Circular rna-based casdelta editing system

Abstract

The present application relates to the field of nucleic acid editing, in particular to a Casdelta editing system based on circular RNA. More specifically, the present application relates to a CRISPR / Cas system for editing a target gene, the system comprising: a Casdelta protein, a reverse transcriptase, a first circular RNA, a first guide nucleic acid, a second circular RNA and a second guide nucleic acid. The present application also relates to a combination of nucleic acid molecules encoding the CRISPR / Cas system, to expression vectors comprising the nucleic acid molecules, and to the use of the CRISPR / Cas system, the combination of nucleic acid molecules, the expression vectors for modifying a target sequence.

Description

Technical Field

[0001] This application relates to the field of nucleic acid editing, specifically to a Casδ-based nucleic acid editing system. More specifically, this application relates to a CRISPR / Cas system for editing target genes, said system comprising: a Casδ protein, a reverse transcriptase, a first circular RNA, a first guide nucleic acid, a second circular RNA, and a second guide nucleic acid. This application also relates to a combination of nucleic acid molecules encoding said CRISPR / Cas system, an expression vector containing said nucleic acid molecules, and the use of said CRISPR / Cas system, the combination of nucleic acid molecules, and the expression vector in modifying target sequences. Background Technology

[0002] Existing traditional guided editing systems mainly include the core PE system (PE1–PE6): PE1 is the initial guided editor, formed by fusing wild-type Moloney murine leukemia virus reverse transcriptase (MMLV-RT) with nCas9 (H840A), which has low editing efficiency; PE2, based on PE1, performs a five-amino acid mutation on MMLV-RT (D200N / L603W / T330P / T306K / W313F), significantly improving the thermostability and continuous synthesis capacity of RT, thus significantly improving editing efficiency; the PE3 system introduces an additional single-guide RNA (sgRNA) to PE2, which is used to create a nick on the unedited DNA strand, using the cell's endogenous mismatch repair mechanism to "stabilize" the edited information onto the non-edited strand. PE3b is designed so that the second nick is only recognized after editing is complete, further reducing the generation of byproducts (such as indels). The PE3 / PE3b strategy significantly improved editing efficiency, but the additional cuts also increased the risk of indel generation. Studies of the DNA mismatch repair (MMR) pathway revealed that MMR inhibits the efficiency of guided editing and promotes the generation of indel byproducts. To address this, PE4 co-expresses an MMR repressor protein (MLH1dn) on top of PE2; PE5, similarly incorporating MLH1dn into PE3, both inhibits the "repair" effect of MMR on the edited product and retains the efficiency advantages of the PE3 strategy. PE4 and PE5 exhibited higher editing efficiency and product purity at multiple sites. The PE6 system represents further optimization of the reverse transcriptase components. By introducing more directed evolution-derived mutations into MMLV-RT (such as optimizations related to the PEmax architecture), PE6 demonstrated superior performance compared to PE2–PE5 at various editing types and target sites, achieving a comprehensive improvement in editing efficiency and product purity. PEmax represents the result of systematic optimization of each component in the PE system, encompassing optimized nCas9 codons, dual-nucleus localization signals (NLS), and improved adapter design. PEmax significantly improves editing efficiency and is one of the most widely used and efficient PE system architectures. Many subsequent optimized systems (such as vPE) are also based on it.

[0003] CPE (Circular RNA-mediated Prime Editor) is a Cas12a-based guided editing system developed by Gao Caixia's team in 2024. The CPE system separates the target sequence from the pegRNA and circularizes the pegRNA containing PBS and RTT to enhance RNA stability. The circular pegRNA recruits reverse transcriptase through the MCP-MS2 recruitment system to perform reverse transcription, thereby writing the target edit into the genome.

[0004] Researchers constructed four CPE systems adapted to different application scenarios: nick enzyme-dependent (niCPE), nuclease-dependent (nuCPE), and their separate guided editing systems (sniCPE and snuCPE). Among them, niCPE and sniCPE showed a significant advantage, achieving 1.1–10.2 times higher editing efficiency at multiple targets compared to nuclease-based guided editing systems. To address the problem of multiple RNAs relying on multiple U6 promoters for expression, resulting in large vector molecules and difficult delivery, researchers utilized the pre-crRNA processing capability of Cas12a to tandemly express crRNA, circular RNA, and nick crRNA, driven by a single U6 promoter, constructing the "one-CPE" system. This system achieved synergistic expression of multiple RNAs and simplified the delivery system. In animal cells, one-CPE (especially one-niCPE3 and one-sniCPE3) achieved higher editing efficiency at multiple targets, outperforming traditional CPE and Cas9-PE2, while also exhibiting some multi-gene editing capabilities.

[0005] Compared to traditional Cas9-based guided editing systems, the CPE system has three core advantages: First, it significantly expands the target range, specifically recognizing T-enriched PAM sequences and precisely editing AT-enriched genomic regions that are difficult for the Cas9 system to cover; second, it possesses powerful multi-target editing capabilities, tandemly executing crRNAs targeting multiple targets within a single circular RNA to achieve simultaneous editing of up to four genes, providing an efficient tool for multi-gene function research; third, the split-type system greatly reduces delivery difficulty, as the two split elements can be delivered in vivo via dual AAV vectors, laying the foundation for the in vivo therapeutic application of guided editing technology.

[0006] Nevertheless, the current CPE system still has certain limitations. On the one hand, its editing efficiency fluctuates significantly across different targets; on the other hand, systematic research on complex editing types such as medium-scale insertions, replacements, and deletions remains insufficient.

[0007] Lai Jinsheng's team identified a novel family of type V CRISPR-Cas effector proteins from massive metagenomic data, named Casδ. Casδ maintains a significant evolutionary distance from all known Cas12 isoforms (such as Cas12a, b, f, n, etc.). This family contains three homologous proteins (Casδ-1, Casδ-2, Casδ-3), with a protein size of only 867-936 amino acids. Among them, Casδ-1 is approximately 24% and 10% smaller than Cas12a and Cas12i, respectively, making it a compact editing tool. Biochemical analysis shows that Casδ-1 requires only one mature crRNA to complete DNA recognition and cleavage, specifically recognizing adjacent 5'-RYR-3' protospacer motifs (R represents A or G, Y represents T or C), which is an important complement to the traditional type V Cas12 family's preference for T-rich PAMs. Systematic testing revealed that Casδ-1 possesses robust double-stranded DNA cleavage activity and target-dependent trans-cleavage activity, enabling efficient cross-species genome editing. It achieved an insertion / deletion mutation rate of up to 60% in human cells and, through stable transformation, yielded homozygous knockout lines in two important monocotyledonous crops (rice and maize). Structural and evolutionary analyses indicate that Casδ plays a key "transitional" role in the evolution of type V CRISPR systems: it may represent an intermediate stage in the evolution from the more primitive Cas12n, which required dual RNA guidance, to the more mature Cas12 effector, which only required single crRNA guidance. Casδ retains some ancestral features (such as the C-terminal domain), while its RuVC domain has been inserted and the NUC domain has been divided into two parts, reflecting the structural features of a more advanced effector.

[0008] To improve its editing efficiency in eukaryotic cells, Lai Jinsheng's team rationally designed and optimized the Casδ-1 protein. By enhancing the interaction between Casδ-1 and PAM double-stranded, crRNA, RNA-DNA heteroduplex, and single-stranded DNA substrates, they promoted the recognition and cleavage of target DNA by Casδ-1. After multiple rounds of screening, they successfully obtained an activity-enhanced variant, enCasδ, containing nine amino acid substitutions. In human cell lines, enCasδ showed 1.3 to 29.3 times greater editing activity at 10 test sites compared to the wild type, with an average editing efficiency of 54.6%. In stable maize transformation lines, the average editing efficiency at TS4 and PSY1 sites reached as high as 80%, and its overall editing performance was comparable to SpCas9 and other Cas12 nucleases. Furthermore, no off-target effects were detected in human cells and maize protoplasts based on randomly selected target site analysis. Summary of the Invention

[0009] The inventors of this application have developed a novel nucleic acid editing system based on the Casδ protein and the Dual-CPE strategy. This system enables precise editing of various types, including point mutations, fragment deletions, and substitutions, with ideal editing efficiency and the ability to achieve precise genome editing at the hundred-base-pair level. Thus, the inventors have completed this invention.

[0010] On one hand, the present invention provides a CRISPR / Cas system for editing target genes, comprising: (1) Casδ protein, the sequence of which is shown in SEQ ID NO: 9; (2) Reverse transcriptase; (3) The first circular RNA, which contains the first primer binding site (PBS) and the first reverse transcriptase template (RTT); (4) A first guide nucleic acid comprising a first crRNA, the first crRNA comprising a first guide sequence that hybridizes to a first target site on the positive strand of the target gene; (5) Second circular RNA, which contains a second primer binding site (PBS) and a second reverse transcriptase template (RTT); (6) A second guide nucleic acid comprising a second crRNA, the second crRNA comprising a second guide sequence that hybridizes to a second target site on the antisense strand of the target gene; Wherein, the first reverse transcriptase template and the second reverse transcriptase template contain complementary regions, which are editing templates; The first primer binding site hybridizes with the 3' primer sequence generated by the cleavage of the Casδ protein and the first crRNA on the antisense strand of the target gene, and the second primer binding site hybridizes with the 3' primer sequence generated by the cleavage of the Casδ protein and the second crRNA on the sense strand of the target gene. The first target and the second target are located at the 3' end of the motif adjacent to the original spacer sequence, and the motif adjacent to the original spacer sequence has the sequence shown in 5'-ATG-3'.

[0011] In some implementations, the guide nucleic acid is guide editing RNA.

[0012] In some implementations, the first RTT is directly connected to the first PBS, and the first RTT is located at the 5' end of the first PBS.

[0013] In some implementations, the second RTT is directly connected to the second PBS, and the second RTT is located at the 5' end of the second PBS.

[0014] In some embodiments, the first PBS hybridizes with a 3' primer sequence generated by cleaving the complex of the fusion protein and the first guide nucleic acid on the antisense strand of the target gene, wherein the 3' primer sequence is generated by cleaving the Casδ protein in the complex and the first crRNA on the antisense strand of the target gene.

[0015] In some embodiments, the second PBS hybridizes with a 3' primer sequence generated by cleavage of the complex formed by the fusion protein and the second guide nucleic acid on the positive strand of the target gene, wherein the 3' primer sequence is generated by cleavage of the Casδ protein in the complex and the second crRNA on the positive strand of the target gene.

[0016] In some embodiments, the distance between the cleavage formed by the Casδ protein and the first crRNA cutting the target gene and the cleavage formed by the Casδ protein and the second crRNA cutting the target gene is 20 bp to 100 bp.

[0017] In some implementations, the editing template includes embellishments, such as missing, replacement, insertion, inversion, repetition, or any combination thereof.

[0018] In some embodiments, the first RTT includes a first edit template sequence and a first homologous arm, the first homologous arm comprising one or more, located at the 5' end and / or 3' end of the first edit template sequence; the second RTT includes a second edit template sequence and a second homologous arm, the second homologous arm comprising one or more, located at the 5' end and / or 3' end of the second edit template sequence; the first edit template sequence and the second edit template sequence are complementary to each other to form an edit template.

[0019] In some embodiments, the reverse transcriptase further comprises an RNA aptamer-binding protein, which is directly or via a peptide linker to the RNA aptamer-binding protein; the first circular RNA and the second circular RNA further comprise one or more RNA aptamers; the RNA aptamers and the RNA aptamer-binding protein are capable of specific binding.

[0020] In some implementations, the RNA aptamer is MS2.

[0021] In some implementations, the RNA aptamer-binding protein is an MCP protein.

[0022] In some embodiments, the MS2 comprises a sequence as shown in SEQ ID NO: 27.

[0023] In some embodiments, the MCP protein comprises the sequence shown in SEQ ID NO: 11.

[0024] In some embodiments, the N-terminus and / or C-terminus of the Casδ protein and / or reverse transcriptase are linked to a nuclear localization signal sequence.

[0025] In some embodiments, the N-terminus and C-terminus of the Casδ protein are connected to nuclear localization signal sequences, and the N-terminus and C-terminus of the reverse transcriptase are connected to nuclear localization signal sequences. In some exemplary embodiments, the NLS sequence comprises the sequence shown in SEQ ID NO: 3.

[0026] In some embodiments, the Casδ protein has an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO: 9; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 amino acids) compared to the sequence shown in SEQ ID NO: 9; or (iii) A sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 9.

[0027] In some embodiments, the reverse transcriptase has an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO: 5; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 amino acids) compared to the sequence shown in SEQ ID NO: 5; or (iii) A sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 5.

[0028] In some embodiments, the peptide linker comprises a glycine residue (G) and / or a serine residue (S). In some exemplary embodiments, the peptide linker comprises a sequence as shown in SEQ ID NO: 7.

[0029] In some embodiments, the first crRNA and the second crRNA are further connected to a unidirectional repeat sequence at their 5' ends. In some embodiments, the unidirectional repeat sequence comprises the sequence shown in SEQ ID NO: 165.

[0030] On the other hand, the present invention provides a nucleic acid molecule combination comprising: (1) A first nucleic acid sequence comprising a nucleotide sequence encoding the Casδ protein of the system of the present invention; (2) A second nucleic acid sequence comprising a nucleotide sequence encoding the reverse transcriptase of the system of the present invention; (3) A third nucleic acid sequence comprising the nucleotide sequence of the first circular RNA for expressing the system of the present invention; (4) A fourth nucleic acid sequence comprising the nucleotide sequence of a first guide nucleic acid for expressing the system of the present invention; (5) A fifth nucleic acid sequence comprising the nucleotide sequence of a second circular RNA for expressing the system of the present invention; (6) A sixth nucleic acid sequence comprising a nucleotide sequence of a second guide nucleic acid for expressing the system of the present invention.

[0031] In some implementations, the first, second, third, fourth, fifth, and sixth nucleic acid sequences are each independently operatively ligated to a promoter at the 5' end and / or operatively ligated to a terminator at the 3' end.

[0032] In some embodiments, the first nucleic acid sequence comprises, from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence encoding an MCP, a nucleotide sequence encoding a peptide linker, a nucleotide sequence encoding a reverse transcriptase, a nucleotide sequence encoding a nuclear localization signal, and a terminator sequence; The second nucleic acid sequence, from the 5' end to the 3' end, includes: a promoter sequence, a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence encoding Casδ, a nucleotide sequence encoding a nuclear localization signal, and a terminator sequence; The third nucleic acid sequence, from the 5' end to the 3' end, comprises: a promoter sequence, a nucleotide sequence encoding a first 5' ribozyme, a nucleotide sequence for expressing a first 5' circumscribed arm, a nucleotide sequence for expressing a first reverse transcriptase template, a nucleotide sequence for expressing a first primer binding site, a nucleotide sequence for expressing a first 3' circumscribed arm, and a nucleotide sequence encoding a first 3' ribozyme; wherein the first 5' circumscribed arm and the first 3' circumscribed arm can be interconnected to form a first circular RNA; The fourth nucleic acid sequence, from the 5' end to the 3' end, includes: a nucleotide sequence for expressing the first crRNA and a terminator sequence; The fifth nucleic acid sequence, from the 5' end to the 3' end, includes: a promoter sequence, a nucleotide sequence encoding a second 5' ribozyme, a nucleotide sequence for expressing a second 5' circumscribed arm, a nucleotide sequence for expressing a second reverse transcriptase template, a nucleotide sequence for expressing a second primer binding site, a nucleotide sequence for expressing a second 3' circumscribed arm, and a nucleotide sequence encoding a second 3' ribozyme; wherein the second 5' circumscribed arm and the second 3' circumscribed arm can be interconnected to form a second circular RNA; The sixth nucleic acid sequence, from the 5' end to the 3' end, includes: a nucleotide sequence for expressing the second crRNA and a terminator sequence.

[0033] In some embodiments, the nucleic acid molecule combination comprises: (1) Nucleic acid molecule A, which contains the first nucleic acid sequence, wherein the nucleic acid molecule A comprises from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence encoding an MCP, a nucleotide sequence encoding a peptide linker, a nucleotide sequence encoding a reverse transcriptase, a nucleotide sequence encoding a nuclear localization signal, and a terminator sequence; (2) Nucleic acid molecule B, which contains the second nucleic acid sequence, wherein the nucleic acid molecule B comprises from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence encoding Casδ, a nucleotide sequence encoding a nuclear localization signal, and a terminator sequence; (3) Nucleic acid molecule C, which contains the third nucleic acid sequence and the fourth nucleic acid sequence, wherein the nucleic acid molecule C contains, from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence for expressing the first 5' ribozyme, a nucleotide sequence for expressing the first 5' circumscribed arm, a nucleotide sequence for expressing the first reverse transcriptase template, a nucleotide sequence for expressing the first primer binding site, a nucleotide sequence for expressing the first 3' circumscribed arm, a nucleotide sequence for expressing the first 3' ribozyme, a nucleotide sequence for expressing the first crRNA, and a terminator sequence; wherein the first 5' circumscribed arm and the first 3' circumscribed arm can be connected to each other to form a first circular RNA; (4) Nucleic acid molecule D, which contains the fifth nucleic acid sequence and the sixth nucleic acid sequence, wherein the nucleic acid molecule D contains, from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence for expressing the second 5' ribozyme, a nucleotide sequence for expressing the second 5' circumscribed arm, a nucleotide sequence for expressing the second reverse transcriptase template, a nucleotide sequence for expressing the second primer binding site, a nucleotide sequence for expressing the second 3' circumscribed arm, a nucleotide sequence for expressing the second 3' ribozyme, a nucleotide sequence for expressing the second crRNA, and a terminator sequence; wherein the second 5' circumscribed arm and the second 3' circumscribed arm can be connected to each other to form a second circular RNA.

[0034] On the other hand, the present invention provides an expression vector comprising the nucleic acid molecule combination of the present invention, wherein the first, second, third, fourth, fifth, and sixth nucleic acid sequences are each independently located in the same or different expression vectors. The vector of the present invention can be a cloning vector or an expression vector. In some embodiments, the vector of the present invention is, for example, a plasmid, granule, bacteriophage, cosmid, etc. In some embodiments, the vector is capable of expressing the system and nucleic acid molecule combination of the present invention in a subject (e.g., a mammal, such as a human).

[0035] On the other hand, the present invention provides a method for modifying a target gene for non-diagnostic and therapeutic purposes, comprising: contacting the target gene with the system, nucleic acid molecule combination or expression vector of the present invention, or delivering it to a cell containing the target gene, wherein the modification includes deletion, substitution, insertion, inversion, duplication or any combination thereof.

[0036] In some embodiments, the method is used to modify a target gene in vitro or ex vivo. In some embodiments, the method is not a method for treating humans or animals as a therapy. In some embodiments, the method does not include the step of modifying human germline genetic characteristics.

[0037] In some embodiments, the target gene is present within a cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is selected from non-human primate, bovine, pig, or rodent cells. In some embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry or fish cell. In some embodiments, the cell is a plant cell, such as a cell found in cultivated plants (e.g., cassava, corn, sorghum, wheat, or rice), algae, trees, or vegetables.

[0038] In some embodiments, the target gene is present in an in vitro nucleic acid molecule (e.g., a plasmid).

[0039] On the other hand, the present invention provides the use of the system, nucleic acid molecule combination or expression vector of the present invention in the preparation of formulations, said formulations being used for: (i) Nucleic acid editing; (ii) In vitro or ex vivo DNA detection; and / or, (iii) Editing target sequences in target loci to modify biological or non-human organisms; The nucleic acid editing includes modifying genes, knocking out genes, altering the expression of gene products, repairing mutations, and / or inserting polynucleotides.

[0040] Beneficial effects of the invention This application describes a novel nucleic acid editing system based on the Casδ protein, which can achieve precise editing of various types such as point mutations, fragment deletions, and substitutions. It has ideal editing efficiency and can achieve precise genome editing at the hundred-base-pair level, showing high application potential. Attached Figure Description

[0041] Figure 1 Structural design of protein expression vectors and RNA expression vectors for Casδ-guided editing systems based on the CPE strategy.

[0042] Figure 2A A schematic diagram of the Casδ guided editing system based on the Dual-CPE strategy.

[0043] Figure 2BStructural design of protein expression vectors and RNA expression vectors for Casδ guided editing systems based on the Dual-CPE strategy.

[0044] Figure 3A CPE strategy and Dual-CPE strategy in VEGFA , TRAC2 , EMX1 , B2M , HPRT The editing efficiency of point mutation editing on genes.

[0045] Figure 3B CPE strategy and Dual-CPE strategy in VEGFA , TRAC2 , EMX1 , B2M , HPRT The level of byproducts of point mutation editing in genes.

[0046] Figure 3C CPE strategy and Dual-CPE strategy in VEGFA , TRAC2 , EMX1 , B2M , HPRT The efficiency of gene deletion editing.

[0047] Figure 3D CPE strategy and Dual-CPE strategy in VEGFA , TRAC2 , EMX1 , B2M , HPRT Levels of byproducts of gene deletion editing.

[0048] Figure 3E CPE strategy and Dual-CPE strategy in VEGFA , TRAC2 , EMX1 , B2M , HPRT The efficiency of gene substitution editing.

[0049] Figure 3F CPE strategy and Dual-CPE strategy in VEGFA , TRAC2 , EMX1 , B2M , HPRT Levels of byproducts of gene substitution editing.

[0050] Figure 4A A comparison of the overall editing efficiency of CPE strategy and Dual-CPE strategy in terms of point mutation, deletion, and replacement.

[0051] Figure 4BComparison of editing byproduct levels of CPE strategy and Dual-CPE strategy in terms of point mutation, deletion, and substitution.

[0052] Sequence information Table 1: Information about the sequences involved in this invention is described in the table below: Detailed Implementation

[0053] The invention will now be described in the following non-limiting embodiments.

[0054] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0055] Example 1: Preparation of a Guided Editing System 1.1 Experimental Supplies 1.1.1 Experimental Materials Animal material: HEK293T cells (human embryonic kidney cells).

[0056] 1.1.2 Strains and vectors used in the experiment DH5α competent E. coli cells (Qingke Biotechnology, TSC-C01) used for vector construction and plasmid propagation.

[0057] The backbone of the protein expression vector in animal cells is pZG, and the backbone of the RNA expression vector is PX458.

[0058] 1.1.3 Experimental reagents (Table 2) Table 2: Biochemical Reagents

[0059] 1.1.4 Main instruments and equipment (Table 3) Table 3: Information related to instruments and equipment

[0060] 1.2 Experimental Methods 1.2.1 Preparation of culture medium and antibiotics (1) Preparation of LB medium LB liquid medium (1L): Accurately weigh 10g peptone, 5g yeast extract, and 10g sodium chloride into an Erlenmeyer flask. Add an appropriate amount of deionized water and stir to dissolve using a magnetic stirrer. Finally, bring the volume to 1L. Seal the Erlenmeyer flask with a sealing film and transfer it to an autoclave. Sterilize at 121℃ for 15 minutes. After sterilization and cooling to room temperature, add the appropriate antibiotic according to the required resistance in a clean bench to obtain the resistance medium. This medium can be used for subsequent shake culture or directly aliquoted and stored at 4℃ for the resuscitation of transformed *E. coli* strains.

[0061] LB solid medium (1L): Add 10g of agar to LB liquid medium, sterilize and cool to about 50℃. In a clean bench, add antibiotics (kanamycin 1:2000; spectinomycin and ampicillin 1:1000; chloramphenicol 1:4000) at the corresponding working concentrations and pour into disposable petri dishes. Let cool and solidify for later use.

[0062] (2) Antibiotic preparation Kanamycin (100 mg / mL): Weigh 1 g of kanamycin, dissolve it in deionized water, and bring the volume to 10 mL. Filter the solution through a 0.22 μm filter membrane for sterilization, aliquot it, and store it at -20 °C for later use.

[0063] Chloramphenicol (100mg / mL): Weigh 1g of chloramphenicol, dissolve it in anhydrous ethanol, and bring the volume to 10mL. Filter the solution through a 0.22μm filter membrane for sterilization, aliquot it, and store it at -20℃ for later use.

[0064] Ampicillin (100mg / mL): Weigh 1g of ampicillin, dissolve it in deionized water, and bring the volume to 10mL. Filter the solution through a 0.22μm filter membrane for sterilization, aliquot it, and store it at -20℃ for later use.

[0065] Spectinomycin (100mg / mL): Weigh 1g of spectinomycin, dissolve it in deionized water, and bring the volume to 10mL. Filter the solution through a 0.22μm filter membrane for sterilization, aliquot, and store at -20℃ for later use.

[0066] 1.2.2 Protoplast Extraction and Transformation Solution Preparation (1) Preparation of mother liquor Table 4: Mother liquor preparation system for protoplasts

[0067] (2) Preparation of enzyme hydrolysate—prepare and use immediately Table 5: Preparation system of enzymatic hydrolysate (50 mL)

[0068] First, mix the reagents in Table 5 thoroughly and transfer them to a 55°C water bath for 10 min. After cooling to room temperature, add 50 μL of 1M CaCl2 and 0.05 g of 0.1% BSA sequentially. Filter the solution through a 0.22 μm filter membrane for sterilization, collect the filtrate in a sterile container, and store it temporarily at 4°C.

[0069] (3) Preparation of W5 solution—prepare and use immediately Table 6: Preparation system of W5 solution (300mL)

[0070] After mixing the reagents in Table 6, filter them through a 0.22 μm filter membrane into a sterile container and store them temporarily at 4°C.

[0071] (4) Preparation of mg solution — prepare and use immediately Table 7: Preparation system of 50 mL of mg solution

[0072] (5) Preparation of PEG solution—prepare and use immediately Table 8: PEG solution (20 mL) preparation system

[0073] After mixing the reagents in Table 8, filter them through a 0.22 μm filter membrane into a sterile container and store them temporarily at 4°C.

[0074] 1.2.3 Preparation of other solutions (1) CTAB extract Table 9: CTAB solution (500 mL) preparation system

[0075] After preparing the system according to Table 9, place it on a magnetic stirrer to dissolve it completely, and store it at room temperature.

[0076] (2) Complete culture medium (50 mL) Table 10: Preparation system of complete culture medium (50 mL)

[0077] 1.2.4 Carrier Construction 1.2.4.1 Casδ-guided editing system based on CPE strategy A dual-plasmid co-transfection strategy using protein and RNA expression vectors was employed. The dual-vector structure was designed as follows: Figure 1 As shown.

[0078] The protein expression vector was designed as follows: The CAG and CMV promoters were used to drive the transcription of reverse transcriptase (RT) and CRISPR-Cas proteins, respectively. An RNA aptamer-binding protein (MS2 phage capsid protein (MCP)) was fused to the N-terminus of the reverse transcriptase. To ensure efficient nuclear translocation of the fusion protein, nuclear localization signals (NLS) were introduced at both the N- and C-termini of its coding frame. The bGH terminator was selected to terminate transcription, utilizing its relatively weak termination efficiency to indirectly enhance the expression level of the target protein. Furthermore, the target gene and the puromycin resistance gene were linked via a T2A cleavage peptide to achieve co-transcription and independent translation of both genes, facilitating subsequent selection of positive cells using puromycin.

[0079] The RNA expression vector was designed as follows: RNA transcription was driven by a human U6 (hU6) RNA polymerase type III promoter. An RNA molecule containing an RNA aptamer (MS2, used to form the RNA stem-loop structure), RTT, and PBS was designed, with circular arms and ribozymes added to both ends of the RNA molecule to form circular RNA. RNA circularization was achieved through ribozyme self-cleavage, and the coding sequence of the circular RNA was tandemly linked with the coding sequence of crRNA, with transcription driven by the same hU6 promoter. Finally, a polyT sequence was used as the transcription termination signal.

[0080] Expression vectors were constructed using either homologous recombination or loop assembly.

[0081] 1.2.4.2 Casδ-guided editing system based on Dual-CPE strategy To improve the editing efficiency of the CPE strategy, a Casδ-guided editing system based on the Dual-CPE strategy was constructed. This system improved the RNA expression vector based on the dual expression vector of the CPE strategy, designing two tandemly expressed RNA molecules: RNA1, driven by the hU6 promoter, contains the coding sequences for circular RNA1 and crRNA1, with a polyT sequence as the transcription termination signal. Circular RNA1 contains a 5' ribozyme, a 5' circular arm, MS2, RTT1, PBS1, a 3' circular arm, and a 3' ribozyme; RNA2, driven by the hU6 promoter, contains the coding sequences for circular RNA2 and crRNA2, with a polyT sequence as the transcription termination signal. Circular RNA2 contains a 5' ribozyme, a 5' circular arm, MS2, RTT2, PBS2, a 3' circular arm, and a 3' ribozyme.

[0082] Specific mechanism diagram and carrier structure are as follows: Figure 2A-2B As shown.

[0083] Expression vectors were constructed using either homologous recombination or loop assembly.

[0084] Example 2: Editing efficiency test of the guided editing system 2.1 Detection Method The CPE editing system and Dual-CPE editing system constructed in Example 1 were tested using the human embryonic kidney cell line HEK293T cells, and five endogenous target sites were selected. VEGFA , TRAC2 , EMX1 , B2M , HPRT Editing is performed, and the editing types are point mutation, deletion, and replacement. See Table 11 below for specific editing types.

[0085] Table 11: Test Targets and Target Editing

[0086] Note: P1 (Position 1) is the first base downstream of PAM.

[0087] Protein expression vectors were designed based on CPE and Dual-CPE editing systems, and different RNA expression vectors were designed based on different target genes and editing types.

[0088] The protein expression vector backbone is pZG. The composition of the protein expression vector insert fragments of the CPE editing system and the Dual-CPE editing system is as follows: CAG promoter (SEQ ID NO: 1) - NLS coding sequence (SEQ ID NO: 4) - MCP coding sequence (SEQ ID NO: 12) - linker coding sequence (SEQ ID NO: 8) - reverse transcriptase coding sequence (SEQ ID NO: 6) - NLS coding sequence (SEQ ID NO: 4) - bGH (SEQ ID NO: 17) - CMV promoter (SEQ ID NO: 2) - NLS coding sequence (SEQ ID NO: 4) - Casδ coding sequence (SEQ ID NO: 10) - NLS coding sequence (SEQ ID NO: 4) - T2A coding sequence (SEQ ID NO: 14) - Puro coding sequence (SEQ ID NO: 16) - bGH (SEQ ID NO: 17).

[0089] The RNA expression vector backbone is PX458. The RNA expression vector insert fragment of the CPE editing system consists of: hU6 promoter (SEQ ID NO: 18) - 5' ribozyme coding sequence (SEQ ID NO: 20) - 5' circular arm coding sequence (SEQ ID NO: 22) - MS2 coding sequence (SEQ ID NO: 28) - RTT coding sequence (SEQ ID NO: 35-39, 50-54, 65-69) - PBS coding sequence (SEQ ID NO: 40-44, 55-59, 70-74) - MS2 coding sequence (SEQ ID NO: 28) - 3' circular arm coding sequence (SEQ ID NO: 24) - 3' ribozyme coding sequence (SEQ ID NO: 26) - homologous repeat coding sequence (SEQ ID NO: 166) - crRNA coding sequence (SEQ ID NO: 30-34, 45-49, 60-64) - poly T (SEQ ID NO: 29);The RNA expression vector insert of the Dual-CPE editing system consists of: hU6 promoter (SEQ ID NO: 18) - 5' ribozyme 1 coding sequence (SEQ ID NO: 20) - 5' circumflex arm 1 coding sequence (SEQ ID NO: 22) - MS2 coding sequence (SEQ ID NO: 28) - RTT1 coding sequence (SEQ ID NO: 80-84, 110-114, 140-144) - PBS1 coding sequence (SEQ ID NO: 85-89, 115-119, 145-149) - MS2 coding sequence (SEQ ID NO: 28) - 3' circumflex arm 1 coding sequence (SEQ ID NO: 24) - 3' ribozyme 1 coding sequence (SEQ ID NO: 26) - direct repeat sequence coding sequence (SEQ ID NO: 166) - crRNA1 coding sequence (SEQ ID NO: 166) 75-79, 105-109, 135-139,)-poly T (SEQ ID NO: 29)-hU6 promoter (SEQ ID NO: 18)-5' ribozyme 2 coding sequence (SEQ ID NO: 20)-5' circumflex arm 2 coding sequence (SEQ ID NO: 22)-MS2 coding sequence (SEQ ID NO: 28)-RTT2 coding sequence (SEQ ID NO: 95-99, 125-129, 155-159)-PBS2 coding sequence (SEQ ID NO: 100-104, 130-134, 160-164)-MS2 coding sequence (SEQ ID NO: 28)-3' circumflex arm 2 coding sequence (SEQ ID NO: 24)-3' ribozyme 2 coding sequence (SEQ ID NO: 26)-homogeneous repeat sequence coding sequence (SEQ ID NO: 166)-crRNA2 coding sequence (SEQ ID NO: 166) 90-94, 120-124, 150-154)-poly T (SEQ ID NO: 29). ;

[0090] A dual plasmid co-transfection strategy using protein and RNA expression vectors was employed, and plasmid delivery was completed via liposome transfection. The specific steps are as follows: (1) Animal cell cryopreservation a. Remove the culture medium from cells in the logarithmic growth phase, add PBS solution to the cells to rinse, and then aspirate the PBS. b. Add 1 mL of trypsin to the culture dish, and then place it in a 37°C cell culture incubator for digestion for about 3 minutes; c. After digestion, pipette the cells to detach them from the culture dish, and add 2 mL of culture medium containing 10% fetal bovine serum to terminate the digestion reaction. d. Transfer the cells to centrifuge tubes, centrifuge at 500 g for 5 min, then add an appropriate amount of cryopreservation buffer (DMSO:FBS:basal medium = 1:3:6) to resuspend the cells, and then aliquot them into cryovials. e. Place the aliquoted cells in a 4°C freezer for 0.5 h, then place them at -20°C for 1 h, then at -80°C overnight, and finally store them in liquid nitrogen.

[0091] (2) Animal cell resuscitation a. Before the experiment, turn on the ultraviolet lamp of the laminar flow hood for 30 minutes and turn on the 37℃ water bath to preheat the basic culture medium. After the ultraviolet sterilization of the laminar flow hood is completed, wipe the laminar flow hood with a cotton ball containing 75% alcohol. b. Remove the frozen cells from liquid nitrogen and immediately place them in a 37°C water bath and shake them rapidly to thaw. Spray the centrifuge tube containing the cells with 75% ethanol for sterilization and place it in a clean bench. First, add 5 mL of basal culture medium to the centrifuge tube, and then immediately transfer the thawed cells to the centrifuge tube. Centrifuge at 500 g for 5 min and remove the supernatant. c. Add an appropriate amount of basal culture medium to the cells, resuspend them by pipetting, add the resuspended cells to the cell culture dish at an appropriate density, then gently shake and examine under a microscope. After the examination, place the dish in a 37°C, 5% CO2 cell culture incubator. Be careful to avoid shaking the culture dish while walking. d. After culturing for 24 h, replace the cell culture medium with complete culture medium.

[0092] (3) Animal cell passage a. When the confluence of cells in the dish reaches 90%, aspirate the culture medium, then add an appropriate amount of PBS solution to the culture dish, gently shake to rinse the cells, and aspirate the PBS solution after rinsing. b. Add trypsin to the culture dish to digest the cells at 37°C for 3 minutes. After digestion, gently shake the culture dish to disperse the cells from the culture dish. c. Add an appropriate amount of basal culture medium containing 10% fetal bovine serum to the culture dish to terminate the reaction; d. Aspirate the cells into a 15 mL centrifuge tube, set the centrifuge parameters to 500 g, and centrifuge for 5 min; e. Aspirate the supernatant, add 5 mL of basal culture medium to the centrifuge tube, resuspend the cells by pipetting, and seed them into new culture dishes at the required density; f. Pass cells every 48 hours, depending on the cell growth rate; (4) Animal cell transfection a. Examine the state of passaged cells under a microscope. When the cell confluence reaches 90%, plant at 2.3 × 10⁶ cells per well. 5 Cells were seeded at a density in 24-well cell culture plates, which were then sprayed with alcohol and placed in an incubator at 37°C and 5% CO2 for approximately 24 hours. b. When the cell confluence reaches 70%-90%, begin transfection. Aspirate the cell culture medium, slowly add 400 μL of DMEM medium to the plate, taking care to avoid upsetting the cells. Return the culture plate to the incubator for later use. c. Cell transfection was performed using liposome transfection, with each well containing 100 μL of DMEM medium, 1.5 μL of transfection reagent (Lipofectamine™ 3000), 750 ng of protein particles, and 250 ng of RNA plasmid. d. Mix the prepared reagents thoroughly and incubate at room temperature for 15-20 min; e. After incubation, add the mixed reagents to the cell culture plate and place the culture plate in a 37°C, 5% CO2 incubator for incubation; f. After culturing for 6-8 h, aspirate 400 μL of culture medium and add 400 μL of complete culture medium (DMEM medium containing antibiotics and 10% fetal bovine serum). g. 24 h after transfection, add Puromycin antibiotic, and collect cells after 48 h of drug screening culture.

[0093] (5) Animal cell collection and extraction a. Preparation of reagents: Dilute 10× cell lysate to 1×, add proteinase K to make a final concentration of 0.2 mg / mL; b. After cell culture is complete, gently shake the culture plate and use a pipette to remove the culture medium; c. Add 60-80 μL of 1× cell lysis buffer containing proteinase K to the cell culture plate and shake the cell culture plate. d. Transfer the cells sequentially to the corresponding 96-well plates, label the sample information, cover with a soft silicone pad, and place in a PCR instrument for lysis reaction. The procedure is shown in Table 12 below.

[0094] Table 12: Animal Cell DNA Extraction Procedure

[0095] The editing efficiency of the collected animal cells was tested, and the specific steps are as follows: (1) Amplify the target gene sequence a. Primer design: Design specific amplification primers approximately 100 bp upstream and downstream of the target sequence. When synthesizing the F primer, add the adapter sequence “ACGACGCTCTTCCGATCT” (SEQ ID NO: 167) to the 5' end. When synthesizing the R primer, add the adapter sequence “ACGTGTGCTCTTCCGATCT” (SEQ ID NO: 168) to the 5' end. The amplified fragment size is approximately 230 bp.

[0096] b. The amplification system is shown in Table 13 below; Table 13: NGS one-round amplification system

[0097] c. The amplification procedure is shown in Table 14 below; Table 14: NGS One-Round Amplification Procedure

[0098] d. Take 3 μL of the amplification product and perform agarose gel electrophoresis. Use gel imaging to analyze whether the amplified band is the target band.

[0099] (2) Second round of amplification with added index sequences Use 1 μL of the first-round amplification product as a template. The amplification system is the same as the first-round amplification, but the number of cycles is reduced to 12. Other procedures are shown in Table 14. Perform agarose gel electrophoresis on the amplification product to detect whether the target band was successfully amplified.

[0100] (3) Constructing a library Eight μL of each of the second-round amplification products was transferred to a centrifuge tube, vortexed to mix thoroughly, and purified using a gel extraction kit. The purified product was the DNA library, which was sent to the company for sequencing. Four biological replicates were set up for each editing experiment.

[0101] (4) Sequencing results analysis Organize sequencing results analysis files, including sample name, first-round amplification sequence, target editing sequence, target information, etc. The company disassembles the data after unloading the sequencing, uploads the FASTQ files to the server, processes and analyzes the files, and obtains the editing efficiency.

[0102] 2.2 Test Results Results of different editing efficiencies and editing by-product levels in various genes are as follows: Figures 3A-3F As shown.

[0103] In point mutation editing ( Figure 3AIn the VEGFA and TRAC2 sites, the Dual-CPE strategy significantly improved the precision editing efficiency compared to the CPE strategy. At the EMX1 and B2M sites, although the difference was not statistically significant, the editing efficiency of Dual-CPE was on average about three times that of CPE. In deletion editing... Figure 3C In the comparison of B2M sites, the Dual-CPE strategy showed more significant optimization effects, with varying degrees of improvement in editing efficiency at all five test targets. The most significant improvement was observed at the B2M site, where the CPE strategy failed to detect quantifiable, precise editing efficiency. The Dual-CPE strategy not only achieved precise editing but also increased editing efficiency by 81.45 times. The editing efficiency improvements at the VEGFA, EMX1, and HPRT sites were all highly significant. Even at the TRAC2 site, although there was no significant difference, the Dual-CPE editing efficiency was still 2.47 times that of the CPE strategy. In replacement editing (… Figure 3E In this study, compared to the CPE strategy, the Dual-CPE strategy achieved a fold-fold improvement in perfect editing efficiency at all target sites. The efficiency improvements at VEGFA, TRAC2, EMX1, B2M, and HPRT sites were 4.21-fold, 3.46-fold, 24.93-fold, 3.19-fold, and 3.14-fold, respectively, with a highly significant difference between EMX1 and B2M sites. At the EMX1 site, the Dual-CPE strategy achieved a precise deletion of a 99 bp fragment with an editing efficiency of approximately 1.5%; further, it mediated a precise insertion of a 30 bp exogenous sequence based on this 99 bp deletion, achieving an editing efficiency of approximately 1%. These results demonstrate that the Dual-CPE strategy possesses the capability to mediate precise editing at the hundred-base-pair level.

[0104] In summary, the Casδ guided editing system based on the Dual-CPE strategy significantly improves editing efficiency compared to the Casδ guided editing system based on the CPE strategy, especially in deletion and substitution editing, where efficiency is improved by tens of times. At the same time, it broadens the editing scope, enabling precise genome editing at the hundred-base-pair level.

[0105] Furthermore, the experimental results were summarized and grouped according to the three editing types, and statistical difference analysis was performed using an independent samples t-test. The results are as follows: Figures 4A-4B As shown.

[0106] like Figure 4AAs shown, the Dual-CPE strategy significantly improved efficiency for all types of editing, with particularly outstanding optimization effects on fragment deletion and replacement editing. Point mutation editing: The editing efficiency of the Dual-CPE strategy was on average 2.87 times that of the CPE strategy, a statistically significant difference (p<0.01); Deletion editing: The editing efficiency of Dual-CPE was increased to 6.95 times that of the CPE strategy (p<0.0001), breaking through the technical bottleneck of the extremely low efficiency of the CPE strategy in fragment deletion editing; Replacement editing: The editing efficiency of the Dual-CPE strategy reached 6.38 times that of the CPE strategy (p<0.01). Most importantly, the CPE strategy is almost unable to achieve precise fragment replacement at the hundred-base-pair level, while the Dual-CPE strategy successfully overcomes this editing length limitation, broadening the editing scope of the CPE strategy.

[0107] like Figure 4B As shown, while the Dual-CPE strategy improves editing efficiency, the amount of byproducts does not increase proportionally: in point mutation editing, the byproduct level of the Dual-CPE strategy is only 1.46 times that of the CPE strategy (p<0.05); in deletion editing, the byproduct level increases by only 1.18 times (p>0.05), with no statistically significant difference; in replacement editing, the byproduct level increases by 1.56 times (p<0.0001), which is far lower than the 20.73-fold increase in editing efficiency.

[0108] In summary, the dual-CPE strategy improves editing efficiency for all three editing types, with optimization effects reaching several times higher, particularly for fragment deletion and substitution editing. Furthermore, the dual-CPE strategy expands the precision editing range from tens of base pairs to hundreds of base pairs, broadening its application scope.

[0109] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A CRISPR / Cas system for editing target genes, comprising: (1) Casδ protein, the sequence of which is shown in SEQ ID NO: 9; (2) Reverse transcriptase; (3) The first circular RNA, which contains the first primer binding site and the first reverse transcriptase template; (4) A first guide nucleic acid comprising a first crRNA, the first crRNA comprising a first guide sequence that hybridizes to a first target site on the positive strand of the target gene; (5) A second circular RNA, which contains a second primer binding site and a second reverse transcriptase template; (6) A second guide nucleic acid comprising a second crRNA, the second crRNA comprising a second guide sequence that hybridizes to a second target site on the antisense strand of the target gene; in, The first reverse transcriptase template and the second reverse transcriptase template contain complementary regions, which are editing templates; The first primer binding site hybridizes with the 3' primer sequence generated by the cleavage of the Casδ protein and the first crRNA on the antisense strand of the target gene, and the second primer binding site hybridizes with the 3' primer sequence generated by the cleavage of the Casδ protein and the second crRNA on the sense strand of the target gene. The first target and the second target are located at the 3' end of the motif adjacent to the original spacer sequence, and the motif adjacent to the original spacer sequence has the sequence shown in 5'-ATG-3'.

2. The system of claim 1, wherein, The reverse transcriptase further comprises an RNA aptamer-binding protein, which is directly or via a peptide linker to the RNA aptamer-binding protein; the first circular RNA and the second circular RNA further comprise one or more RNA aptamers; the RNA aptamers and the RNA aptamer-binding protein are capable of specific binding.

3. The system of claim 2, wherein, The RNA aptamer is MS2, and the RNA aptamer-binding protein is MCP protein.

4. The system of claim 1, wherein, The N-terminus and / or C-terminus of the Casδ protein and / or reverse transcriptase are connected to nuclear localization signal sequences.

5. A combination of nucleic acid molecules comprising: (1) A first nucleic acid sequence comprising a nucleotide sequence encoding the Casδ protein of any one of claims 1-4; (2) A second nucleic acid sequence comprising a nucleotide sequence encoding the reverse transcriptase of any one of claims 1-4; (3) A third nucleic acid sequence comprising a nucleotide sequence for expressing the first circular RNA of any one of claims 1-4; (4) A fourth nucleic acid sequence comprising a nucleotide sequence for expressing a first guide nucleic acid of any one of claims 1-4; (5) A fifth nucleic acid sequence comprising a nucleotide sequence for expressing a second circular RNA of any one of claims 1-4; (6) A sixth nucleic acid sequence comprising a nucleotide sequence for expressing a second guide nucleic acid of any one of claims 1-4.

6. The nucleic acid molecule combination according to claim 5, wherein, The first, second, third, fourth, fifth, and sixth nucleic acid sequences are each independently operably linked to a promoter at the 5' end and / or operably linked to a terminator at the 3' end.

7. The nucleic acid molecule combination of claim 6, comprising: (1) Nucleic acid molecule A, which contains the first nucleic acid sequence, wherein the nucleic acid molecule A comprises from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence encoding an MCP, a nucleotide sequence encoding a peptide linker, a nucleotide sequence encoding a reverse transcriptase, a nucleotide sequence encoding a nuclear localization signal, and a terminator sequence; (2) Nucleic acid molecule B, which contains the second nucleic acid sequence, wherein the nucleic acid molecule B comprises from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence encoding Casδ, a nucleotide sequence encoding a nuclear localization signal, and a terminator sequence; (3) Nucleic acid molecule C, comprising the third nucleic acid sequence and the fourth nucleic acid sequence, wherein the nucleic acid molecule C comprises, from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence for expressing the first 5' ribozyme, a nucleotide sequence for expressing the first 5' cyclic arm, a nucleotide sequence for expressing the first reverse transcriptase template, a nucleotide sequence for expressing the first primer binding site, a nucleotide sequence for expressing the first 3' cyclic arm, a nucleotide sequence for expressing the first 3' ribozyme, a nucleotide sequence for expressing the first crRNA, and a terminator sequence; wherein, The first 5' end loop arm and the first 3' end loop arm can connect with each other to form a first circular RNA; (4) Nucleic acid molecule D, which contains the fifth nucleic acid sequence and the sixth nucleic acid sequence, wherein the nucleic acid molecule D contains, from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence for expressing the second 5' ribozyme, a nucleotide sequence for expressing the second 5' circumscribed arm, a nucleotide sequence for expressing the second reverse transcriptase template, a nucleotide sequence for expressing the second primer binding site, a nucleotide sequence for expressing the second 3' circumscribed arm, a nucleotide sequence for expressing the second 3' ribozyme, a nucleotide sequence for expressing the second crRNA, and a terminator sequence; wherein the second 5' circumscribed arm and the second 3' circumscribed arm can be connected to each other to form a second circular RNA.

8. An expression vector comprising the nucleic acid molecule combination according to any one of claims 5-7, wherein, The first, second, third, fourth, fifth, and sixth nucleic acid sequences are each independently located in the same or different expression vectors.

9. A method for modifying a target gene for non-diagnostic therapeutic purposes, comprising: The system of any one of claims 1-4, the combination of nucleic acid molecules of any one of claims 5-7, or the expression vector of claim 7 is contacted with the target gene or delivered to a cell containing the target gene, wherein the modification includes deletion, substitution, insertion, inversion, duplication, or any combination thereof.

10. Use of the system according to any one of claims 1-4, the nucleic acid molecule combination according to any one of claims 5-7, or the expression vector according to claim 8 in the preparation of a formulation, wherein the formulation is used for: (i) Nucleic acid editing; (ii) In vitro or ex vivo DNA detection; and / or, (iii) Editing target sequences in target loci to modify biological or non-human organisms; in, The nucleic acid editing includes modifying genes, knocking out genes, altering the expression of gene products, repairing mutations, and / or inserting polynucleotides.

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