Casδ-based nucleic acid editing system

Abstract

The present application relates to the field of nucleic acid editing, in particular to a Casδ based nucleic acid editing system. More specifically, the present application relates to a CRISPR / Cas system for editing a target gene, the system comprising: a fusion protein comprising a reverse transcriptase and a Casδ protein, a first guide nucleic acid and a second guide nucleic acid. The present application also relates to a combination of nucleic acid molecules encoding the CRISPR / Cas system, expression vectors comprising the nucleic acid molecules, and the use of the CRISPR / Cas system, the combination of nucleic acid molecules, the expression vectors in 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, the system comprising: a fusion protein containing a reverse transcriptase and a Casδ protein, a first guide nucleic acid, and a second guide nucleic acid. This application also relates to a nucleic acid molecule combination encoding the CRISPR / Cas system, an expression vector containing the nucleic acid molecules, and the use of the CRISPR / Cas system, the nucleic acid molecule combination, 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] Previous research by Aaron W. Hummel's team revealed that the design architecture of traditional Cas9-based guided editing systems cannot be directly used in the Cas12a system. Even after experimenting with various strategies, including different Cas12a-RT fusion methods, PBS / RTT lengths, RT recruitment strategies, and creating nicks on the target strand, effective and precise editing at the target site could not be detected. The reason for this is speculated to be the significant spatial conformational difference in the R-loop structure formed after Cas12a and Cas9 bind to the target DNA. This prevents the reverse transcriptase (RT) from effectively binding to the linear pegRNA and the cleaved DNA ends, hindering the reverse transcription process. To overcome this spatial limitation, the team modified the traditional pegRNA design: in the traditional PE system, the RT reverse transcribes the target edit to the non-target strand (NTS) containing PAM, while in the REDRAW system (RNA-encoded DNA replacement of alleles with CRISPR), the RT directly reverse transcribes the target edit to the target strand (TS) complementary to the crRNA. The core components of the REDRAW system include the Cas12a protein, reverse transcriptase (RT), and 3' extended tagRNA (target alleleguide RNA). The tagRNA is based on crRNA and incorporates PBS and RTT sequences complementary to the target strand. After Cas12a cleaves the target DNA to form a double-strand break (DSB), the PBS sequence of the tagRNA pairs complementary to the free end of the target strand. The reverse transcriptase uses the RTT sequence in this RNA as a template to reverse transcribe the target editing information into the target strand. Finally, through endogenous DNA repair mechanisms, the edited information is stably integrated into the genome.

[0004] This system is compatible with various CRISPR nucleases, including Type II (Cas9) and Type V (Cas12a). The LbCas12a-based REDRAW system can achieve all types of single-base substitution, precise insertion, and deletion editing, with a targeting window of 8–10 bp. By fusing the Brex27 peptide motif at the N- or C-terminus to stabilize single-stranded DNA, editing efficiency can be increased by 1.8 or 1.3 times. Adding the RNA structural motif PP7 to the end of the tagRNA to enhance RNA stability can increase editing efficiency by an average of 2.3 times (Kim et al., 2022). This study establishes for the first time a guided editing technology adapted to Cas12 family nucleases that does not rely on traditional PE design patterns, revealing a novel molecular mechanism of RT-mediated guided editing and providing a new technical pathway for the development of PE systems for non-Cas9 nucleases. However, the REDRAW system's editing capabilities are mainly concentrated on small fragments, and there is a lack of systematic research on the precise editing of medium-length fragments; editing efficiency is relatively low, and control of editing byproducts is insufficient.

[0005] 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.

[0006] 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

[0007] The inventors of this application have developed a novel nucleic acid editing system based on the Casδ protein and the Dual-REDRAW 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.

[0008] On one hand, the present invention provides a CRISPR / Cas system for editing target genes, comprising:

[0009] (1) A fusion protein comprising a reverse transcriptase and a Casδ protein, wherein the reverse transcriptase is linked to the N-terminus or C-terminus of the Casδ protein via a peptide linker;

[0010] (2) A first guide nucleic acid comprising: (i) a first crRNA containing a first guide sequence that hybridizes to a first target site on the positive strand of the target gene; and (ii) an extension containing a first primer binding site (PBS) and a first reverse transcriptase template (RTT);

[0011] (3) A second guide nucleic acid comprising: (i) a second crRNA containing a second guide sequence that hybridizes to a second target site on the antisense strand of the target gene; and (ii) an extension containing a second primer binding site (PBS) and a second reverse transcriptase template (RTT);

[0012] Wherein, the first RTT and the second RTT contain complementary regions, and the regions are editing templates;

[0013] The first PBS hybridizes with the 3' primer sequence generated by the cleavage of the Casδ protein and the second crRNA on the target gene, and the second PBS hybridizes with the 3' primer sequence generated by the cleavage of the Casδ protein and the first crRNA on the target gene.

[0014] The first and second target sites are located at the 3' end of the adjacent motif (PAM) of the original spacer sequence, and the PAM has the sequence shown in 5'-ATG-3'.

[0015] In some implementations, the guide nucleic acid is guide editing RNA.

[0016] In some embodiments, the first PBS hybridizes with a 3' primer sequence generated by cleaving the complex formed by the fusion protein and the second guide nucleic acid on the target gene, wherein the 3' primer sequence is generated by cleaving the Casδ protein in the complex and the second crRNA on the target gene.

[0017] In some embodiments, the second PBS hybridizes with a 3' primer sequence generated by cleaving the complex formed by the fusion protein and the first guide nucleic acid on the target gene, wherein the 3' primer sequence is generated by cleaving the Casδ protein in the complex and the first crRNA on the target gene.

[0018] 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.

[0019] In some implementations, the editing template includes embellishments, such as missing, replacement, insertion, inversion, repetition, or any combination thereof.

[0020] 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.

[0021] In some embodiments, the fusion protein further comprises a single-stranded DNA-binding protein and a nuclear localization signal (NLS) sequence, wherein the single-stranded DNA-binding protein is selected from Brex27, hRad51, hRad52, BsRecA, EcRecA, and T4SSB.

[0022] In some exemplary embodiments, the single-stranded DNA binding protein comprises a sequence as shown in SEQ ID NO: 10.

[0023] In some embodiments, the fusion protein comprises one or more NLS sequences. In some embodiments, the NLS is linked to the N-terminus or C-terminus of the Casδ protein. In some exemplary embodiments, the NLS sequence comprises the sequence shown in SEQ ID NO: 2.

[0024] In some embodiments, the Casδ protein has an amino acid sequence selected from the following:

[0025] (i) The sequence shown in SEQ ID NO: 8;

[0026] (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: 8; or

[0027] (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: 8.

[0028] In some embodiments, the 3' ends of the first and second guide nucleic acids also include stem-loop structures (e.g., PP7 stem-loop structures) or pseudoknot structures.

[0029] In some embodiments, the reverse transcriptase has an amino acid sequence selected from the following:

[0030] (i) The sequence shown in SEQ ID NO: 4;

[0031] (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: 4; or

[0032] (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: 4.

[0033] 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: 6.

[0034] 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: 155.

[0035] On the other hand, the present invention provides a nucleic acid molecule assembly comprising:

[0036] (1) A first nucleic acid sequence comprising a nucleotide sequence encoding a fusion protein of the system of the present invention;

[0037] (2) A second nucleic acid sequence comprising a nucleotide sequence of a first guide nucleic acid for expressing the system of the present invention;

[0038] (3) A third nucleic acid sequence, which contains a nucleotide sequence of a second guide nucleic acid for expressing the system of the present invention.

[0039] In some implementations, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are each independently operatively ligated to a promoter at the 5' end and operatively ligated to a terminator at the 3' end.

[0040] In some embodiments, the first nucleic acid sequence comprises, from the 5' end to the 3' end: a promoter sequence, a nucleotide sequence encoding NLS, a nucleotide sequence encoding reverse transcriptase, a nucleotide sequence encoding a peptide linker, a nucleotide sequence encoding Casδ, a nucleotide sequence encoding NLS, a nucleotide sequence encoding Brex27, and a terminator sequence.

[0041] The second nucleic acid sequence from the 5' end to the 3' end includes: a promoter sequence, a nucleotide sequence for expressing the first crRNA, a nucleotide sequence for expressing the first RTT, a nucleotide sequence for expressing the first PBS, a nucleotide sequence for expressing the stem-loop structure or pseudoknot structure, and a terminator sequence;

[0042] The third nucleic acid sequence, from the 5' end to the 3' end, includes: a promoter sequence, a nucleotide sequence for expressing the second crRNA, a nucleotide sequence for expressing the second RTT, a nucleotide sequence for expressing the second PBS, a nucleotide sequence for expressing the stem-loop structure or pseudoknot structure, and a terminator sequence.

[0043] On the other hand, the present invention provides an expression vector comprising the nucleic acid molecule combination of the present invention, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence 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, a granule, a bacteriophage, a 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).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In some embodiments, the target gene is present in an in vitro nucleic acid molecule (e.g., a plasmid).

[0048] 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:

[0049] (i) Nucleic acid editing;

[0050] (ii) In vitro or ex vivo DNA detection; and / or,

[0051] (iii) Editing target sequences in target loci to modify biological or non-human organisms;

[0052] The nucleic acid editing includes modifying genes, knocking out genes, altering the expression of gene products, repairing mutations, and / or inserting polynucleotides.

[0053] Beneficial effects of the invention

[0054] 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

[0055] Figure 1 Structural design of protein expression vectors and RNA expression vectors for the Casδ-guided editing system based on the REDRAW strategy.

[0056] Figure 2A A schematic diagram of the Casδ guided editing system based on the Dual-REDRAW strategy.

[0057] Figure 2B Structural design of protein expression vectors and RNA expression vectors based on the Dual-REDRAW strategy Casδ guided editing system.

[0058] Figure 3A Editing efficiency of REDRAW strategy and Dual-REDRAW strategy in point mutation editing of VEGFA, TRAC2, EMX1, B2M and HPRT genes.

[0059] Figure 3B The byproduct levels of point mutation editing in the VEGFA, TRAC2, EMX1, B2M, and HPRT genes using the REDRAW and Dual-REDRAW strategies.

[0060] Figure 3C Editing efficiency of REDRAW strategy and Dual-REDRAW strategy in deletion editing of VEGFA, TRAC2, EMX1, B2M and HPRT genes.

[0061] Figure 3D The levels of byproducts of deletion editing in the VEGFA, TRAC2, EMX1, B2M, and HPRT genes under the REDRAW and Dual-REDRAW strategies.

[0062] Figure 3E Editing efficiency of REDRAW strategy and Dual-REDRAW strategy in substitution editing of VEGFA, TRAC2, EMX1, B2M and HPRT genes.

[0063] Figure 3F The levels of byproducts of deletion editing in the VEGFA, TRAC2, EMX1, B2M, and HPRT genes under the REDRAW and Dual-REDRAW strategies.

[0064] Figure 4A A comparison of the overall editing efficiency of the REDRAW strategy and the Dual-REDRAW strategy in terms of point mutations, deletions, and replacements.

[0065] Figure 4B Comparison of editing byproducts of the REDRAW strategy and the Dual-REDRAW strategy in terms of point mutations, deletions, and substitutions.

[0066] Sequence information

[0067] Table 1: Information about the sequences involved in this invention is described in the table below: Detailed Implementation

[0068] The invention will now be described in the following non-limiting embodiments.

[0069] 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.

[0070] Example 1: Preparation of a Guided Editing System

[0071] 1.1 Experimental Supplies

[0072] 1.1.1 Experimental Materials

[0073] Animal material: HEK293T cells (human embryonic kidney cells).

[0074] 1.1.2 Strains and vectors used in the experiment

[0075] DH5α competent E. coli cells (Qingke Biotechnology, TSC-C01) were used for vector construction and plasmid propagation; EHA105 competent Agrobacterium cells (Qingke Biotechnology, TSC-A03) were used for stable transformation of maize.

[0076] The backbone of the protein expression vector in animal cells is pZG, and the backbone of the RNA expression vector is PX458.

[0077] 1.1.3 Experimental reagents (Table 2)

[0078] Table 2: Biochemical Reagents

[0079] 1.1.4 Main instruments and equipment (Table 3)

[0080] Table 3: Information related to instruments and equipment

[0081] 1.2 Experimental Methods

[0082] 1.2.1 Preparation of culture medium and antibiotics

[0083] (1) Preparation of LB medium

[0084] 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. After sealing the Erlenmeyer flask with a sealing film, transfer it to an autoclave and 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 dispensed and stored at 4℃ for the resuscitation of E. coli transformed strains.

[0085] 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.

[0086] (2) Antibiotic preparation

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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 it, and store it at -20℃ for later use.

[0091] 1.2.2 Protoplast Extraction and Transformation Solution Preparation

[0092] (1) Preparation of mother liquor

[0093] Table 4: Mother liquor preparation system for protoplasts

[0094] (2) Preparation of enzyme hydrolysate—prepare and use immediately

[0095] Table 5: Preparation system of enzymatic hydrolysate (50 mL)

[0096] 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.

[0097] (3) Preparation of W5 solution—prepare and use immediately

[0098] Table 6: Preparation system of W5 solution (300mL)

[0099] 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.

[0100] (4) Preparation of mg solution — prepare and use immediately

[0101] Table 7: Preparation system of Mg solution (50 mL)

[0102] (5) Preparation of PEG solution—prepare and use immediately

[0103] Table 8: PEG solution (20 mL) preparation system

[0104] 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.

[0105] 1.2.3 Preparation of other solutions

[0106] (1) CTAB extract

[0107] Table 9: CTAB solution (500 mL) preparation system

[0108] After preparing the system according to Table 9, place it on a magnetic stirrer to dissolve it completely, and store it at room temperature.

[0109] (2) Complete culture medium (50 mL)

[0110] Table 10: Preparation system of complete culture medium (50 mL)

[0111] 1.2.4 Carrier Construction

[0112] 1.2.4.1 Casδ Guided Editing System Based on REDRAW Strategy

[0113] 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.

[0114] The protein expression vector was designed as follows: a single CMV promoter was used to drive the transcription of the target gene, with the coding sequence being NLS-reverse transcriptase-linker-Casδ-NLS-Brex27 (single-stranded DNA binding protein).

[0115] The RNA expression vector was designed as follows: the hU6 promoter was selected to drive stagRNA transcription. The stagRNA contained sequences such as crRNA, RTT, PBS, and PP7. A PP7 stem-loop structure was added to the 3' end of the RNA to resist intracellular exonuclease degradation and improve RNA stability. Finally, the polyT sequence was used as the transcription termination signal.

[0116] Expression vectors were constructed using either homologous recombination or loop assembly.

[0117] 1.2.4.2 Casδ Guided Editing System Based on Dual-REDRAW Strategy

[0118] To improve the editing efficiency of the REDRAW strategy, a Casδ-guided editing system based on the Dual-REDRAW strategy was constructed. This system improves upon the dual-expression vector of the REDRAW strategy by designing two tandemly expressed RNA molecules: RNA1 uses the hU6 promoter to drive stagRNA transcription. The stagRNA contains sequences such as crRNA1, RTT1, PBS1, and PP7. A PP7 stem-loop structure is added to the 3' end of the RNA to resist intracellular exonuclease degradation and enhance RNA stability. The polyT sequence is ultimately used as the transcription termination signal. RNA2 also uses the hU6 promoter to drive stagRNA transcription. The stagRNA contains sequences such as crRNA1, RTT1, PBS1, and PP7. A PP7 stem-loop structure is added to the 3' end of the RNA to resist intracellular exonuclease degradation and enhance RNA stability. The polyT sequence is ultimately used as the transcription termination signal.

[0119] The specific mechanism diagram and carrier structure are shown in Figure 2.

[0120] Expression vectors were constructed using either homologous recombination or loop assembly.

[0121] Example 2: Editing efficiency test of the guided editing system

[0122] 2.1 Detection Method

[0123] The REDRAW editing system and Dual-REDRAW editing system constructed in Example 1 were tested using the human embryonic kidney cell line HEK293T cells. Five endogenous targets (VEGFA, TRAC2, EMX1, B2M, HPRT) were selected for editing. The editing types were point mutation, deletion, and substitution. The specific editing types are shown in Table 11 below.

[0124] Table 11: Test Targets and Target Editing

[0125] Note: P1 (Position 1) is the first base downstream of PAM.

[0126] Protein expression vectors were designed based on the REDRAW editing system and the Dual-REDRAW editing system, and different RNA expression vectors were designed based on different target genes and editing types.

[0127] The protein expression vector backbone is pZG. The composition of the protein expression vector insert fragments of the REDRAW editing system and the Dual-REDRAW editing system is as follows: CMV promoter (SEQ ID NO: 1) - NLS (SEQ ID NO: 3) - reverse transcriptase (SEQ ID NO: 5) - linker (SEQ ID NO: 7) - Casδ coding sequence (SEQ ID NO: 9) - NLS (SEQ ID NO: 3) - Brex27 coding sequence (SEQ ID NO: 11) - T2A (SEQ ID NO: 13) - Puro (SEQ ID NO: 15) - bGH (SEQ ID NO: 16).

[0128] The RNA expression vector backbone is PX458. The RNA expression vector insert for the REDRAW editing system consists of: hU6 promoter (SEQ ID NO: 17) - homologous repeat coding sequence (SEQ ID NO: 156) - crRNA (SEQ ID NO: 20-24, 35-39, 50-54) - RTT (SEQ ID NO: 25-29, 40-44, 55-59) - PBS (SEQ ID NO: 30-34, 45-49, 60-64) - PP7 (SEQ ID NO: 18) - poly T (SEQ ID NO: 19); the RNA expression vector insert for the Dual-REDRAW editing system consists of: hU6 promoter (SEQ ID NO: 17) - homologous repeat coding sequence (SEQ ID NO: 156) - crRNA1 (SEQ ID NO: 65-69, 95-99, 125-129) - RTT1 (SEQ ID NO: 19). NO: 70-74, 100-104, 130-134)-PBS1 (SEQ ID NO: 75-79, 105-109, 135-139)-PP7 (SEQ ID NO: 18)-poly T (SEQ ID NO: 19)-hU6 promoter (SEQ ID NO: 17)-coding sequence of the same direction repeat sequence (SEQ ID NO: 156)-crRNA2 (SEQ ID NO: 80-84, 110-114, 140-144)-RTT2 (SEQ ID NO: 85-89, 115-119, 145-149)-PBS2 (SEQ ID NO: 90-94, 120-124, 150-154)-PP7 (SEQ ID NO: 18)-poly T (SEQ ID NO: 19).

[0129] 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:

[0130] (1) Animal cell cryopreservation

[0131] 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.

[0132] 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;

[0133] 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.

[0134] 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.

[0135] 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.

[0136] (2) Animal cell resuscitation

[0137] 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.

[0138] 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.

[0139] 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.

[0140] d. After culturing for 24 h, replace the cell culture medium with complete culture medium.

[0141] (3) Animal cell passage

[0142] 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.

[0143] 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.

[0144] c. Add an appropriate amount of basal culture medium containing 10% fetal bovine serum to the culture dish to terminate the reaction;

[0145] d. Aspirate the cells into a 15 mL centrifuge tube, set the centrifuge parameters to 500 g, and centrifuge for 5 min;

[0146] 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;

[0147] f. Pass cells every 48 hours, depending on the cell growth rate;

[0148] (4) Animal cell transfection

[0149] 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.

[0150] 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.

[0151] 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.

[0152] d. Mix the prepared reagents thoroughly and incubate at room temperature for 15-20 min;

[0153] 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;

[0154] 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).

[0155] g. 24 h after transfection, add Puromycin antibiotic, and collect cells after 48 h of drug screening culture.

[0156] (5) Animal cell collection and extraction

[0157] a. Preparation of reagents: Dilute 10× cell lysis buffer to 1×, add proteinase K to make a final concentration of 0.2 mg / mL;

[0158] b. After cell culture is complete, gently shake the culture plate and use a pipette to remove the culture medium;

[0159] c. Add 60-80 μL of 1× cell lysis buffer containing proteinase K to the cell culture plate and shake the cell culture plate.

[0160] 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.

[0161] Table 12: Animal Cell DNA Extraction Procedure

[0162] The editing efficiency of the collected animal cells was tested, and the specific steps are as follows:

[0163] (1) Amplify the target gene sequence

[0164] 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” to the 5' end. When synthesizing the R primer, add the adapter sequence “ACGTGTGCTCTTCCGATCT” to the 5' end. The amplified fragment size is approximately 230 bp.

[0165] b. The amplification system is shown in Table 13 below;

[0166] Table 13: NGS one-round amplification system

[0167] c. The amplification procedure is shown in Table 14 below;

[0168] Table 14: NGS One-Round Amplification Procedure

[0169] 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.

[0170] (2) Second round of amplification with added index sequences

[0171] 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.

[0172] (3) Constructing a library

[0173] 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.

[0174] (4) Sequencing results analysis

[0175] 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.

[0176] 2.2 Test Results

[0177] Results of different editing efficiencies and editing by-product levels in various genes are as follows: Figures 3A-3F As shown.

[0178] The REDRAW strategy detected accurate point mutation editing events at all five test targets, but its overall editing efficiency was relatively low. The Dual-REDRAW strategy, on the other hand, showed significantly higher editing efficiency than the REDRAW strategy across all five tested targets. Specifically, in point mutation editing ( Figure 3A The editing efficiency of three targets was significantly higher than that of the REDRAW strategy. The most significant difference was observed at the EMX1 site, where the precision editing efficiency improved from an average of 0.18% to an average of 0.73%. This was followed by the VEGFA target, which saw an improvement from an average of 0.25% to an average of 0.63%, a change of approximately 2.52-fold. The TRAC2 site showed a 2.02-fold increase in editing efficiency compared to its original state (P<0.05). In deletion editing (… Figure 3C In the replacement editing type () the optimization effect of the Dual-REDRAW strategy is very significant, with editing efficiency of nearly tens of times improved for all targets, and the differences are all highly significant. Among them, VEGFA site improved by 7.41 times, TRAC2 site by 55.77 times, EMX1 site by 28.78 times, B2M site by 10.11 times, and HPRT site by 23.35 times. Figure 3EIn this study, compared to the guided editing system mediated by the REDRAW strategy, which only achieved precise editing efficiency at the B2M site, the Dual-REDRAW strategy achieved precise editing at all target sites with a several-fold improvement. The differences were highly significant at the VEGFA, EMX1, and B2M sites, with precise editing efficiencies increasing by 28.48 times, 52.79 times, and 18.53 times, respectively. The TRAC2 and HPRT sites also saw improvements of 32.3 times and 8.89 times, respectively, all at a statistically significant level. At the EMX1 site, the Dual-REDRAW strategy achieved a precise deletion of a 99 bp fragment with an editing efficiency of approximately 2.5%; further, it mediated a precise insertion of a 30 bp exogenous sequence based on this 99 bp deletion, with an editing efficiency of approximately 0.8%. These results demonstrate that the Dual-REDRAW strategy possesses the ability to mediate precise editing at the base pair level.

[0179] 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.

[0180] like Figure 4A As shown, the Dual-REDRAW 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-REDRAW strategy was on average 2.47 times that of the REDRAW strategy, a statistically significant difference (p<0.01); Deletion editing: The editing efficiency of Dual-REDRAW was increased to 13.46 times that of the REDRAW strategy (p<0.0001), breaking through the technical bottleneck of the extremely low efficiency of the REDRAW strategy in fragment deletion editing; Replacement editing: The editing efficiency of the Dual-REDRAW strategy reached 20.73 times that of the REDRAW strategy (p<0.001). Most importantly, the REDRAW strategy is almost unable to achieve precise fragment replacement at the hundred-base-pair level, while the Dual-REDRAW strategy successfully overcomes this editing length limitation, broadening the editing scope of the REDRAW strategy.

[0181] like Figure 4B As shown, while the Dual-REDRAW strategy improves editing efficiency, the amount of byproducts does not increase proportionally: in point mutation editing, there is no statistically significant difference in byproduct levels between the Dual-REDRAW and REDRAW strategies (p>0.05); in deletion editing, byproducts increase by only 1.67 times (p<0.001), far lower than the 13.46-fold increase in editing efficiency; in replacement editing, byproduct levels increase by 1.66 times (p<0.001), also far lower than the 20.73-fold increase in editing efficiency.

[0182] In summary, the dual-REDRAW strategy improves editing efficiency for all three editing types, especially for fragment deletion and substitution editing, where the optimization effect can be dozens of times greater. Furthermore, the dual-REDRAW strategy expands the precision editing range from tens of base pairs to hundreds of base pairs, broadening its application scope.

[0183] 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) A fusion protein comprising a reverse transcriptase and a Casδ protein, wherein the reverse transcriptase is linked to the N-terminus or C-terminus of the Casδ protein via a peptide linker, and the sequence of the Casδ protein is shown in SEQ ID NO: 8; (2) A first guide nucleic acid comprising: (i) a first crRNA containing a first guide sequence that hybridizes to a first target site on the positive strand of the target gene; and (ii) an extension containing a first primer binding site and a first reverse transcriptase template; (3) A second guide nucleic acid comprising: (i) a second crRNA containing a second guide sequence that hybridizes to a second target site on the antisense strand of the target gene; and (ii) an extension containing a second primer binding site and a second reverse transcriptase template; 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 second crRNA on 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 first crRNA on the target gene. The first and second target sites 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 fusion protein further comprises a single-stranded DNA-binding protein and a nuclear localization signal sequence, wherein the single-stranded DNA-binding protein is selected from Brex27, hRad51, hRad52, BsRecA, EcRecA, and T4SSB.

3. The system according to claim 1 or 2, wherein, The 3' ends of the first and second guide nucleic acids also contain stem-loop structures or pseudoknot structures.

4. A combination of nucleic acid molecules comprising: (1) A first nucleic acid sequence comprising a nucleotide sequence encoding a fusion protein of any one of claims 1-3; (2) A second nucleic acid sequence comprising a nucleotide sequence for expressing a first guide nucleic acid of any one of claims 1-3; (3) A third nucleic acid sequence comprising a nucleotide sequence for expressing a second guide nucleic acid of any one of claims 1-3.

5. The nucleic acid molecule combination according to claim 4, wherein, The first, second, and third nucleic acid sequences are each operatively operably linked to a promoter at the 5' end and operatively linked to a terminator at the 3' end.

6. The nucleic acid molecule combination according to claim 5, wherein, 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 a reverse transcriptase, a nucleotide sequence encoding a peptide linker, a nucleotide sequence encoding Casδ, a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence encoding Brex27, and a terminator sequence. The second nucleic acid sequence from the 5' end to the 3' end includes: a promoter sequence, a nucleotide sequence for expressing the first crRNA, 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 stem-loop structure or pseudoknot structure, and a terminator sequence; The third nucleic acid sequence, from the 5' end to the 3' end, includes: a promoter sequence, a nucleotide sequence for expressing the second crRNA, 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 stem-loop structure or pseudoknot structure, and a terminator sequence.

7. An expression vector comprising the nucleic acid molecule combination according to any one of claims 4-6, wherein, The first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are each independently located in the same or different expression vectors.

8. A method for modifying a target gene for non-diagnostic therapeutic purposes, comprising: The system of any one of claims 1-3, the combination of nucleic acid molecules of any one of claims 4-6, or the expression vector of claim 7 are 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.

9. Use of the system according to any one of claims 1-3, the nucleic acid molecule combination according to any one of claims 4-6, or the expression vector according to claim 7 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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