, preferably 30–60 nt, and more preferably 38–56 nt.

3. The RNA editing method according to claim 1, characterized in that, The bases added or missing by the gRNA relative to the target RNA are located within the cleavage window of the type III CRISPR complex. Preferably, the bases added or deleted by the gRNA relative to the target RNA are located between the 3rd and 4th bases within the cleavage window.

4. The RNA editing method according to claim 1, characterized in that, The gRNA has 3n+1 or 3n+2 bases added or deleted relative to the target RNA, where n is an integer ≥ 0; Preferably, the gRNA has 1 to 60 bases added or deleted relative to the target RNA; Preferably, the gRNA is increased or decreased by 1 nt, 2 nt, 4 nt, 5 nt, 7 nt, 8 nt or 10 nt relative to the target RNA.

5. The RNA editing method according to any one of claims 1 to 4, characterized in that, In the gRNA, the portion other than the deleted and / or added bases has a matching rate with the target RNA of not less than 30%.

6. The RNA editing method according to any one of claims 1 to 5, characterized in that, The type III CRISPR system includes Csm family complexes, Cas7-11 family proteins, or other types of type III CRISPR systems; Preferably, the type III CRISPR system is a Csm complex, which includes: Csm1 subunit, Csm2 subunit, Csm3 subunit, Csm4 subunit and Csm5 subunit; preferably, the amino acid sequence of the Csm is shown in any one of SEQ ID NO:104 to 108; Preferably, the type III CRISPR Cas7-11 system comprises a single Cas7-11 protein; preferably, the amino acid sequence of the Cas7-11 system is shown in SEQ ID NO:

109.

7. The RNA editing method according to any one of claims 1 to 6, characterized in that, The method includes: transfecting cells containing target RNA with the gRNA and type III CRISPR complex; Preferably, the cells are animal cells, plant cells, and / or microbial cells.

8. The RNA editing method according to claim 7, characterized in that, The target RNA is the RNA of a reporter gene and / or disease-related RNA.

9. Biomaterials, including any one of the following: I) The gRNA described in any one of claims 1 to 8; II) A composition comprising the gRNA and type III CRISPR complex as described in any one of claims 1 to 8; III) Plasmid vector, comprising the gRNA as described in any one of claims 1 to 8; or comprising the gRNA as described in any one of claims 1 to 8 and a type III CRISPR complex; IV), host, transformation or transfection of the plasmid vector described in III).

10. An RNA editing reagent comprising at least one of the biological materials of claim 9.

11. The RNA editing reagent according to claim 10, characterized in that, It also includes reagents, adjuvants, or excipients required for transfection and / or transformation; The reagents include electroporation transfection reagents, iontophoresis transfection reagents, and / or liposome transfection reagents; The adjuvants include at least one of the following: cytokines, co-stimulatory molecules, emulsifying adjuvants, Freund's adjuvants, saponins, monophosphate acyl lipids, TLR agonists, CpG oligonucleotides, oligodeoxynucleotides, and / or immunomodulatory proteins. The excipients include polyethylene glycol and / or polylysine.

12. The use of at least one of the biomaterials of claim 9 or the RNA editing reagent of claim 10 or 11 in the preparation of a treatment for the prevention and / or treatment of RNA-related diseases.

13. The application according to claim 12, characterized in that, The RNA-related diseases include tumors, neurological diseases, immune system diseases, metabolic diseases, and / or infectious diseases; Preferably, the tumor includes malignant tumors and benign tumors, and is selected from at least one of breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, bladder cancer, and skin melanoma; The neurological diseases include at least one of Parkinson's disease, Alzheimer's disease, stroke, multiple sclerosis, meningitis, spinal cord injury, and brain tumor; The immune system diseases mentioned include at least one of rheumatoid arthritis, systemic lupus erythematosus, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, scleroderma, and systemic sclerosis; The metabolic diseases mentioned include at least one of diabetes, obesity, hypertension, hyperlipidemia, hyperthyroidism, hypothyroidism, and osteoporosis. The infectious diseases mentioned include at least one of the following: AIDS, hepatitis B, hepatitis C, tuberculosis, malaria, dengue fever, cholera, influenza, and pneumonia.

14. A medicament for the prevention and / or treatment of RNA-related diseases, comprising at least one of the biological materials of claim 9 or the RNA editing reagent of claim 10 or 11.

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