RNA editing method and reagent

Abstract

The invention relates to the technical field of nucleic acid editing, in particular to an RNA editing method and reagent. According to the method provided by the invention, target RNA molecules are edited by using a III type CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) complex and a raised ring structure which introduces at least one basic group on gRNA. According to the method, the characteristic of a cutting rule based on six nucleotides can be bypassed, so that RNA base cutting and connection of a non-6nt multiple are realized.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid editing technology, and in particular to methods and reagents for RNA editing. Background Art

[0002] Precision nucleic acid editing technologies, both at the DNA and RNA levels, are of great value to biological research and treatment. CRISPR RNA-guided endonucleases have enabled programmable DNA cutting and promoted the development of new therapies. The first generation of CRISPR genome editing used the Cas9 nuclease to create double-stranded DNA breaks, which were then edited at the break site by cellular repair. Although DNA editing has the potential to cure genetic diseases, it can cause unexpected changes in the genome and activate cellular stress responses that can be toxic. In contrast, RNA editing technology is able to regulate biological functions by changing the cell's gene expression program without introducing heritable mutations in DNA.

[0003] CRISPR-mediated selective DNA binding or cleavage has revolutionized DNA editing, including base editing, prime editing, and manipulation of large DNA fragments. However, progress in RNA editing tools has lagged behind that of DNA editing. Cas13 is a type VI CRISPR RNA-guided endoribonuclease that has been used to knock down cellular transcripts, but its target recognition also activates non-sequence-specific nuclease activity and degrades non-target RNA. Nuclease-inactive mutants of Cas13 (dCas13) have been applied to induce exon skipping, trans-splicing, or delivery of specific base editors to achieve conversions of adenosine to inosine or cytosine to uracil. However, RNA programmable deletions introduced by DNA editors have not been described in a similar manner.

[0004] To meet the need for RNA versatility and ease of manipulation, Nemudraia developed a technology for sequence-specific RNA editing in living cells. The technology uses the type III CRISPR system to programmably cut the target RNA. Unlike Cas13, which has non-sequence-specific nuclease activity, the type III CRISPR system only cuts the target RNA in units of 6-nt intervals. Nemudraia used this precise cutting activity to excise 6-nt interval fragments from human transcripts and showed that the resulting RNA fragments can be repaired.

[0005] The current type III CRISPR system relies on 6-nt intervals for RNA shearing. When applied to the CDS region, it can only produce in-frame deletions, so it cannot repair frameshift mutations, and is mainly suitable for repairing nonsense mutations. The use of alternative tools such as ADAR base editing, engineered tRNA, and programmable RNA pseudouridylation can also achieve similar nonsense mutation repair at the RNA level. This fixed 6-nt interval limits its ability to precisely regulate genetic information, and it is impossible to perform non-6-nt interval shearing to adjust the open reading frame or repair specific types of genetic mutations, thus limiting the widespread application of this tool. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method and reagent for RNA editing based on the type III CRISPR system, bypassing the characteristics of the cutting rule based on 6 nucleotides, in order to cut sequences that are not integer multiples of 6 nucleotides.

[0007] The RNA editing method provided by the present invention comprises: editing a target RNA molecule using a type III CRISPR system and gRNA;

[0008] The gRNA targets the target RNA and introduces a protruding loop of at least 1 base at different positions.

[0009] The method provided by the present invention is a selective cutting and intramolecular splicing technology of RNA, referred to as SCISSOR. The method is based on the principle that the type III CRISPR system calculates the base length in the gRNA according to the 5' rule, and then cuts the target RNA at 6-nt intervals in the opposite position. By adding or deleting bases during the engineering gRNA design, it produces a raised ring in the target binding region (the raised ring is located on the target RNA side, or on the gRNA side), thereby allowing non-6-nt multiple length shearing on the RNA molecule, thereby providing greater flexibility and being able to accurately regulate the editing of RNA molecules. This is particularly important for applications that require fine adjustment of the open reading frame or repair of specific genetic mutations.

[0010] In the present invention, the sequence of the gRNA is reverse complementary to the target RNA, and its total length is 20 to 100 nt. Experiments show that the editing efficiency increases with the increase of gRNA length, and the optimal editing efficiency is obtained in 50-nt and 56-nt gRNAs.

[0011] Preferably, the length is 30 to 60 nt, more preferably, the length is 38 to 56 nt. For example, in an embodiment of the present invention, the length of the gRNA is 38 nt, 44 nt, 50 nt or 56 nt.

[0012] In the present 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.

[0013] In the present invention, the gRNA is a sequence that adds or deletes one or more bases in the reverse complementary sequence of the target RNA.

[0014] Preferably, the base added or deleted by the gRNA relative to the target RNA is located between the 3rd and 4th bases in the cutting window.

[0015] As a feasible example, the deleted or added base is located at a distance of 1 to 25 nt from the 5' end, preferably, it is 10 to 25 nt from the 5' end; preferably, it is 10 to 20 nt from the 5' end; more preferably, it is 10 to 15 nt, or 12 to 17 nt, 15 to 20 nt, or 17 to 22 nt from the 5' end. For example, in the gRNA, the base deleted or added relative to the target RNA is 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.

[0016] In other cases, the deleted or added base is located at a distance of 1 to 25 nt from the 3' end, preferably, it is 10 to 25 nt from the 3' end; preferably, it is 10 to 20 nt from the 3' end; more preferably, it is 10 to 15 nt, or 12 to 17 nt, 15 to 20 nt, or 17 to 22 nt from the 3' end. For example, in the gRNA, the base deleted or added relative to the target RNA is 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.

[0017] In the present invention, the bases added or deleted by the gRNA relative to the target RNA are located within the cutting window of the type III CRISPR system, such as the Csm complex or the Cas7-11 system.

[0018] The Csm complex starts from the 5' end of the gRNA, and every 6 bases are a cutting window. The present invention verifies the position of the raised loop, and RNA cutting can be achieved regardless of where it is located in the cutting window, but when the raised loop is located in the middle of the cutting window, its editing efficiency is the highest. Preferably, the base added or deleted by the gRNA relative to the target RNA is located between the 3rd and 4th bases in the cutting window.

[0019] In the present invention, in order to achieve the cutting of the target RNA with a length that is not a multiple of 6-nt, the gRNA increases or loses 3n+1 or 3n+2 bases relative to the target RNA, and 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 or 1 or 2. In an embodiment of the present invention, the gRNA increases or loses 1nt, 2nt, 4nt, 5nt, 7nt, 8nt or 10nt relative to the target RNA.

[0020] In the present invention, as another feasible scheme, RNA is flexibly edited, and the gRNA increases or loses 3 to 60 bases relative to the target RNA. By designing gRNA, a 3-nt to 30-nt raised loop is introduced, which can also knock down the target RNA and has good editing efficiency. In an embodiment of the present invention, the gRNA increases or loses 3 to 30 bases relative to the target RNA, and preferably, the gRNA increases or loses 3nt, 6nt, 9nt, 18nt, 24nt or 30nt relative to the target RNA.

[0021] The present invention verifies that the raised loop is located on the gRNA side or on the target RNA side. Preferably, the raised loop is located on the target RNA side, which is more conducive to improving the editing efficiency. Therefore, the gRNA lacks at least one base relative to the target RNA. Moreover, the effect of lacking 1nt is better than that of 2nt.

[0022] The present invention also verifies the gRNA with mismatches. In the embodiment, the mismatch occurs in the part of the gRNA other than the missing and / or added bases. The results show that even if some bases are mismatched, a good editing effect can still be achieved. However, there is a negative correlation between the number of mismatches and the knockout efficiency. Preferably, in the gRNA, the part other than the missing and / or added bases has a matching rate with the target RNA of not less than 30%. In a specific embodiment, in a gRNA with a length of 38nt, the number of mismatched bases does not exceed 6, preferably, does not exceed 5, does not exceed 4, does not exceed 3, does not exceed 2 or 1.

[0023] The type III CRISPR system Csm complex includes: Csm1 subunit, Csm2 subunit, Csm3 subunit, Csm4 subunit and Csm5 subunit; preferably, the amino acid sequence of Csm is shown in any one of SEQ ID NOs: 104 to 108.

[0024] The type III CRISPR Cas7-11 system comprises a single Cas7-11 protein. Preferably, the amino acid sequence of the Cas7-11 system is as shown in SEQ ID NO:109.

[0025] The present invention does not limit the proteins in the type III CRISPR system, and any protein with similar functions can be used to implement the scheme of the present invention.

[0026] In the present invention, the type III CRISPR system includes Cas7 protein, Cas10 protein, Csm protein complex or Cmr protein complex. For example, the Csm protein complex includes at least one of Csm1 subunit, Csm2 subunit, Csm3 subunit, Csm4 subunit, Csm5 subunit and Csm6 subunit, and the Cmr protein complex includes at least one of Cmr1 subunit, Cmr2 subunit, Cmr3 subunit, Cmr4 subunit, Cmr5 subunit and Cmr6 subunit.

[0027] As a feasibility case, the type III CRISPR system is a Csm complex, and the Csm complex includes: a Csm1 subunit, a Csm2 subunit, a Csm3 subunit, a Csm4 subunit and a Csm5 subunit; preferably, the amino acid sequence of the Csm is shown in any one of SEQ ID NOs: 104 to 108;

[0028] In another case, the type III CRISPR Cas7-11 system comprises a single Cas7-11 protein; preferably, the amino acid sequence of the Cas7-11 system is as shown in SEQ ID NO:109.

[0029] Specifically, the RNA editing method described in 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.

[0030] The RNA editing method provided by the present invention has a wide range of applications in RNA editing of organisms. Preferably, the cell is a eukaryotic cell. In the present invention, the cell is an animal cell, a plant cell and / or a microbial cell. For example, it is a cell, embryo and / or tissue from an animal. Or, for example, it is a cell, tissue, seed or embryo from a plant. Or, for example, it is a cell or spore from a eukaryotic microorganism. In other embodiments, the organism is a prokaryote, which is a cell of a prokaryotic microorganism. In an embodiment of the present invention, human cells were used as subjects for efficacy verification, but in fact, the method of the present invention can achieve good RNA editing effects on a variety of biological cells.

[0031] As a feasible example, the animal is a human, a primate, a rodent, an even-toed ungulate, an avian, a cat and / or a canine. The plant is an algae, a moss, a pteridophyte, an angiosperm and / or a gymnosperm. For example, it is a cyanobacteria, a green algae, a spirulina, a rice, a soybean, a wheat, an oat, a corn, a sorghum, an apple, a pear, a peach, a cabbage, a radish, a carrot, a tobacco or an Arabidopsis. The microorganism is a yeast, an Escherichia coli, a Bacillus thuringiensis, a Lactobacillus or a Bifidobacterium.

[0032] In the present invention, the type of the target RNA is not limited, and the targeted RNA is selected from at least one of pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA), small nuclear RNA (snRNA), microinterfering RNA (miRNA) and Piwi interacting RNA (piRNA). In an embodiment of the present invention, the target RNA is RNA of a reporter gene and / or disease-related RNA. According to the method described in the present invention, any RNA can be edited. As a feasibility case, the target RNA molecule is a GFP reporter gene, a coding gene of Nano-Luc luciferase and / or endogenous transcripts PARK7, HEXA, RNF43, TCERG1, CKB. In a specific embodiment of the present invention, the gRNA has a nucleic acid sequence shown in any one of SEQ ID NO: 1 to 103.

[0033] The present invention also provides the gRNA as described in the RNA editing method described above.

[0034] Furthermore, the present invention also provides a composition comprising the gRNA as described above and a protein complex of the type III CRISPR system.

[0035] Furthermore, the present invention also provides a plasmid vector, which includes the gRNA as described above; or includes the gRNA as described above and a protein complex of the type III CRISPR system.

[0036] The plasmid vector of the present invention may include only gRNA, or may include a protein complex of gRNA and a type III CRISPR system, which is not limited by the present invention. Preferably, the plasmid vector of the present 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).

[0037] The present invention also provides a host transformed or transfected with the plasmid vector as described above.

[0038] The host described in the present invention is used for the storage or transfection of the plasmid vector as described above, and the present invention is not limited to this. For example, the host can be Escherichia coli, yeast, 293T cells, etc.

[0039] Furthermore, the present invention also provides an RNA editing reagent, which includes at least one of the following:

[0040] i) gRNA as described above;

[0041] ii), a composition as described above;

[0042] iii), a plasmid vector as described above;

[0043] iv) A host cell as described above.

[0044] In an embodiment of the present invention, the RNA editing reagent further includes reagents, adjuvants or auxiliary materials required for transfection and / or transformation;

[0045] The reagents include electroporation transfection reagents, ion transfection reagents and / or liposome transfection reagents; for example, the electroporation transfection reagents include trypsin, PBS buffer, sodium chloride, potassium chloride, lactic acid, acetic acid, Tween, poloxamer, and span; the ion transfection reagents include calcium phosphate transfection reagents; the liposome transfection reagents include 3000, Lipofectamine RNAiMAX, Lipo8000, etc.

[0046] The adjuvant includes: at least one of cytokines (such as 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, etc., and other cytokines such as TNF and GM-CSF, etc.), co-stimulatory molecules (such as B7-1 and B7-2), emulsified adjuvants (Titer MaxGold (TMG)), Freund's adjuvant, saponin, and monophosphate lipids.

[0047] The auxiliary materials include polyethylene glycol and / or polylysine.

[0048] Furthermore, the present invention also provides at least one of the following uses in the preparation of a method for preventing and / or treating RNA-related diseases:

[0049] i) gRNA as described above;

[0050] ii), a composition as described above;

[0051] iii), a plasmid vector as described above;

[0052] iv), a host cell as described above;

[0053] v) RNA editing reagents as described above.

[0054] In the present invention, the RNA-related diseases include tumors, nervous system diseases, immune system diseases, metabolic diseases and / or infectious diseases.

[0055] For example, the tumor includes malignant tumors and benign tumors, 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 nervous system 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; the infectious disease includes at least one of AIDS, hepatitis B, hepatitis C, tuberculosis, malaria, dengue fever, cholera, influenza, and pneumonia.

[0056] Furthermore, the present invention also provides a drug for preventing and / or treating RNA-related diseases, comprising at least one of the following:

[0057] i) gRNA as described above;

[0058] ii), a composition as described above;

[0059] iii), a plasmid vector as described above;

[0060] iv), a host cell as described above;

[0061] v) RNA editing reagents as described above.

[0062] Furthermore, the present invention also provides a method for preventing and / or treating RNA-related diseases, which comprises administering the above-mentioned drug to a subject.

[0063] The method provided by the present invention uses a type III CRISPR system and a raised loop structure that introduces at least one base on the gRNA to edit the target RNA molecule. The method can bypass the characteristics of the 6-nucleotide cutting rule, thereby achieving RNA base cutting and connection that is not a multiple of 6nt. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1Figure 1 shows the design principle of SCISSOR, which uses type III CRISPR systems, such as Csm complexes and engineered gRNAs to flexibly excise RNA, where:

[0065] The top diagram shows the Csm-mediated RNA cleavage model. The gRNA associated with the Csm complex can recognize the target RNA with a complementary sequence, which forms a gRNA-target RNA duplex. The Csm3 subunit cleaves the target RNA at intervals of 6 nt. In the duplex structure, the gRNA chain is on the top and the target RNA chain is on the bottom.

[0066] The bottom diagram shows that the addition of a 1-nt raised loop in the target RNA or gRNA can lead to cleavage of the target RNA by the Csm3 subunit with 7-nt or 5-nt;

[0067] Figure 2 The following is a diagram showing the construction of the SCISSOR reporting system:

[0068] (A) Top: Schematic diagram of three types of stop-GFP reporter genes; Bottom: Definition of editing modes;

[0069] (B) 48 hours after transfection, cells were imaged using a fluorescence microscope; no GFP signal was observed for the three types of stop-GFP reporter genes;

[0070] (C) shows that 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 gene;

[0071] (D) Schematic diagram showing the SCISSOR method for repairing frameshifted GFP;

[0072] (E) shows a proposed method for introducing a 1-nt bulge 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 bulge loops opposite to positions 1 to 6 of the second window are shown as examples; TL, gRNA with bulge loop introduced on the target side;

[0073] Figure 3 This demonstrates the validation of SCISSOR on a reporting system where:

[0074] (A) shows the relative GFP RNA expression level in HEK293T cells co-transfected with a GFP reporter gene and a Csm complex expressing a gRNA with a 1-nt target bulge loop; qPCR signals were normalized to the puromycin gene expressed by the GFP reporter plasmid, and the non-targeting 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 a non-targeting gRNA; note that due to sequence composition, in window 2, TL1 = TL2, TL5 = TL6; in window 3, TL2 = TL3;

[0075] (B) Editing efficiency of samples in (A) quantified by targeted RNA-seq; error bars represent the mean standard deviation of three biological replicates;

[0076] (C) Target deletion patterns of engineered gRNAs that introduced a 1-nt target bulge loop are shown; the deletion frequency of each pattern was quantified as the average of three biological replicates; diagonal stripes indicate deletions that include the target window; Del, deletion; p, position;

[0077] (D) Same as in (A), but the engineered gRNA introduced a 1-nt gRNA bulge loop;

[0078] (E) Same as in (B), but the engineered gRNA introduced a 1-nt gRNA bulge loop;

[0079] (F) Same as in (C), but the engineered gRNA introduced a 1-nt gRNA bulge loop;

[0080] (G) Proposed method to introduce a 2-nt bulge loop into the target to generate an 8-nt cleavage in the target RNA;

[0081] (H) Same as in (A), but engineered gRNAs introduced a 2-nt target bulge loop;

[0082] (I) Same as in (B), but engineered gRNAs introduced a 2-nt target bulge loop;

[0083] (J) Same as in (C), but engineered gRNAs introduced a 2-nt target bulge loop;

[0084] (K) Relative GFP RNA expression levels in HEK293T cells co-transfected with a GFP reporter and a Csm complex expressing a gRNA with a 2-nt target bulge loop; error bars represent the mean standard deviation of three biological replicates; note that due to sequence composition, in window 2, TL2 = TL3;

[0085] Figure 4Schematic diagram of introducing bulge loops of different lengths into gRNA, where:

[0086] (AB) shows the cutting mode of introducing raised rings of different lengths into the target;

[0087] (C) Relative GFP RNA expression levels in HEK293T cells co-transfected with a GFP reporter gene and Csm complexes expressing gRNAs with different lengths of target bulge loops are shown; error bars represent the mean standard deviation of three biological replicates;

[0088] (D) Editing efficiency of samples in (C) quantified by targeted RNA-seq; error bars represent the mean standard deviation of three biological replicates;

[0089] (E) Deep sequencing results showing the use of engineered gRNAs to introduce raised loops of different lengths into the target;

[0090] (F) shows that the gRNA with a large bulge ring restored the protein expression of the stop GFP reporter gene;

[0091] Figure 5 The effect after extending the gRNA is shown, where:

[0092] (A) shows the design of gRNAs with extended length;

[0093] (B) Relative GFP RNA expression levels in HEK293T cells co-transfected with a GFP reporter gene and a Csm complex expressing an extended length gRNA; error bars represent the mean standard deviation of three biological replicates;

[0094] (C) Editing efficiency of samples in (B) quantified by targeted RNA-seq; error bars represent the mean standard deviation of three biological replicates;

[0095] Figure 6 The effect of incorporating a 5' mismatch into the gRNA is shown, where:

[0096] (A) shows the design of incorporating 5' mismatches between gRNA and target mRNA;

[0097] (B) Relative GFP RNA expression levels in HEK293T cells co-transfected with a GFP reporter gene and Csm complexes expressing gRNAs; error bars represent the mean standard deviation of three biological replicates.

[0098] (C) Editing efficiency of samples in (B) quantified by targeted RNA-seq; error bars represent the mean standard deviation of three biological replicates;

[0099] (D) shows the deep sequencing results of engineered gRNAs with 5' mismatches introduced in different windows, and each row represents a biological replicate;

[0100] Figure 7 The results show that the Cas7-11 system with engineered gRNAs can perform flexible cleavage, where:

[0101] (A) Schematic diagram showing GFP-stop-Luc-fs1 and GFP-stop-Luc-fs2 reporter genes; GFP was used to confirm transfection efficiency;

[0102] (B) shows the relative reporter gene expression levels of HEK293T cells co-transfected with reporter genes and gRNAs expressing Cas7-11; error bars represent the mean standard deviation of three biological replicates;

[0103] (C) Editing efficiency of samples in (B) quantified by targeted RNA-seq; error bars represent the mean standard deviation of three biological replicates;

[0104] (D) Representative examples of RNA editing results for selected gRNAs are shown; for each gRNA, the top three most common RNA editing results are shown; the dashed lines indicate the positions of RNA breaks; the deletion frequency is quantified as the mean standard deviation of three biological replicates;

[0105] Figure 8 The figure shows the flexible cleavage of endogenous targets mediated by SCISSOR, where:

[0106] (A) shows gRNAs designed to introduce flexible excision of PARK7 transcripts; target bulge loops were introduced at different positions in window 2;

[0107] (B) PARK7 RNA expression levels in HEK293T cells transfected with Csm complexes 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;

[0108] (C) PARK7 editing efficiency, quantified by targeted RNA-seq; error bars represent the mean standard deviation of three biological replicates;

[0109] (D) shows the targeted deletion patterns of engineered gRNAs. The deletion frequency of each pattern is the average of three biological replicates;

[0110] Fig. 9 Figure 2 shows the flexible cleavage of the endogenous target CKB mediated by SCISSOR, where:

[0111] (A) gRNAs were designed to introduce flexible excision of CKB transcripts. A bulge loop was introduced in the middle of window 3, and five gRNAs were designed;

[0112] (B) Expression levels of CKB RNA in HEK293T cells transfected with Csm complexes expressing gRNAs, error bars represent the mean standard deviation of three biological replicates;

[0113] (C) CKB editing efficiency, quantified by targeted RNA-seq, error bars represent the mean standard deviation of three biological replicates;

[0114] (D) Targeted deletion patterns of engineered gRNAs. The deletion frequency of each pattern is the average of three biological replicates.

[0115] Fig.10 SCISSOR-mediated repair of pathogenic frameshift mutations, where:

[0116] (A) Top: Schematic diagram of the HEXA4bp insertion mutation and the introduction of a premature stop codon caused by this mutation;

[0117] Bottom: gRNA design to repair frameshift mutations. We introduced a raised loop in the middle of window 2 to design 5 gRNAs.

[0118] (B) RNA expression levels of HEXA reporter gene in HEK293T cells, HEK293T cells were co-transfected with HEXA reporter gene and Csm complex expressing 1-nt target bulge loop gRNA, error bars represent the mean standard deviation of three biological replicates;

[0119] (C) HEXA editing efficiency, quantified by targeted RNA-seq, error bars represent the mean standard deviation of three biological replicates;

[0120] (D) Targeted deletion patterns of engineered gRNAs. The deletion frequency of each pattern is the average of three biological replicates.

[0121] (E) Western blot showing HEXA reporter protein obtained by SCISSOR editing using gRNA H1, NT, non-targeting gRNA, GAPDH as internal reference gene, and HA-tag antibody to detect HEXA-HA fusion protein;

[0122] (F) Top: Schematic diagram of HEK293T cells generating HEXA mutations using the CRISPR / Cas9 system;

[0123] Bottom: Sanger results indicate successful 4-bp insertion in mutant cells;

[0124] (G) RNA expression levels of HEXA endogenous transcripts in 1278ins4 mutant HEK293T cells transfected with Csm complex expressing gRNAH1, error bars represent the mean standard deviation of three biological replicates;

[0125] (H) Editing efficiency of HEXA endogenous transcripts, error bars represent the mean standard deviation of three biological replicates;

[0126] (I) Same as (D), but targeting HEXA endogenous transcripts;

[0127] Fig.11 It was shown that SCISSOR can produce immunogenic frameshift multiple epitopes, among which:

[0128] Upper panel: Schematic diagram of the reporter plasmid for detecting SCISSOR-edited transcripts at the protein level via HA-tag;

[0129] Bottom: For each gene, we introduced a raised loop in the middle of window 2 to design 5 gRNAs;

[0130] (B) RNA expression levels of TCERG1 and RNF43 reporter plasmids in HEK293T cells, error bars represent the mean standard deviation of three biological replicates;

[0131] (C) Editing efficiency of TCERG1 and RNF43 reporter plasmids quantified by targeted RNA-seq, error bars represent the mean standard deviation of three biological replicates;

[0132] (D) Deletion patterns of engineered gRNA targeting TCERG1 reporter plasmid, and the deletion frequency of each pattern was quantified as the average of three biological replicates;

[0133] (E) Same as (D) but for the RNF43 reporter plasmid;

[0134] (F) Western blot showing the expression of frameshift multi-epitopes from TCERG1 and RNF43 transcripts, PTC, positive control; NT, non-targeting gRNA; FS, 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 reference protein, and HA-tag antibody is used to detect frameshift multi-epitope-HA fusion protein;

[0135] (G) RNA expression levels of TCERG1 and RNF43 endogenous transcripts in HEK293T cells transfected with Csm complex expressing gRNAs T2 and R3, error bars represent the mean standard deviation of three biological replicates;

[0136] (H) Editing efficiency of TCERG1 and RNF43 endogenous transcripts, error bars represent the mean standard deviation of three biological replicates;

[0137] (I) Same as (D), but targeting TCERG1 and RNF43 endogenous transcripts;

[0138] (J) Same as (G), but for the U2OS cell line;

[0139] (K) Same as (H), but for the U2OS cell line;

[0140] (L) Same as (I) but for the U2OS cell line. DETAILED DESCRIPTION

[0141] The present invention provides methods and reagents for RNA editing, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0142] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0143] In the present invention, "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0144] In the present invention, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of a single item or multiple items.

[0145] In the present invention, "include", "comprising" and "having" are used interchangeably, and are intended to indicate the inclusiveness of the scheme, meaning that the scheme may have other elements besides the listed elements. At the same time, it should be understood that the descriptions of "include", "comprising" and "having" used in this article also provide "consisting of..." schemes.

[0146] In the present invention, "RNA" stands for ribonucleic acid, which is a long chain molecule formed by condensation of ribonucleotides through phosphodiester bonds. In the present invention, the bases of RNA are mainly A adenine, G guanine, C cytosine, and U uracil. Generally, there are three major types of RNA molecules, namely 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.

[0147] In the present invention, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) refers to regularly spaced clustered short palindromic repeats. CRISPR is a natural immune system, and the CRISPR-Cas system can be used for genome editing, by complementary pairing of guide RNA with target DNA or RNA sequences, thereby achieving precise editing of specific sites on the genome.

[0148] In the present invention, SCISSOR (Selective Cleavage and Intramolecular Stitches of RNA) refers to the selective cleavage and intramolecular splicing of RNA.

[0149] In the present invention, CDS refers to the coding region of a gene, also known as a coding sequence, which is the portion of the DNA or RNA of a gene that encodes a protein.

[0150] In the present invention, inDel refers to Insertions and Deletions, which insert one or more DNA bases into the genome, or delete one or more DNA bases in the genome.

[0151] In the present invention, gRNA refers to guide RNA (gRNA) or single guide RNA (sgRNA), which is a short sequence of RNA that can serve as a guide for Cas9-nuclease or other Cas proteins that can cut double-stranded DNA and can thus be used for gene editing.

[0152] In the present invention, frameshift mutation refers to a gene mutation caused by the insertion or deletion (indel) of nucleotides that are not multiples of three. Since the codons are composed of three nucleotides during gene expression, such insertion or deletion will change the reading frame, thereby causing the mRNA to translate into a completely different protein after this mutation. The more upstream the frameshift mutation occurs in the open reading frame, the greater the impact on the protein.

[0153] In the present invention, nonsense mutation refers to a specific codon for an amino acid in a gene that is converted to a termination codon for polypeptide chain synthesis due to base substitution or frameshift mutation, resulting in interruption of polypeptide chain synthesis and production of a protein product without function.

[0154] In the present invention, an open reading frame refers to an open reading frame, which refers to a sequence that does not contain a stop codon in a given reading frame. This sequence is a part of the genome of an individual organism that may serve as a protein coding sequence.

[0155] In the present invention, the cleavage window refers to starting from the 5' end of the gRNA of the Csm complex, with every 6 bases being a cleavage window.

[0156] In the present invention, "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects who already have a condition or disease, as well as subjects who are susceptible to a condition or disease or subjects who intend to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0157] In the present invention, "drug" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to a subject to whom the pharmaceutical composition is administered.

[0158] In the present invention, the "drug" can be preferably administered by injection, enteral administration, or topical 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 topical administration includes, but is not limited to, spray inhalation, nasal administration, buccal administration, vaginal administration, etc.

[0159] In the present invention, "subject" refers to an organism that receives treatment for a specific disease or condition as described in the present invention. Exemplarily, "subject" includes mammals that receive treatment for a disease or condition, and the mammals include bovines, equines, ovines, swine, canines, felines, rodents, primates, wherein the preferred mammal is a human, cat, dog or pig.

[0160] As used herein, an "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, is effective in preventing or alleviating a disease symptom or the progression of the disease, to a cell, tissue, or other subject. An "effective amount" also refers to an amount of a compound sufficient to relieve symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevent, or alleviate these conditions. When an active ingredient is administered to an individual alone, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0161] In the present invention, the drug can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. Preferably, the dosage form of the drug depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, a sterile injection solution can be prepared by the following method: incorporating the necessary dose of the gRNA of the present invention into an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, a sterile injection solution can be prepared as a sterile lyophilized powder (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), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0162] In the present invention, "cancer" and "tumor" are not mutually exclusive. "Cancer" refers to or describes a physiological condition in mammals that is typically 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 precancerous and cancerous cells and tissues. More specifically, the tumor or tumor refers to a tumor or tumor associated with RNA modification.

[0163] In the present invention, the design and modification of gRNA based on CRISPR-Csm: by introducing 1nt, 2nt and multiple base nt length deletions at specific positions of gRNA, non-6nt cutting and ligation on mRNA is achieved. This new type of RNA editing can have a wide range of application prospects, such as treating diseases caused by certain frameshift mutations, or introducing frameshift mutations for tumor treatment. Furthermore, a longer base pairing sequence is introduced at one or both ends of the gRNA to enhance the binding to the target, thereby enhancing the efficiency of cutting and ligation; further, by introducing mismatched bases at the target cutting site, the cutting at this position is reduced or eliminated, further improving the purity of the product. The design principle and mechanism of the SCISSOR technology of the present invention are: Structural analysis shows that the type III CRISPR complex calculates the base length in the gRNA according to the 5' rule, and then cuts the target RNA at 6-nt intervals at the opposite position. Therefore, we believe that by designing the gRNA to produce a raised loop in the gRNA and target binding region, it is possible to enable the type III CRISPR system to cut the target RNA in cells at a length that is not a multiple of 6-nt ( Figure 1 ).

[0164] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0165] The present invention will be further described below in conjunction with embodiments:

[0166] Example

[0167] 1. Experimental steps

[0168] Cell culture

[0169] HEK293T cell line and U2OS cell line were purchased from the Cell Bank of the Center for Type Culture Collection of the Chinese Academy of Sciences (CBTCCCAS). Their identity was verified by CBTCCCAS using short tandem repeat (STR) analysis, which included the simultaneous amplification of 17 STR markers plus amelogenin to confirm the identity of the cells. Both cell lines were maintained in DMEM (Gibco) supplemented with 10% FBS (HyClone). Mycoplasma contamination has been checked by CBTCCCAS, and mycoplasma has been routinely tested by PCR detection of conditioned medium.

[0170] 1.2. Construction of type III CRISPR complex plasmids and their reporter plasmids

[0171] The gRNA fragments were synthesized by Suzhou Hesheng Biotechnology Co., Ltd. and subcloned into Csm plasmids (from pDAC627 (Addgene, Plasmid #195240)) or Cas 7-11gRNA plasmids (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, Luc luciferase, and HA-tag were integrated into the GFP gene using overlapping PCR.

[0172] To generate the reporter plasmids HEXA-HA (wild type) and HEXA-frameshift-HA (1278ins4), the wild type gene fragment was synthesized by Suzhou Synthetic Biotechnology Co., Ltd. Overlapping PCR was used to introduce the 1278ins4 mutation and retain introns 11-13.

[0173] 1.3. Plasmid transfection for qPCR and targeted RNA-seq experiments

[0174] HEK293T cells were seeded one day before the experiment and transfected when they reached 60%-80% confluence. Transfection was performed using Lipofectamine3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. For experiments editing the GFP reporter gene, 700ng of plasmid DNA encoding the Csm complex and gRNA or Cas7-11 and gRNA was co-transfected with 300ng of plasmid DNA encoding the GFP reporter gene into a 12-well plate. Cells were harvested 48 hours after transfection. For experiments editing HEXA and frameshift multi-epitope host reporter genes, 700ng of plasmid DNA encoding the Csm complex and gRNA was co-transfected with 300ng of plasmid DNA encoding the target gene into a 12-well plate. Cells were harvested 48 hours after transfection. For experiments editing endogenously expressed genes, 500ng of plasmid DNA encoding the Csm complex and gRNA was transfected into a 24-well plate. 24 hours after transfection, cells were detached and plated in 12-well plates in medium supplemented with puromycin (1 g / mL). Puromycin resistance selection Cells were harvested 72 hours later.

[0175] 1.4.qPCR

[0176] Total cellular RNA was extracted using TRIzol reagent and Direct-zol RNA kit (Zymo Research). RNA was treated with DNaseI and reverse transcribed using HiScript IIQ RT Super Mix (Vazyme) with random primers. Three PCRs were performed for each cDNA sample using ChamQ SYBR qPCR Master Mix (Vazyme). For quantification of the GFP reporter gene, a primer pair for amplifying aminoglycoside phosphotransferase in the reporter gene plasmid was used as an internal control. The same internal control was used for quantification of HEXA and frameshift multi-epitope reporter genes. For endogenous gene quantification, the housekeeping gene GAPDH was used as an internal control.

[0177] Targeted RNA-seq

[0178] 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 72°C for 1 minute. Next, the PCR product was purified with DNA Clean Beads (Vazyme) and dissolved in 10 μl of water. 1 μl of the purified product was amplified for 5 cycles using the same procedure as above using sequencing adapters. Finally, the PCR product was recovered and sequenced using the Zymoclean gel DNA recovery kit (ZymoResearch).

[0179] 1.6.Western blot

[0180] HEK293T cells were seeded in 6-well plates one day before the experiment. In the experiment of editing HEXA reporter gene, the experimental group was transfected with 1.2μg plasmid DNA encoding mutant HEXA reporter gene and 0.8μg plasmid DNA encoding Csm complex and gRNA, respectively. The negative control group was transfected with 1.2μg plasmid DNA encoding mutant HEXA reporter gene and 0.8μg plasmid DNA encoding Csm complex and non-targeting gRNA, respectively. The positive control group was transfected with 0.2μg plasmid DNA encoding wild-type HEXA reporter gene and 1.8μg plasmid DNA encoding Csm complex and non-targeting gRNA, respectively. For the experiment of editing TCERG1 and RNF43 reporter genes, the positive control group was transfected with 0.9μg and 0.2μg, respectively, and the experimental group was transfected with 1.8μg reporter gene.

[0181] All transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. After 48 hours, cells were rinsed with chilled 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. The cell lysate was 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% nonfat dry milk (Bio-Rad) at room temperature for 1 hour and incubated overnight at 4°C with a 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)-conjugated anti-mouse antibodies were used as secondary antibodies, and proteins were visualized using an Odyssey CLx Imager (LI-COR).

[0182] Generation of mutant cell lines

[0183] HEXA mutant cell lines were generated by CRISPR / cas9-induced knock-in using ssDNA templates. The sgRNA targeting the HEXA gene was cloned into lentiCRISPRv2 (Addgene plasmid #52961), and the ssDNA template was synthesized by Suzhou Synbiotechnologies. 500ng of sgRNA plasmid and 500ng of ssDNA were transfected in 12-well plates. After 48 hours, cells were selected with puromycin, and surviving cells were screened for integration by Sanger sequencing.

[0184] 1.8. Sequencing data analysis

[0185] First, Cutadapt (version 3.7) was used to trim low-quality bases (quality score < 20) and sequencing adapter sequences. A reference genome was established using STAR (version 2.7.10a). Reads were mapped to the genome using STAR using the following parameters: "--runThreadN 30 --outSAMprimaryFlag AllBestScore --alignEndsType EndToEnd --scoreDelOpen0 --scoreDelBase0".

[0186] The aligned BAM files were sorted using Samtools (version 1.6), and the editing levels were analyzed using a custom Python script. In brief, the deletion type of each read was identified by examining the CIGAR value located at the reference target gene. The editing level was calculated by determining the ratio of the number of reads that repaired or introduced frameshift mutations at the target site relative to the total number of reads.

[0187] Based on the above steps, the following experiments are performed:

[0188] 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, thereby generating 1-nt and 2-nt frameshifted GFP variants (stop-GFP-fs1 and stop-GFP-fs2) ( Figure 2 After transfecting these stop-GFP-fs1 and stop-GFP-fs2 plasmids into cells, we did not observe GFP signal ( Figure 2 In contrast, a plasmid with the same sequence but without the stop codon and deletion produced strong GFP fluorescence ( Figure 2 Next, we designed gRNAs to remove the stop codon and correct the frameshift mutation ( Figure 2 We designed gRNAs to introduce a 1-nt bulge in one of the windows, with the goal of achieving a 7-nt or 5-nt deletion in the target RNA. We selected a 32-nt gRNA that performed best and designed it to have a bulge opposite to positions 1 to 6 in the selected window ( Figure 2 E), the sequences of gRNAs are shown in Table 1, and are recorded as window2_1nt_TL1~6 and window3_1nt_TL1~6. The results showed that the editing effect of setting the raised loop between the 3rd and 4th positions was the best.

[0189] 2.2 The integrated plasmid expressing the Csm complex and gRNA was transfected into HEK293T cells together with the reporter plasmid, and the cells were collected 48 hours after transfection. We found that the engineered gRNA with a 1-nt raised loop on the target side resulted in a more than 90% reduction in target RNA ( Figure 3 The cleavage window is excised in 17–45% of transcripts, depending on the position of the raised loop ( Figure 3 Most importantly, all deletions spanning the cutting window result in the removal of exactly 7 nt within the window ( Figure 3 For the gRNA with a gRNA side bulge loop, which edits the stop-GFP-fs2 reporter gene, the target window is excised in 2%-35% of the transcripts ( Figure 3In addition, all deletions spanning the target window resulted in the removal of 5-nt within the window ( Figure 3 We also explored the possibility of rescuing the 2-nt frameshift by introducing a 2-nt target-side bulge loop in the second or third window ( Figure 3 This strategy successfully knocked out the target RNA ( Figure 3 Similarly, all deletions spanning the target window resulted in the removal of the 8-nt within the window ( Figure 3 IJ), which restored stopGFP-fs2 protein expression ( Figure 3 K in the middle). The sequences of the gRNAs with the bulge loop on the target gene are shown in Table 1, and are recorded as window2_2nt_TL1~6 and window3_2nt_TL1~6. The sequences of the gRNAs with the bulge loop on the gRNA are shown in Table 1, and are recorded as window2_2nt_GL1~6 and window3_2nt_GL1~6.

[0190] 2.3 Design of gRNAs targeting the frameshifted GFP reporter gene and introduction of bulged loops from 3-nt to 30-nt opposite position 4 of the third window (3-, 6-, 9-, 18-, 24-, and 30-nt, Figure 4 To ensure adequate binding of 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 ( Figure 4 C) and has high editing efficiency ( Figure 4 GFP signals were detected in all groups after editing ( Figure 4 (F). The sequence of gRNA is shown in Table 1 and is denoted as window3_(3 / 6 / 9 / 18 / 24 / / 30)nt_TL.

[0191] 2.4 Study on the design of the target side raised loop. First, we tried to increase the gRNA length. We extended the gRNA length in 6-nt units, ranging from 1 to 3 additional units ( Figure 5 Notably, both gRNAs showed significantly increased editing efficiency with increasing length, reaching peaks of 73% and 87% at 12-nt and 18-nt stretches ( Figure 5 The sequences of gRNAs are shown in Table 1 and are denoted as w3d2-3+(0 / 6 / 12 / 18) or w3d1-2+(0 / 6 / 12 / 18).

[0192] 2.5 Design a series of gRNAs with 5' mismatches ( Figure 6 We found a negative correlation between the number of mismatches and knockout efficiency ( Figure 6 Although the introduction of a 5-position mismatch effectively inhibits the cutting of the corresponding window, it also reduces the editing efficiency ( Figure 6 CD). The sequences of gRNAs are shown in Table 1 and are denoted as 1-(0 / 1 / 2 / 3)-PS or 2-(0 / 1 / 2 / 3)-PS.

[0193] 2.6 Cas7-11 with engineered gRNAs for flexible cleavage: Development of plasmids expressing GFP and Nano-Luc luciferase linked to the viral 2A peptide. We introduced a stop codon and 2-nt or 1-nt deletions before the start codon of Nano-Luc to generate 1-nt and 2-nt frameshifted Nano-Luc variants (GFP-stop-Luc-fs1 and GFP-stop-Luc-fs2) ( Figure 7 Next, we designed gRNAs to remove the stop codon and correct the frameshift. Plasmids expressing Cas7-11 and gRNA were co-transfected into HEK293T cells, and cells were collected 48 hours after transfection. We observed that all engineered gRNAs resulted in successful RNA knockout ( Figure 7 Deep sequencing of the target RNA amplicons confirmed that the majority of the samples contained RNA deletions that corrected the frameshift mutation ( Figure 7 CD). The sequences of gRNAs are shown in Table 1 and are denoted as Luc-TG (1 / 2 / 3 / 4 / 5)-1nt or Luc-TG (1 / 2 / 3 / 4 / 5)-2nt or Luc-GL (1 / 2 / 3 / 4 / 5)-1nt.

[0194] 2.7 SCISSOR-mediated flexible cleavage of endogenous targets: We targeted the endogenous transcript PARK7 for testing ( Figure 8 Middle A). qPCR showed that different gRNAs resulted in different levels of knockdown of PARK7 transcripts ( Figure 8 Middle B). Deep sequencing confirmed the presence of 7-nt and 8-nt deletions ( Figure 8 CD). The editing efficiency of gRNAs with 1-nt bulge loops was 8.3%, and that of gRNAs with 2-nt bulge loops was 1.4%. The sequences of gRNAs are shown in Table 1 and are denoted as P1-(1~5) or P2-(1~5).

[0195] 2.8 SCISSOR applied to endogenous transcripts CKB ( Fig. 9 Middle A). Positioning the raised loop at position 4 of the CKB gRNA, qPCR showed that different gRNAs resulted in different levels of knockdown of the CKB transcript ( Fig. 9The results showed that SCISSOR successfully achieved 7-nt and 8-nt deletions on CKB transcripts, with the best performing gRNAs having editing efficiencies of 4.7% and 4%, respectively. Fig. 9 Medium CD).

[0196] 2.9 Excision of pathogenic frameshift mutations in RNA by SCISSOR can restore protein expression:

[0197] To determine the therapeutic potential of SCISSOR to repair frameshift mutations, a clinically relevant HEXA gene frameshift mutation was targeted. This mutation is a 4 bp insertion (TATC,1278ins4) in exon 11 of the HEXA gene and is the most common mutation causing Tay-Sachs disease (TSD), accounting for approximately 80% of TSD cases. This mutation results in the generation of a premature stop codon downstream, and the mutant transcript is rapidly degraded through the nonsense-mediated messenger RNA (mRNA) degradation (NMD) pathway.

[0198] First, the HEXA-1278ins4 reporter plasmid was constructed, which encodes the HEXA minigene fused to a C-terminal HA tag ( Fig.10 Only the repaired transcripts can express the C-terminal HA-tag, which is convenient for detecting the repaired transcripts at the protein level. Five gRNAs with 1nt target protrusion loops were designed ( Fig.10 A). The reporter gene and the Csm complex expressing gRNA were co-transfected into HEK293T cells. Among them, gRNA H1 had the highest editing efficiency, reaching 53% ( Fig.10 Co-transfection of the reporter gene and the Csm complex expressing gRNAH1 successfully restored the C-terminus of the HEXA protein ( Fig.10 Middle E).

[0199] To further evaluate the ability of SCISSOR to correct this frameshift mutation in endogenous HEXA transcripts, a HEK293T cell line containing the 1278ins4 insertion was constructed using the CRISPR / Cas9 system ( Fig.10 Middle F). The efficiency of editing the mutant HEXA endogenous transcript using the Csm complex expressing gRNAH1 reached 56% ( Fig.10 Medium GI).

[0200] 2.10 SCISSOR can produce immunogenic frameshift poly-epitopes (fs-poly-epitopes)

[0201] Two frameshift polytopes that have been shown to be highly immunogenic in microsatellite instability tumors were selected for testing. These two frameshift polytopes are derived from frameshift mutations in the TCERG1 (CA>C, position 2861) and RNF43 (GT>T, position 1,975) genes, respectively.

[0202] First, TCERG1 and RNF43 reporter plasmids were generated. Each plasmid expressed GFP and flanking regions around the TCERG1 or RNF43 frameshift mutation site, and an HA-tag ( Fig.11 Only transcripts that produce a defined frameshift can produce frameshift polytopes and C-terminal HA-tags, thus facilitating detection of frameshift polytopes at the protein level. Five gRNAs with 1-nt target bulge loops were designed for each of the two genes ( Fig.11 (A). The reporter gene and the Csm complex expressing the gRNA were co-transfected into HEK293T cells. The best performing gRNAs achieved editing efficiencies of 32% and 9%, respectively ( Fig.11 BC), and successfully generated a frameshift ( Fig.11 Consistent with this, Western blot analysis confirmed that the edited transcripts produced frameshift polytopes ( Fig.11 Middle F).

[0203] Finally, SCISSOR was applied to endogenously expressed TCERG1 and RNF43 transcripts in HEK293T cells, achieving 30% and 19% editing efficiencies, respectively, using the best performing gRNAs identified above. Fig.11 We also applied SCISSOR to endogenously expressed TCERG1 and RNF43 transcripts in U2OS cells and achieved editing efficiencies of 7% and 6%, respectively, using the best performing gRNAs identified above ( Fig.11 Chinese JL).

[0204] Table 1 SCISSOR gRNA design

[0205]

[0206]

[0207]

[0208]

[0209] Table 2 CSM protein amino acid sequence

[0210]

[0211] Table 3 Cas7-11 protein amino acid sequence

[0212]

[0213]

[0214] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as 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 increases or deletes at least one base relative to the target RNA.

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

3. The RNA editing method according to claim 1, characterized in that 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; Preferably, the base added or deleted by the gRNA relative to the target RNA is located between the 3rd and 4th bases in the cutting window.

4. The RNA editing method according to claim 1, characterized in that The gRNA adds or deletes 3n+1 or 3n+2 bases relative to the target RNA, where n is an integer ≥0; Preferably, the gRNA increases or loses 1 to 60 bases relative to the target RNA; Preferably, the gRNA increases or decreases by 1nt, 2nt, 4nt, 5nt, 7nt, 8nt or 10nt 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 comprises a Csm family complex, a Cas7-11 family protein, or other types of type III CRISPR systems; Preferably, the type III CRISPR system is a Csm complex, and the Csm complex comprises: a Csm1 subunit, a Csm2 subunit, a Csm3 subunit, a Csm4 subunit and a Csm5 subunit; preferably, the amino acid sequence of the Csm is shown in any one of SEQ ID NOs: 104 to 108; Preferably, the type III CRISPRCas7-11 system comprises a single Cas7-11 protein; preferably, the amino acid sequence of the Cas7-11 system is as 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 comprises: transfecting a cell containing a target RNA with the gRNA and a type III CRISPR complex; Preferably, the cell is an animal cell, a plant cell and / or a microbial cell.

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

9. Biological materials, including any of the following: I), the gRNA described in any one of claims 1 to 8 of the RNA editing method; II), a composition comprising the gRNA and a type III CRISPR complex as described in any one of claims 1 to 8; III), a plasmid vector comprising the gRNA described in any one of claims 1 to 8; or comprising the gRNA described in any one of claims 1 to 8 and a type III CRISPR complex; IV), host, transform or transfect the plasmid vector described in III).

10. An RNA editing agent 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 auxiliary materials required for transfection and / or transformation; The reagents include electroporation transfection reagents, ion transfection reagents and / or liposome transfection reagents; The adjuvant comprises: at least one of cytokines, costimulatory molecules, emulsified adjuvants, Freund's adjuvant, saponin, monophosphoryl lipids, TLR agonists, CpG oligonucleotides, oligodeoxynucleotides and / or immunomodulatory proteins; The auxiliary materials include polyethylene glycol and / or polylysine.

12. Use of at least one of the biomaterials according to claim 9 or the RNA editing reagent according to claim 10 or 11 in the preparation of a method for preventing and / or treating RNA-related diseases.

13. The use according to claim 12, characterized in that: The RNA-related diseases include tumors, nervous system 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 nervous system 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; The infectious disease includes at least one of AIDS, hepatitis B, hepatitis C, tuberculosis, malaria, dengue fever, cholera, influenza, and pneumonia.

14. A drug for preventing and / or treating RNA-related diseases, comprising at least one of the biological materials according to claim 9 or the RNA editing agent according to claim 10 or 11.

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