Gene editing system and use thereof

Abstract

Provided is a trackable, robust and universal prime editing system TRU-PE, which comprises a vector. The vector comprises a gene encoding a guide RNA (gRNA) targeting a target gene locus, a tRNA (Transfer RNA), and a gene encoding a selection marker. The selection marker does not comprise a resistance gene. In the system, cells expressing editing elements are enriched by means of fluorescence guidance, such that the editing efficiency can be significantly improved when DNA or viral delivery methods are used. By using a split-PE design and a pegRNA architecture processed by hCtRNA, the TRU-PE system achieves simultaneous editing of up to 10 gene loci, with the editing efficiency being improved by up to 20 fold, effectively expanding the application range in hard-to-transfect cell types and complex gene loci. The gene editing system overcomes the application bottlenecks of prime editing technology in terms of size, delivery efficiency and multiplex editing. Moreover, the system is compatible with a variety of prime editor architectures and has wide applicability.

Description

A gene editing system and its application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a gene editing system and its applications. Background Technology

[0002] Gene editing technology has demonstrated immense potential in basic research and clinical applications, particularly in the treatment of genetic diseases. However, existing gene editing technologies, especially multi-site editing, still face several key challenges. First, the large size of gene editing vectors leads to lower delivery efficiency in different cell lines, thus limiting the overall effectiveness of gene editing. Furthermore, while traditional screening strategies (such as enriching cells successfully delivered with gene-editing elements using the antibiotic puromycin) can identify some successfully transfected cells, this process is time-consuming, generates high background noise, and may activate the p53 pathway, further reducing the efficiency of Prime Editor (PE)-mediated gene editing.

[0003] More importantly, existing PE-based multi-site editing technologies achieve multi-site editing by using hCtRNA to drive the co-expression of multiple pegRNAs. However, their effectiveness in mammalian cells is limited, typically only able to process a maximum of three sites. This limitation makes it difficult to meet the multi-gene editing needs of studying complex polygenic diseases (such as coronary heart disease, Parkinson's disease, and myelodysplastic syndromes), thus hindering in-depth research into the pathogenesis and gene interactions of these diseases. Summary of the Invention

[0004] The first aspect of this invention aims to provide the use of fluorescent proteins as screening markers in the preparation of gene editing systems.

[0005] The second aspect of this invention is to provide a gene editing system.

[0006] A third aspect of the present invention aims to provide a delivery system.

[0007] The fourth aspect of the present invention is to provide a cell.

[0008] The fifth aspect of this invention aims to provide the gene editing system of the second aspect of this invention, the delivery system of the third aspect of this invention, or the application of the cell of the fourth aspect of this invention.

[0009] The sixth aspect of this invention aims to provide a product.

[0010] The seventh aspect of this invention aims to provide a method for editing nucleic acid molecules.

[0011] An eighth aspect of the present invention aims to provide a method for constructing plant, animal or cell models.

[0012] The ninth aspect of this invention aims to provide a method for treating gene-related diseases.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A first aspect of the invention provides the use of fluorescent proteins as, or in the preparation of, screening markers for gene editing systems.

[0015] In some embodiments, the fluorescent protein comprises green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, AzamiGreen, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent protein (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent protein (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent protein (e.g., CFP, eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), and red fluorescent protein (e.g., RFP, eRFP, mKate, m...). At least one of Kate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, monomer Kusabira-Orange, mTangerine, tdTomato), and luciferase; further comprising at least one of eGFP, RFP, and BFP.

[0016] In some embodiments, the gene editing system includes a pilot editing (PE) system.

[0017] In some embodiments, the pilot editing system includes at least one of PE1, PE2, PE3, PEmax, PE4, PE5, PE6 (e.g., PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, PE6g), and PE7; further, it includes at least one of PE5max, PE6e, and PE7.

[0018] A second aspect of the present invention provides a gene editing system comprising a vector;

[0019] The vector contains: a gene encoding a guide RNA (gRNA) targeting a specific gene site, a tRNA (transfer RNA), and a gene encoding a selection marker;

[0020] The selection marker does not contain resistance genes.

[0021] In this invention, tRNA is used to drive the expression of genes encoding guide editing RNA (pegRNA) targeting target gene sites and / or nicking sgRNA (nicking RNA) targeting target gene sites.

[0022] This invention utilizes tRNA (Transfer RNA) and genes encoding selection markers (excluding resistance genes) in a gene editing system to screen successfully transfected cells using flow cytometry. This offers significant advantages over traditional puromycin resistance screening: it significantly improves the accuracy and efficiency of gene-edited cell screening without activating intracellular signaling pathways such as p53 and minimizing cytotoxicity. By using tRNA to drive the expression of one or more guide RNAs (pegRNAs) encoding target gene sites, this lead editing system can achieve gene editing at up to 10 sites. Compared to reported MPE systems, it not only achieves higher gene editing completion (meaning a higher proportion of Homozygous genotype) but also more efficiently enables simultaneous editing of multiple gene sites.

[0023] In some embodiments, the pilot editing system is the pilot editing system of the first aspect of the present invention.

[0024] In some embodiments, the gene editing system comprises a CRISPR / Cas9 system; further comprises at least one of a cytosine base editing system, an adenine base editing system, a guanine base editing system, and a lead editing system; and even further comprises a lead editing system.

[0025] In some embodiments, the screening marker is a fluorescent protein or a mutant or truncated form thereof.

[0026] In some embodiments, the fluorescent protein is the fluorescent protein of the first aspect of the present invention.

[0027] In some embodiments, the tRNA is hCtRNA (human cysteine ​​tRNA).

[0028] In some embodiments, the nucleotide sequence of the hCtRNA comprises:

[0029] a1)SEQ ID NO:253; or

[0030] a2) A nucleotide sequence of SEQ ID NO:253 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:253.

[0031] In some embodiments, the vector comprises: an expression cassette encoding a gene encoding a Cas9 nickase (nCas9), a gene encoding a reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding a fluorescent protein or a mutant or truncated form thereof.

[0032] The expression cassette of the gene encoding RNA targeting the target gene site includes: tRNA (Transfer RNA) and a gene encoding guide RNA (pegRNA) targeting the target gene site.

[0033] In this invention, a fluorescent protein mutant refers to a mutant that cannot express fluorescent protein on its own, but can express fluorescent protein after being edited with pegRNA targeting the fluorescent protein mutant.

[0034] In some embodiments, when the screening marker includes a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0035] In some embodiments, when the screening marker includes a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes a promoter and a pegRNA targeting the fluorescent protein mutant.

[0036] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0037] In this invention, fluorescent protein mutants can be used to screen samples that have undergone gene editing events, thereby improving the editing efficiency of the samples.

[0038] In some embodiments, the pilot editing system includes at least one of PE1, PE2, PE3, PEmax, PE4, PE5, PE6 (e.g., PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, PE6g), and PE7; further, it includes at least one of PE5max, PE6e, and PE7.

[0039] “PE1” refers to a PE complex containing a fusion protein with the following structure: the fusion protein contains Cas9(H840A) and wild-type MMLVRT: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]+ pegRNA targeting the target gene site;

[0040] "PE2" refers to a PE complex containing a fusion protein with the following structure: the fusion protein contains Cas9(H840A) and the variant MMLV_RT:(NLS)-[(Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+ pegRNA targeting the target gene site;

[0041] “PE3” refers to PE2 plus a second-strand nicking guide RNA (nicking RNA that targets the target gene site). It combines with PE2 and introduces a nick into the non-edited DNA strand to induce preferential substitution of the edited strand.

[0042] “PE4” refers to a system containing trans-expressed PE2 plus MLH1 dominant-negative protein (i.e., wild-type MLH1 with amino acid 754-756 truncated, which may be referred to as “MLH1Δ754-756” or “MLH1dn” in this article);

[0043] “PE5” refers to a system that includes trans-expressed PE3 plus MLH1 dominant-negative protein (i.e., wild-type MLH1 with amino acid 754-756 truncated, which may be called “MLH1Δ754-756” or “MLH1dn”).

[0044] "PEmax" refers to a PE complex containing a fusion protein, which includes Cas9 (R221K, N39K, H840A) and the variant MMLV_RT five mutants (D200N, T306K, W313F, T330P, L603W), and has the following structure: [Bis-NLS]-[Cas9(R221K)(N394K)(H840A)]-[Linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[Bis-NLS]-[NLS]+pegRNA targeting the target gene site;

[0045] "PE5max" refers to PE5 using the PEmax architecture;

[0046] “PE6” is a small-volume reverse transcriptase that replaces MMLV with a highly efficient one and is further optimized by mutating Cas9 (Doman, JL, et al. (2023). "Phage-assisted evolution and protein engineering yield compact, efficient prime editors." Cell 186(18):3983-4002.e3926.);

[0047] “PE7” was created by binding the N-terminal domain of the La protein (specifically amino acid sequence 1 to 194, denoted as La(1-194)) to PEmax (Yan, J., Oyler-Castrillo, P., Ravisankar, P. et al. Improving prime editing with an endogenous small RNA-binding protein. Nature 628, 639–647 (2024)).

[0048] In some implementations...

[0049] 1) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in a vector, said vector containing genes encoding one or more fluorescent proteins or their mutants or truncated forms; or

[0050] 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in two vectors;

[0051] 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing an expression cassette encoding RNA targeting a gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0052] 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or

[0053] 3) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in three vectors;

[0054] 31) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein; the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins; the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0055] 32) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) further contains a gene encoding fragment 2 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; fragment 1 of the first fluorescent protein or a mutant of the first fluorescent protein and fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein constitute the complete first fluorescent protein or a mutant of the first fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein and the second fluorescent protein are different (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0056] In some embodiments, the carrier comprises: a first carrier;

[0057] 1) The first vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a target gene site, and a gene encoding a fluorescent protein or a mutant or a truncated form of it (preferably a fluorescent protein mutant);

[0058] 2) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and a gene encoding RNA targeting a specific gene site; fragment 1 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein or its mutant or its truncated form.

[0059] In some embodiments, the carrier comprises: a first carrier and a second carrier;

[0060] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant); the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant), and an expression cassette encoding RNA targeting the target gene site; fragment 1 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) constitute the complete fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant); or

[0061] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and an expression cassette encoding RNA targeting the target gene site; the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), and a gene encoding fragment 2 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant); fragment 1 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) constitute the complete fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant);

[0062] 3) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), a gene encoding reverse transcriptase (RT), and a gene encoding fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein); fragment 1 of the first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein) and fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein) constitute the complete first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0063] 4) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein or a truncated form of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0064] In this invention, by fusing fragment 1 and fragment 2 of fluorescent protein, which can form a complete fluorescent protein, with Cas9 nickase (nCas9) or reverse transcriptase (RT) respectively, samples containing the complex of Cas9 nickase (nCas9) and reverse transcriptase (RT) can be screened by fluorescent protein, thereby improving the efficiency of sample editing.

[0065] In some embodiments, the carrier includes: a first carrier, a second carrier, and a third carrier;

[0066] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0067] The second vector contains (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT) and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0068] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein (preferably a mutant of the third fluorescent protein);

[0069] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths);

[0070] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0071] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT) and a gene encoding segment 2 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0072] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0073] The first fluorescent protein or fragment 1 of the first fluorescent protein mutant and fragment 2 of the first fluorescent protein or the first fluorescent protein mutant constitute the complete first fluorescent protein or the first fluorescent protein mutant; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein and the second fluorescent protein are different (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0074] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0075] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0076] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0077] In some embodiments, the vector further comprises a gene encoding the MLH1dn protein (when the lead editing system includes PE4, PE5 (e.g., PE5max)).

[0078] In some embodiments, the vector comprises: an expression cassette encoding a gene encoding a Cas9 nickase (nCas9), a gene encoding a reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, a gene encoding an MLH1dn protein, and a gene encoding a fluorescent protein or a mutant or truncated form thereof.

[0079] The expression cassette of the gene encoding RNA targeting the target gene site includes the gene encoding guide editing RNA (pegRNA) targeting the target gene site and tRNA;

[0080] 1) An expression cassette containing a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding MLH1dn protein, wherein the vector contains genes encoding one or more fluorescent proteins, mutants thereof, or truncated forms thereof; or

[0081] 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), the gene encoding RNA targeting the target gene site, and the gene encoding MLH1dn protein are located in two vectors.

[0082] 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the vector encoding MLH1dn protein further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and the vector containing an expression cassette encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein or a truncated form of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0083] 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or

[0084] 3) The expression cassettes of the genes encoding Cas9 nickase (nCas9), reverse transcriptase (RT), RNA targeting the target gene site, and MLH1dn protein are located in three vectors.

[0085] 31) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) and the vector encoding MLH1dn protein further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein; the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins; the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0086] 32) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) and the vector encoding MLH1dn protein further contains a gene encoding fragment 2 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein, wherein the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0087] In some embodiments, the carrier comprises: a first carrier;

[0088] 1) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, a gene encoding RNA targeting the target gene site, and a gene encoding a fluorescent protein or a mutant or a truncated form of the fluorescent protein (preferably a fluorescent protein mutant).

[0089] 2) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette of: a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant), a gene encoding MLH1dn protein, and a gene encoding RNA targeting the target gene site; fragment 1 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant) and fragment 2 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant) constitute the complete fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant).

[0090] In some embodiments, the carrier comprises: a first carrier and a second carrier;

[0091] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant); the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant), a gene encoding MLH1dn protein, and an expression cassette encoding RNA targeting the target gene site; fragment 1 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein; or

[0092] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and an expression cassette encoding RNA targeting the target gene site; the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and a gene encoding MLH1dn protein; fragment 1 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein;

[0093] 3) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 encoding a first fluorescent protein or a first fluorescent protein mutant (preferably a first fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 encoding the first fluorescent protein or a first fluorescent protein mutant (preferably a first fluorescent protein mutant), and a gene encoding MLH1dn protein; the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, a gene encoding a second fluorescent protein or The gene of a second fluorescent protein mutant (preferably a second fluorescent protein mutant); fragment 1 of the first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant) and fragment 2 of the first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant) constitute the complete first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0094] 4) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0095] In some embodiments, the carrier includes: a first carrier, a second carrier, and a third carrier;

[0096] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0097] The second vector contains (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0098] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein (preferably a mutant of the third fluorescent protein);

[0099] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths);

[0100] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0101] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and a gene encoding MLH1dn protein;

[0102] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein or a mutant or truncated form of it (preferably a mutant of the second fluorescent protein);

[0103] The first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0104] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0105] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0106] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0107] In some embodiments, the vector further comprises: a gene encoding a nicking sgRNA (nicking RNA) targeting a specific gene site (when the lead editing system includes PE3, PE5, PE6 (e.g., PE6e), PE7).

[0108] In some embodiments, the vector comprises: an expression cassette encoding a gene encoding a Cas9 nickase (nCas9), a gene encoding a reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, a gene encoding an MLH1dn protein, and a gene encoding a fluorescent protein or a mutant or truncated form thereof.

[0109] The expression cassette of the gene encoding RNA targeting the target gene site includes a gene encoding a guide editing RNA (pegRNA) targeting the target gene site, a gene encoding a nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA;

[0110] 1) An expression cassette containing a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding MLH1dn protein, wherein the vector contains genes encoding one or more fluorescent proteins, mutants thereof, or truncated forms thereof; or

[0111] 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), the gene encoding RNA targeting the target gene site, and the gene encoding MLH1dn protein are located in two vectors.

[0112] 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the vector encoding MLH1dn protein further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and the vector containing an expression cassette encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein or a truncated form of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0113] 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or

[0114] 3) The expression cassettes of the genes encoding Cas9 nickase (nCas9), reverse transcriptase (RT), RNA targeting the target gene site, and MLH1dn protein are located in three vectors.

[0115] 31) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) and the vector encoding MLH1dn protein further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein; the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins; the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0116] 32) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a first fluorescent protein mutant fragment 1; the vector containing the gene encoding reverse transcriptase (RT) and the vector encoding MLH1dn protein further contains a gene encoding a first fluorescent protein or a first fluorescent protein mutant fragment 2; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a second fluorescent protein mutant, wherein the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0117] In some embodiments, the carrier comprises: a first carrier;

[0118] 1) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, a gene encoding RNA targeting the target gene site, and a gene encoding a fluorescent protein or a mutant or a truncated form of the fluorescent protein (preferably a fluorescent protein mutant).

[0119] 2) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette of: a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant), a gene encoding MLH1dn protein, and a gene encoding RNA targeting the target gene site; fragment 1 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant) and fragment 2 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant) constitute the complete fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant).

[0120] In some embodiments, the carrier comprises: a first carrier and a second carrier;

[0121] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant); the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant), a gene encoding MLH1dn protein, and an expression cassette encoding RNA targeting the target gene site; fragment 1 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein; or

[0122] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and an expression cassette encoding RNA targeting the target gene site; the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and a gene encoding MLH1dn protein; fragment 1 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein;

[0123] 3) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and a gene encoding MLH1dn protein; the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein. Or the gene of a second fluorescent protein mutant (preferably a second fluorescent protein mutant); fragment 1 of the first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant) and fragment 2 of the first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant) constitute the complete first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0124] 4) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0125] In some embodiments, the carrier includes: a first carrier, a second carrier, and a third carrier;

[0126] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0127] The second vector contains (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0128] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein (preferably a mutant of the third fluorescent protein);

[0129] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths).

[0130] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0131] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and a gene encoding MLH1dn protein;

[0132] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein or a mutant or truncated form of it (preferably a mutant of the second fluorescent protein);

[0133] The first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0134] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0135] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0136] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0137] In some implementations, the pilot editing system is PE5max.

[0138] In some embodiments, the carrier includes: a first carrier; (the corresponding pre-editing system is named uPE5max)

[0139] The first vector comprises: an expression cassette encoding a gene for Cas9 nickase (nCas9), a gene for reverse transcriptase (RT), a gene for RNA that targets a gene site, a gene for MLH1dn protein, and a gene for fluorescent protein or a mutant or truncated form thereof.

[0140] The expression cassette of the gene encoding RNA targeting the target gene site includes: a gene encoding guide editing RNA (pegRNA) targeting the target gene site, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA.

[0141] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, a gene encoding RNA targeting a specific gene site, and a gene encoding a fluorescent protein or a mutant or truncated form thereof (preferably a fluorescent protein mutant).

[0142] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end) an expression cassette of: a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or a mutant or a truncated form (preferably a fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or a mutant or a truncated form (preferably a fluorescent protein mutant), a gene encoding MLH1dn protein, and a gene encoding RNA targeting a specific gene site; wherein fragment 1 of the fluorescent protein or a mutant or a truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or a mutant or a truncated form (preferably a fluorescent protein mutant) constitute the complete fluorescent protein or a mutant or a truncated form (preferably a fluorescent protein mutant).

[0143] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0144] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0145] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0146] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, and a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site.

[0147] In some embodiments, the vector comprises: a first vector; the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, an expression cassette encoding a Cas9 nickase (nCas9), a gene encoding a reverse transcriptase (RT), a gene encoding an MLH1dn protein, a gene encoding RNA targeting a specific gene site, and a gene encoding a fluorescent protein or a mutant or truncated form thereof.

[0148] In some embodiments, the vector comprises: a first vector; the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, an expression cassette encoding RNA targeting a specific gene site, and a gene encoding a fluorescent protein.

[0149] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (ttttttt).

[0150] In some embodiments, the first promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, at least one of the following: CMV promoter and EF-1α promoter (SEQ ID NO: 297); and even further, a CMV promoter.

[0151] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0152] b1)SEQ ID NO:244; or

[0153] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0154] In some embodiments, the fluorescent protein is EGFP.

[0155] In some embodiments, the nucleotide sequence of the EGFP comprises:

[0156] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[0157] c1)SEQ ID NO:256; or

[0158] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[0159] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0160] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[0161] d1)SEQ ID NO:246; or

[0162] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[0163] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0164] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[0165] e1)SEQ ID NO:248; or

[0166] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[0167] In some embodiments, the nucleotide sequence of the gene encoding the MLH1dn protein comprises:

[0168] f0) The coding sequence of the MLH1dn protein, wherein the amino acid sequence of the MLH1dn protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:252; or

[0169] f1)SEQ ID NO:252; or

[0170] f2) A nucleotide sequence of SEQ ID NO:252 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:252.

[0171] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the fluorescent protein further include (preferably from the 5' end to the 3' end): a second promoter, an resistance gene, and a first 2A peptide.

[0172] In some embodiments, the second promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0173] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0174] g1)SEQ ID NO:254; or

[0175] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0176] In some embodiments, the resistance gene is Puro.

[0177] In some embodiments, the nucleotide sequence of the Puro comprises:

[0178] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0179] h1)SEQ ID NO:255; or

[0180] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0181] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0182] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0183] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0184] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0185] j1)SEQ ID NO:245; or

[0186] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0187] In some embodiments, a linker is further included between the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT).

[0188] In some embodiments, the nucleotide sequence of the linker comprises:

[0189] The coding sequence of the linker (k0), wherein the amino acid sequence of the linker is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:247; or

[0190] k1)SEQ ID NO:247; or

[0191] k2) A nucleotide sequence of SEQ ID NO:247 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:247.

[0192] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding MLH1dn protein further include (preferably from the 5' end to the 3' end): a second NLS, a third NLS, and a second 2A peptide.

[0193] In some implementations, the second NLS is a second SV40 NLS.

[0194] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0195] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0196] l1)SEQ ID NO:249; or

[0197] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0198] In some implementations, the third NLS is a C-Myc NLS.

[0199] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0200] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0201] n1)SEQ ID NO:250; or

[0202] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0203] In some embodiments, the first 2A peptide and the second 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, both the first 2A peptide and the second 2A peptide are P2A peptides.

[0204] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0205] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0206] i1)SEQ ID NO:251; or

[0207] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0208] In some implementations, the target gene loci are one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0209] In some embodiments, when there are multiple target gene loci, the expression frame of the gene encoding RNA targeting the target gene locus includes N expression frames of genes encoding RNA targeting the target gene locus, where N is the number of target gene loci; for example, the expression frame of the gene encoding RNA targeting the target gene locus includes: an expression frame of a gene encoding RNA targeting a first target gene locus, an expression frame of a gene encoding RNA targeting a second target gene locus, ..., an expression frame of a gene encoding RNA targeting the Nth target gene locus.

[0210] In some embodiments, the target gene site is FANCF, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:210, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:227.

[0211] In some embodiments, the target gene site is DNMT1, the sequence of the guide editing guide RNA (pegRNA) targeting the site-directed mutation (site-directed mutation + 5G / T) of the target gene site is shown in SEQ ID NO:215, and the sequence of the nicking sgRNA targeting the site-directed mutation (site-directed mutation + 5G / T) of the target gene site is shown in SEQ ID NO:232.

[0212] In some embodiments, the target gene site is ERCC6, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:211, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:228.

[0213] In some embodiments, the target gene site is EMX1, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:212, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:229.

[0214] In some embodiments, the carrier comprises: a first carrier and a second carrier; (the corresponding pre-editing system is named PE5maxGR)

[0215] 1) The first vector comprises: a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein;

[0216] The second vector comprises: an expression cassette encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; or

[0217] 2) The first vector comprises: a gene encoding Cas9 nickase (nCas9), a gene encoding a first fluorescent protein or a fragment 1 of a first fluorescent protein mutant, a gene encoding reverse transcriptase (RT), a gene encoding a first fluorescent protein or a fragment 2 of a first fluorescent protein mutant, and a gene encoding MLH1dn protein; the second vector comprises: an expression cassette encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein or a second fluorescent protein mutant; fragment 1 of the first fluorescent protein or its mutant or truncated form and fragment 2 of the first fluorescent protein or its mutant or truncated form constitute the complete first fluorescent protein or its mutant or truncated form; and

[0218] The expression cassette of the gene encoding RNA targeting the target gene site includes: a gene encoding guide editing RNA (pegRNA) targeting the target gene site, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA;

[0219] The first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0220] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding a first fluorescent protein or a fragment 1 of a first fluorescent protein mutant (preferably a first fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding a first fluorescent protein or a fragment 2 of a first fluorescent protein mutant (preferably a first fluorescent protein mutant), and a gene encoding MLH1dn protein; the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second The gene of a fluorescent protein or a mutant of a second fluorescent protein (preferably a mutant of a second fluorescent protein); fragment 1 of the first fluorescent protein or its mutant or its truncated form (preferably a mutant of a first fluorescent protein) and fragment 2 of the first fluorescent protein or its mutant or its truncated form (preferably a mutant of a first fluorescent protein) constitute the complete first fluorescent protein or its mutant or its truncated form (preferably a mutant of a first fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0221] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting a target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0222] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0223] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0224] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0225] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a first fluorescent protein.

[0226] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein.

[0227] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, and a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site.

[0228] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (ttttttt).

[0229] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): EF-1α promoter (SEQ ID NO:297), tRNA, gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (when the vector is a viral vector (e.g., a lentiviral vector)).

[0230] In some embodiments, the 5' end of the expression frame of the gene encoding RNA targeting the target gene site further includes: an EF-1α promoter ((SEQ ID NO:297) when the vector is a viral vector (e.g., a lentiviral vector)).

[0231] In some embodiments, the first promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, at least one of the following: CMV promoter and EF-1α promoter (SEQ ID NO: 297); and even further, a CMV promoter.

[0232] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0233] b1)SEQ ID NO:244; or

[0234] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0235] In some embodiments, the first fluorescent protein is EGFP.

[0236] In some embodiments, the nucleotide sequence of the EGFP comprises:

[0237] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[0238] c1)SEQ ID NO:256; or

[0239] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[0240] In some embodiments, the second fluorescent protein is RFP.

[0241] In some embodiments, the nucleotide sequence of the RFP comprises:

[0242] The encoding sequence of m0)RFP, wherein the amino acid sequence of the RFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:260; or

[0243] m1)SEQ ID NO:260; or

[0244] m2) A nucleotide sequence of SEQ ID NO:260 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:260.

[0245] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0246] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[0247] d1)SEQ ID NO:246; or

[0248] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[0249] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0250] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[0251] e1)SEQ ID NO:248; or

[0252] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[0253] In some embodiments, the nucleotide sequence of the gene encoding the MLH1dn protein comprises:

[0254] f0) The coding sequence of the MLH1dn protein, wherein the amino acid sequence of the MLH1dn protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:259; or

[0255] f1)SEQ ID NO:259; or

[0256] f2) A nucleotide sequence of SEQ ID NO:259 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:259.

[0257] In some embodiments, the gene encoding the MLH1dn protein and the gene encoding the first fluorescent protein further include a first 2A peptide.

[0258] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the second fluorescent protein further include (preferably from the 5' end to the 3' end): a second promoter, an resistance gene, and a second 2A peptide.

[0259] In some embodiments, the second promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0260] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0261] g1)SEQ ID NO:254; or

[0262] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0263] In some embodiments, the resistance gene is Puro.

[0264] In some embodiments, the nucleotide sequence of the Puro comprises:

[0265] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0266] h1)SEQ ID NO:255; or

[0267] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0268] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0269] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0270] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0271] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0272] j1)SEQ ID NO:245; or

[0273] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0274] In some embodiments, a linker is further included between the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT).

[0275] In some embodiments, the nucleotide sequence of the linker comprises:

[0276] The coding sequence of the linker (k0), wherein the amino acid sequence of the linker is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:247; or

[0277] k1)SEQ ID NO:247; or

[0278] k2) A nucleotide sequence of SEQ ID NO:247 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:247.

[0279] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding MLH1dn protein further include (preferably from the 5' end to the 3' end): a second NLS, a third NLS, and a third 2A peptide.

[0280] In some implementations, the second NLS is a second SV40 NLS.

[0281] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0282] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0283] l1)SEQ ID NO:249; or

[0284] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0285] In some implementations, the third NLS is a C-Myc NLS.

[0286] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0287] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0288] n1)SEQ ID NO:250; or

[0289] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0290] In some embodiments, the first 2A peptide, the second 2A peptide, and the third 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, and the third 2A peptide are all P2A peptides.

[0291] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0292] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0293] i1)SEQ ID NO:251; or

[0294] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0295] In some implementations, the target gene loci are one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0296] In some embodiments, when there are multiple target gene loci, the expression frame of the gene encoding RNA targeting the target gene locus includes N expression frames of genes encoding RNA targeting the target gene locus, where N is the number of target gene loci; for example, the expression frame of the gene encoding RNA targeting the target gene locus includes: an expression frame of a gene encoding RNA targeting a first target gene locus, an expression frame of a gene encoding RNA targeting a second target gene locus, ..., an expression frame of a gene encoding RNA targeting the Nth target gene locus.

[0297] In some embodiments, the target gene site is FANCF, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:210, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:227.

[0298] In some embodiments, the target gene site is DNMT1, the sequence of the guide editing guide RNA (pegRNA) targeting the site-directed mutation (site-directed mutation + 5G / T) of the target gene site is shown in SEQ ID NO:215, and the sequence of the nicking sgRNA targeting the site-directed mutation (site-directed mutation + 5G / T) of the target gene site is shown in SEQ ID NO:232.

[0299] In some embodiments, the target gene site is ERCC6, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:211, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:228.

[0300] In some embodiments, the target gene site is EMX1, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:212, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:229.

[0301] In some embodiments, the carrier comprises: a first carrier, a second carrier, and a third carrier; (the corresponding pilot editing systems are named TRU-PE5max and TRU-PE5max-N).

[0302] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein;

[0303] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein;

[0304] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein;

[0305] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0306] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein;

[0307] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a first fluorescent protein or a mutant of the first fluorescent protein, and a gene encoding MLH1dn protein;

[0308] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein or a mutant or truncated form thereof;

[0309] Fragment 1 of the first fluorescent protein or the first fluorescent protein mutant and fragment 2 of the first fluorescent protein or the first fluorescent protein mutant together constitute the complete first fluorescent protein or the first fluorescent protein mutant;

[0310] The first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); and

[0311] The expression cassette of the gene encoding RNA targeting the target gene site includes: a gene encoding guide editing RNA (pegRNA) targeting the target gene site, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA.

[0312] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0313] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0314] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0315] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding fragment 1 of a second fluorescent protein.

[0316] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): a second promoter, a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a second fluorescent protein, and a gene encoding MLH1dn protein.

[0317] In some embodiments, the third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein or a mutant or truncated form thereof.

[0318] In some embodiments, the first promoter and the second promoter are each independently selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; furthermore, the first promoter and the second promoter are both EF-1α promoter (SEQ ID NO:297) or CMV promoter; even further, the first promoter and the second promoter are both CMV promoters.

[0319] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0320] b1)SEQ ID NO:244; or

[0321] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0322] In some embodiments, the nucleotide sequence of the gene encoding fragment 1 of the second fluorescent protein comprises:

[0323] x0) The coding sequence of fragment 1 of the second fluorescent protein, wherein the amino acid sequence of fragment 1 of the second fluorescent protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:329; or

[0324] x1)SEQ ID NO:329; or

[0325] x2) A nucleotide sequence of SEQ ID NO:329 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:329.

[0326] In some embodiments, the nucleotide sequence of the gene encoding fragment 2 of the second fluorescent protein comprises:

[0327] y0) The coding sequence of fragment 2 of the second fluorescent protein, wherein the amino acid sequence of fragment 2 of the second fluorescent protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:330; or

[0328] y1)SEQ ID NO:330; or

[0329] y2) A nucleotide sequence of SEQ ID NO:330 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:330.

[0330] In some embodiments, the third fluorescent protein is RFP.

[0331] In some embodiments, the nucleotide sequence of the RFP comprises:

[0332] The encoding sequence of m0)RFP, wherein the amino acid sequence of the RFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:260; or

[0333] m1)SEQ ID NO:260; or

[0334] m2) A nucleotide sequence of SEQ ID NO:260 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:260.

[0335] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0336] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[0337] d1)SEQ ID NO:246; or

[0338] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[0339] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0340] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[0341] e1)SEQ ID NO:248; or

[0342] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[0343] In some embodiments, the nucleotide sequence of the gene encoding the MLH1dn protein comprises:

[0344] f0) The coding sequence of the MLH1dn protein, wherein the amino acid sequence of the MLH1dn protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:259; or

[0345] f1)SEQ ID NO:259; or

[0346] f2) A nucleotide sequence of SEQ ID NO:259 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:259.

[0347] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0348] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0349] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0350] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0351] j1)SEQ ID NO:245; or

[0352] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0353] In some embodiments, the gene encoding the Cas9 nickase (nCas9) and the gene encoding fragment 1 of the second fluorescent protein further include (preferably from the 5' end to the 3' end): a second NLS and a first 2A peptide.

[0354] In some implementations, the second NLS is a second SV40 NLS.

[0355] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0356] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:261; or

[0357] l1)SEQ ID NO:261; or

[0358] l2) A nucleotide sequence of SEQ ID NO:261 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:261.

[0359] In some embodiments, a third NLS is also included between the second promoter and the gene encoding reverse transcriptase (RT).

[0360] In some implementations, the third NLS (Nuclear localization sequence) is the third SV40 NLS.

[0361] In some embodiments, the nucleotide sequence of the third SV40 NLS comprises:

[0362] j0) The coding sequence of the third SV40 NLS, wherein the amino acid sequence of the third SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0363] j1)SEQ ID NO:245; or

[0364] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0365] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding MLH1dn protein further include (preferably from the 5' end to the 3' end): a fourth NLS, a fifth NLS, and a second 2A peptide.

[0366] In some implementations, the fourth NLS is a fourth SV40 NLS.

[0367] In some embodiments, the nucleotide sequence of the fourth SV40 NLS comprises:

[0368] l0) The coding sequence of the fourth SV40 NLS, wherein the amino acid sequence of the fourth SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0369] l1)SEQ ID NO:249; or

[0370] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0371] In some implementations, the fifth NLS is a C-Myc NLS.

[0372] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0373] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0374] n1)SEQ ID NO:250; or

[0375] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0376] In some embodiments, the gene encoding the MLH1dn protein and the gene encoding fragment 2 of the second fluorescent protein further include a third 2A peptide.

[0377] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the third fluorescent protein mutant further include (preferably from the 5' end to the 3' end): a third promoter, an resistance gene, and a fourth 2A peptide.

[0378] In some embodiments, the third promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0379] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0380] g1)SEQ ID NO:254; or

[0381] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0382] In some embodiments, the resistance gene is Puro.

[0383] In some embodiments, the nucleotide sequence of the Puro comprises:

[0384] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0385] h1)SEQ ID NO:255; or

[0386] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0387] In some embodiments, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are all P2A peptides.

[0388] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0389] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0390] i1)SEQ ID NO:251; or

[0391] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0392] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a first fluorescent protein.

[0393] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): a second promoter, a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein.

[0394] In some embodiments, the third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein mutant.

[0395] In some embodiments, the first promoter and the second promoter are each independently selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; furthermore, the first promoter and the second promoter are both EF-1α promoter (SEQ ID NO:297) or CMV promoter; even further, the first promoter and the second promoter are both CMV promoters.

[0396] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0397] b1)SEQ ID NO:244; or

[0398] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0399] In some embodiments, the first fluorescent protein is BFP.

[0400] In some embodiments, the nucleotide sequence of the BFP comprises:

[0401] c0) The coding sequence of BFP, wherein the amino acid sequence of BFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:262; or

[0402] c1)SEQ ID NO:262; or

[0403] c2) A nucleotide sequence of SEQ ID NO:262 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:262.

[0404] In some embodiments, the second fluorescent protein is EGFP.

[0405] In some embodiments, the nucleotide sequence of the EGFP comprises:

[0406] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[0407] c1)SEQ ID NO:256; or

[0408] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[0409] In some embodiments, the third fluorescent protein is RFP.

[0410] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0411] y1)SEQ ID NO:328; or

[0412] y2) A nucleotide sequence of SEQ ID NO:328 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:328.

[0413] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site further includes: pegRNA targeting the third fluorescent protein mutant.

[0414] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the third fluorescent protein mutant.

[0415] In some embodiments, the promoter and the pegRNA targeting the third fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0416] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:326.

[0417] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0418] y1)SEQ ID NO:331; or

[0419] y2) A nucleotide sequence of SEQ ID NO:331 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:331.

[0420] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:363.

[0421] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0422] y1)SEQ ID NO:332; or

[0423] y2) A nucleotide sequence of SEQ ID NO:332 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:332.

[0424] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:364.

[0425] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0426] y1)SEQ ID NO:333; or

[0427] y2) A nucleotide sequence of SEQ ID NO:333 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:333.

[0428] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:365.

[0429] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0430] y1)SEQ ID NO:334; or

[0431] y2) A nucleotide sequence of SEQ ID NO:334 with one or more nucleotide substitutions, deletions and / or additions, and having the same function as the nucleic acid molecule shown in SEQ ID NO:334.

[0432] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:366.

[0433] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0434] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[0435] d1)SEQ ID NO:246; or

[0436] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[0437] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0438] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[0439] e1)SEQ ID NO:248; or

[0440] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[0441] In some embodiments, the nucleotide sequence of the gene encoding the MLH1dn protein comprises:

[0442] f0) The coding sequence of the MLH1dn protein, wherein the amino acid sequence of the MLH1dn protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:259; or

[0443] f1)SEQ ID NO:259; or

[0444] f2) A nucleotide sequence of SEQ ID NO:259 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:259.

[0445] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0446] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0447] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0448] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0449] j1)SEQ ID NO:245; or

[0450] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0451] In some embodiments, the gene encoding the Cas9 nickase (nCas9) and the gene encoding the first fluorescent protein further include (preferably from the 5' end to the 3' end): a second NLS and a first 2A peptide.

[0452] In some implementations, the second NLS is a second SV40 NLS.

[0453] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0454] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:261; or

[0455] l1)SEQ ID NO:261; or

[0456] l2) A nucleotide sequence of SEQ ID NO:261 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:261.

[0457] In some embodiments, a third NLS is also included between the second promoter and the gene encoding reverse transcriptase (RT).

[0458] In some implementations, the third NLS (Nuclear localization sequence) is the third SV40 NLS.

[0459] In some embodiments, the nucleotide sequence of the third SV40 NLS comprises:

[0460] j0) The coding sequence of the third SV40 NLS, wherein the amino acid sequence of the third SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0461] j1)SEQ ID NO:245; or

[0462] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0463] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding MLH1dn protein further include (preferably from the 5' end to the 3' end): a fourth NLS, a fifth NLS, and a second 2A peptide.

[0464] In some implementations, the fourth NLS is a fourth SV40 NLS.

[0465] In some embodiments, the nucleotide sequence of the fourth SV40 NLS comprises:

[0466] l0) The coding sequence of the fourth SV40 NLS, wherein the amino acid sequence of the fourth SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0467] l1)SEQ ID NO:249; or

[0468] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0469] In some implementations, the fifth NLS is a C-Myc NLS.

[0470] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0471] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0472] n1)SEQ ID NO:250; or

[0473] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0474] In some embodiments, a third 2A peptide is further included between the gene encoding the MLH1dn protein and the gene encoding the second fluorescent protein.

[0475] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the third fluorescent protein mutant further include (preferably from the 5' end to the 3' end): a third promoter, an resistance gene, and a fourth 2A peptide.

[0476] In some embodiments, the third promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0477] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0478] g1)SEQ ID NO:254; or

[0479] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0480] In some embodiments, the resistance gene is Puro.

[0481] In some embodiments, the nucleotide sequence of the Puro comprises:

[0482] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0483] h1)SEQ ID NO:255; or

[0484] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0485] In some embodiments, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are all P2A peptides.

[0486] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0487] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0488] i1)SEQ ID NO:251; or

[0489] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0490] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a first fluorescent protein.

[0491] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): a second promoter, a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein.

[0492] In some embodiments, the third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein mutant.

[0493] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, and a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site.

[0494] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (ttttttt).

[0495] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): EF-1α promoter (SEQ ID NO:297), tRNA, gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (when the vector is a viral vector (e.g., a lentiviral vector)).

[0496] In some embodiments, the 5' end of the expression frame of the gene encoding RNA targeting the target gene site further includes: an EF-1α promoter ((SEQ ID NO:297) when the vector is a viral vector (e.g., a lentiviral vector)).

[0497] In some embodiments, the first promoter and the second promoter are each independently selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; furthermore, the first promoter and the second promoter are both EF-1α promoter (SEQ ID NO:297) or CMV promoter; even further, the first promoter and the second promoter are both CMV promoters.

[0498] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0499] b1)SEQ ID NO:244; or

[0500] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0501] In some embodiments, the first fluorescent protein is RFP.

[0502] In some embodiments, the nucleotide sequence of the RFP comprises:

[0503] The encoding sequence of m0)RFP, wherein the amino acid sequence of the RFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:260; or

[0504] m1)SEQ ID NO:260; or

[0505] m2) A nucleotide sequence of SEQ ID NO:260 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:260.

[0506] In some embodiments, the second fluorescent protein is EGFP.

[0507] In some embodiments, the nucleotide sequence of the EGFP comprises:

[0508] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[0509] c1)SEQ ID NO:256; or

[0510] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[0511] In some embodiments, the third fluorescent protein is BFP.

[0512] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0513] c1)SEQ ID NO:396; or

[0514] c2) A nucleotide sequence of SEQ ID NO:396 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:396.

[0515] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site further includes: pegRNA targeting the third fluorescent protein mutant.

[0516] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the third fluorescent protein mutant.

[0517] In some embodiments, the promoter and the pegRNA targeting the third fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0518] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:393.

[0519] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0520] y1)SEQ ID NO:397; or

[0521] y2) A nucleotide sequence of SEQ ID NO:397 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:397.

[0522] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:394.

[0523] In some embodiments, the nucleotide sequence of the third fluorescent protein mutant comprises:

[0524] y1)SEQ ID NO:398; or

[0525] y2) A nucleotide sequence of SEQ ID NO:398 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:398.

[0526] In some embodiments, the sequence of the pegRNA targeting the third fluorescent protein mutant is shown in SEQ ID NO:395.

[0527] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0528] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[0529] d1)SEQ ID NO:246; or

[0530] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[0531] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0532] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[0533] e1)SEQ ID NO:248; or

[0534] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[0535] In some embodiments, the nucleotide sequence of the gene encoding the MLH1dn protein comprises:

[0536] f0) The coding sequence of the MLH1dn protein, wherein the amino acid sequence of the MLH1dn protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:259; or

[0537] f1)SEQ ID NO:259; or

[0538] f2) A nucleotide sequence of SEQ ID NO:259 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:259.

[0539] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0540] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0541] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0542] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0543] j1)SEQ ID NO:245; or

[0544] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0545] In some embodiments, the gene encoding the Cas9 nickase (nCas9) and the gene encoding the first fluorescent protein further include (preferably from the 5' end to the 3' end): a second NLS and a first 2A peptide.

[0546] In some implementations, the second NLS is a second SV40 NLS.

[0547] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0548] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:261; or

[0549] l1)SEQ ID NO:261; or

[0550] l2) A nucleotide sequence of SEQ ID NO:261 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:261.

[0551] In some embodiments, a third NLS is also included between the second promoter and the gene encoding reverse transcriptase (RT).

[0552] In some implementations, the third NLS (Nuclear localization sequence) is the third SV40 NLS.

[0553] In some embodiments, the nucleotide sequence of the third SV40 NLS comprises:

[0554] j0) The coding sequence of the third SV40 NLS, wherein the amino acid sequence of the third SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0555] j1)SEQ ID NO:245; or

[0556] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0557] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding MLH1dn protein further include (preferably from the 5' end to the 3' end): a fourth NLS, a fifth NLS, and a second 2A peptide.

[0558] In some implementations, the fourth NLS is a fourth SV40 NLS.

[0559] In some embodiments, the nucleotide sequence of the fourth SV40 NLS comprises:

[0560] l0) The coding sequence of the fourth SV40 NLS, wherein the amino acid sequence of the fourth SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0561] l1)SEQ ID NO:249; or

[0562] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0563] In some implementations, the fifth NLS is a C-Myc NLS.

[0564] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0565] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0566] n1)SEQ ID NO:250; or

[0567] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0568] In some embodiments, a third 2A peptide is further included between the gene encoding the MLH1dn protein and the gene encoding the second fluorescent protein.

[0569] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the third fluorescent protein further include (preferably from the 5' end to the 3' end): a third promoter, an resistance gene, and a fourth 2A peptide.

[0570] In some embodiments, the third promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0571] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0572] g1)SEQ ID NO:254; or

[0573] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0574] In some embodiments, the resistance gene is Puro.

[0575] In some embodiments, the nucleotide sequence of the Puro comprises:

[0576] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0577] h1)SEQ ID NO:255; or

[0578] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0579] In some embodiments, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are all P2A peptides.

[0580] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0581] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0582] i1)SEQ ID NO:251; or

[0583] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0584] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a first fluorescent protein.

[0585] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): a second promoter, a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein.

[0586] In some embodiments, the third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein.

[0587] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, and a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site.

[0588] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (ttttttt).

[0589] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): EF-1α promoter (SEQ ID NO:297), tRNA, gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (when the vector is a viral vector (e.g., a lentiviral vector)).

[0590] In some embodiments, the 5' end of the expression frame of the gene encoding RNA targeting the target gene site further includes: an EF-1α promoter ((SEQ ID NO:297) when the vector is a viral vector (e.g., a lentiviral vector)).

[0591] In some embodiments, the first promoter and the second promoter are each independently selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; furthermore, the first promoter and the second promoter are both EF-1α promoter (SEQ ID NO:297) or CMV promoter; even further, the first promoter and the second promoter are both CMV promoters.

[0592] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0593] b1)SEQ ID NO:244; or

[0594] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0595] In some embodiments, the first fluorescent protein is BFP.

[0596] In some embodiments, the nucleotide sequence of the BFP comprises:

[0597] c0) The coding sequence of BFP, wherein the amino acid sequence of BFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:262; or

[0598] c1)SEQ ID NO:262; or

[0599] c2) A nucleotide sequence of SEQ ID NO:262 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:262.

[0600] In some embodiments, the second fluorescent protein is EGFP.

[0601] In some embodiments, the nucleotide sequence of the EGFP comprises:

[0602] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[0603] c1)SEQ ID NO:256; or

[0604] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[0605] In some embodiments, the third fluorescent protein is RFP.

[0606] In some embodiments, the nucleotide sequence of the RFP comprises:

[0607] The encoding sequence of m0)RFP, wherein the amino acid sequence of the RFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:260; or

[0608] m1)SEQ ID NO:260; or

[0609] m2) A nucleotide sequence of SEQ ID NO:260 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:260.

[0610] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0611] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[0612] d1)SEQ ID NO:246; or

[0613] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[0614] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0615] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[0616] e1)SEQ ID NO:248; or

[0617] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[0618] In some embodiments, the nucleotide sequence of the gene encoding the MLH1dn protein comprises:

[0619] f0) The coding sequence of the MLH1dn protein, wherein the amino acid sequence of the MLH1dn protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:259; or

[0620] f1)SEQ ID NO:259; or

[0621] f2) A nucleotide sequence of SEQ ID NO:259 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:259.

[0622] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0623] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0624] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0625] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0626] j1)SEQ ID NO:245; or

[0627] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0628] In some embodiments, the gene encoding the Cas9 nickase (nCas9) and the gene encoding the first fluorescent protein further include (preferably from the 5' end to the 3' end): a second NLS and a first 2A peptide.

[0629] In some implementations, the second NLS is a second SV40 NLS.

[0630] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0631] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:261; or

[0632] l1)SEQ ID NO:261; or

[0633] l2) A nucleotide sequence of SEQ ID NO:261 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:261.

[0634] In some embodiments, a third NLS is also included between the second promoter and the gene encoding reverse transcriptase (RT).

[0635] In some implementations, the third NLS (Nuclear localization sequence) is the third SV40 NLS.

[0636] In some embodiments, the nucleotide sequence of the third SV40 NLS comprises:

[0637] j0) The coding sequence of the third SV40 NLS, wherein the amino acid sequence of the third SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0638] j1)SEQ ID NO:245; or

[0639] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0640] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding MLH1dn protein further include (preferably from the 5' end to the 3' end): a fourth NLS, a fifth NLS, and a second 2A peptide.

[0641] In some implementations, the fourth NLS is a fourth SV40 NLS.

[0642] In some embodiments, the nucleotide sequence of the fourth SV40 NLS comprises:

[0643] l0) The coding sequence of the fourth SV40 NLS, wherein the amino acid sequence of the fourth SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0644] l1)SEQ ID NO:249; or

[0645] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0646] In some implementations, the fifth NLS is a C-Myc NLS.

[0647] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0648] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0649] n1)SEQ ID NO:250; or

[0650] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0651] In some embodiments, a third 2A peptide is further included between the gene encoding the MLH1dn protein and the gene encoding the second fluorescent protein.

[0652] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the third fluorescent protein further include (preferably from the 5' end to the 3' end): a third promoter, an resistance gene, and a fourth 2A peptide.

[0653] In some embodiments, the third promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0654] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0655] g1)SEQ ID NO:254; or

[0656] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0657] In some embodiments, the resistance gene is Puro.

[0658] In some embodiments, the nucleotide sequence of the Puro comprises:

[0659] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0660] h1)SEQ ID NO:255; or

[0661] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0662] In some embodiments, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are all P2A peptides.

[0663] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0664] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0665] i1)SEQ ID NO:251; or

[0666] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0667] In some implementations, the target gene loci are one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0668] In some embodiments, when there are multiple target gene loci, the expression frame of the gene encoding RNA targeting the target gene locus includes N expression frames of genes encoding RNA targeting the target gene locus, where N is the number of target gene loci; for example, the expression frame of the gene encoding RNA targeting the target gene locus includes: an expression frame of a gene encoding RNA targeting a first target gene locus, an expression frame of a gene encoding RNA targeting a second target gene locus, ..., an expression frame of a gene encoding RNA targeting the Nth target gene locus.

[0669] In some embodiments, the target gene site is FANCF, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:210, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:227.

[0670] In some embodiments, the target gene site is DNMT1, the sequence of the guide editing guide RNA (pegRNA) targeting the site-directed mutation (site-directed mutation + 5G / T) of the target gene site is shown in SEQ ID NO:215, and the sequence of the nicking sgRNA targeting the site-directed mutation (site-directed mutation + 5G / T) of the target gene site is shown in SEQ ID NO:232.

[0671] In some embodiments, the target gene site is DNMT1, the sequence of the guide editing RNA (pegRNA) targeting the deletion mutation (del1-15) at the target gene site is shown in SEQ ID NO:226, and the sequence of the nicking sgRNA targeting the deletion mutation (del1-15) at the target gene site is shown in SEQ ID NO:243.

[0672] In some embodiments, the target gene site is ERCC6, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:211, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:228.

[0673] In some embodiments, the target gene site is EMX1, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:212, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:229.

[0674] In some embodiments, the target gene site is KRAS, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:219, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:236.

[0675] In some embodiments, the target gene site is LRRK2, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:218, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:235.

[0676] In some embodiments, the target gene site is NMB, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:214, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:231.

[0677] In some embodiments, the target gene site is BAG3, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:213, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:230.

[0678] In some embodiments, the target gene site is GBA1, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:217, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:234.

[0679] In some embodiments, the target gene site is TP53, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:216, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:233.

[0680] In some embodiments, the target gene site is RNF2, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:220, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:237.

[0681] In some embodiments, the target gene sites are BAG3, NMB, and FANCF; wherein the sequence of the guide editing guide RNA (pegRNA) targeting BAG3 is shown in SEQ ID NO:213, the sequence of the nicking sgRNA targeting BAG3 is shown in SEQ ID NO:230, the sequence of the guide editing guide RNA (pegRNA) targeting NMB is shown in SEQ ID NO:214, the sequence of the nicking sgRNA targeting NMB is shown in SEQ ID NO:231, the sequence of the guide editing guide RNA (pegRNA) targeting FANCF is shown in SEQ ID NO:210, and the sequence of the nicking sgRNA targeting FANCF is shown in SEQ ID NO:227.

[0682] In some embodiments, the target gene sites are DNMT1, EMX1, and ERCC6; wherein, the sequence of the guide editing guide RNA (pegRNA) targeting the DNMT1 site-directed mutation (site-directed mutation + 5G / T) is shown in SEQ ID NO:215, the sequence of the nicking sgRNA targeting the DNMT1 site-directed mutation (site-directed mutation + 5G / T) is shown in SEQ ID NO:232, the sequence of the guide editing guide RNA (pegRNA) targeting EMX1 is shown in SEQ ID NO:212, the sequence of the nicking sgRNA targeting EMX1 is shown in SEQ ID NO:229, the sequence of the guide editing guide RNA (pegRNA) targeting ERCC6 is shown in SEQ ID NO:211, and the sequence of the nicking sgRNA targeting ERCC6 is shown in SEQ ID NO:228.

[0683] In some embodiments, the target gene sites are GATA2, SETBP1, and ASXL1; wherein the sequence of the guide editing guide RNA (pegRNA) targeting GATA2 is shown in SEQ ID NO:221, the sequence of the nicking sgRNA targeting GATA2 is shown in SEQ ID NO:238, the sequence of the guide editing guide RNA (pegRNA) targeting SETBP1 is shown in SEQ ID NO:222, the sequence of the nicking sgRNA targeting SETBP1 is shown in SEQ ID NO:239, the sequence of the guide editing guide RNA (pegRNA) targeting ASXL1 is shown in SEQ ID NO:223, and the sequence of the nicking sgRNA targeting ASXL1 is shown in SEQ ID NO:240.

[0684] In some embodiments, the target gene sites are BAG3, NMB, FANCF, DNMT1, EMX1, and ERCC6; wherein the sequence of the guide editing guide RNA (pegRNA) targeting BAG3 is shown in SEQ ID NO:213, the sequence of the nicking sgRNA targeting BAG3 is shown in SEQ ID NO:230, the sequence of the guide editing guide RNA (pegRNA) targeting NMB is shown in SEQ ID NO:214, the sequence of the nicking sgRNA targeting NMB is shown in SEQ ID NO:231, the sequence of the guide editing guide RNA (pegRNA) targeting FANCF is shown in SEQ ID NO:210, the sequence of the nicking sgRNA targeting FANCF is shown in SEQ ID NO:227, and the sequence of the guide editing guide RNA (pegRNA) targeting the site-directed mutation (site-directed mutation + 5G / T) of DNMT1 is shown in SEQ ID NO:227. As shown in NO:215, the sequence of the nicking sgRNA targeting the DNMT1 site-directed mutation (site-directed mutation +5G / T) is shown in SEQ ID NO:232, the sequence of the guide editing RNA (pegRNA) targeting EMX1 is shown in SEQ ID NO:212, the sequence of the nicking sgRNA targeting EMX1 is shown in SEQ ID NO:229, the sequence of the guide editing RNA (pegRNA) targeting ERCC6 is shown in SEQ ID NO:211, and the sequence of the nicking sgRNA targeting ERCC6 is shown in SEQ ID NO:228.

[0685] In some embodiments, the target gene sites are BAG3, NMB, FANCF, DNMT1, EMX1, ERCC6, TP53, GBA1, LRRK2, and KRAS; wherein the sequence of the guide editing guide RNA (pegRNA) targeting BAG3 is shown in SEQ ID NO:213, the sequence of the nicking sgRNA targeting BAG3 is shown in SEQ ID NO:230, the sequence of the guide editing guide RNA (pegRNA) targeting NMB is shown in SEQ ID NO:214, the sequence of the nicking sgRNA targeting NMB is shown in SEQ ID NO:231, the sequence of the guide editing guide RNA (pegRNA) targeting FANCF is shown in SEQ ID NO:210, the sequence of the nicking sgRNA targeting FANCF is shown in SEQ ID NO:227, and the sequence of the guide editing guide RNA (pegRNA) targeting the site-directed mutation (site-directed mutation + 5G / T) of DNMT1 is shown in SEQ ID NO:227. As shown in NO:215, the sequence of the nicking sgRNA targeting the DNMT1 site-directed mutation (site-directed mutation + 5G / T) is shown in SEQ ID NO:232; the sequence of the guide editing RNA (pegRNA) targeting EMX1 is shown in SEQ ID NO:212; the sequence of the nicking sgRNA targeting EMX1 is shown in SEQ ID NO:229; the sequence of the guide editing RNA (pegRNA) targeting ERCC6 is shown in SEQ ID NO:211; the sequence of the nicking sgRNA targeting ERCC6 is shown in SEQ ID NO:228; the sequence of the guide editing RNA (pegRNA) targeting TP53 is shown in SEQ ID NO:216; the sequence of the nicking sgRNA targeting TP53 is shown in SEQ ID NO:233; the sequence of the guide editing RNA (pegRNA) targeting GBA1 is shown in SEQ ID NO:217; and the sequence of the nicking sgRNA targeting GBA1 is shown in SEQ ID NO:218. As shown in NO:234, the sequence of the guide editing RNA (pegRNA) targeting LRRK2 is shown in SEQ ID NO:218, the sequence of the nicking sgRNA targeting LRRK2 is shown in SEQ ID NO:235, the sequence of the guide editing RNA (pegRNA) targeting KRAS is shown in SEQ ID NO:219, and the sequence of the nicking sgRNA targeting KRAS is shown in SEQ ID NO:236.

[0686] In some implementations, the pilot editing system is PE6.

[0687] In some embodiments, the vector comprises: an expression cassette encoding a gene encoding a Cas9 nickase (nCas9), a gene encoding a reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding a fluorescent protein or a mutant or truncated form thereof.

[0688] The expression cassette of the gene encoding RNA targeting the target gene site includes a gene encoding a guide editing RNA (pegRNA) targeting the target gene site, a gene encoding a nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA;

[0689] 1) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in a vector, said vector containing genes encoding one or more fluorescent proteins or their mutants or truncated forms; or

[0690] 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in two vectors;

[0691] 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0692] 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or

[0693] 3) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in three vectors;

[0694] 31) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein; the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins; the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0695] 32) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) further contains a gene encoding fragment 2 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; fragment 1 of the first fluorescent protein or a mutant of the first fluorescent protein and fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein constitute the complete first fluorescent protein or a mutant of the first fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein and the second fluorescent protein are different (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0696] In some embodiments, the carrier comprises: a first carrier;

[0697] 1) The first vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a target gene site, and a gene encoding a fluorescent protein or a mutant or a truncated form of it (preferably a fluorescent protein mutant);

[0698] 2) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and a gene encoding RNA targeting a specific gene site; fragment 1 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein or its mutant or its truncated form.

[0699] In some embodiments, the carrier comprises: a first carrier and a second carrier;

[0700] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant); the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant), and an expression cassette encoding RNA targeting the target gene site; fragment 1 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) constitute the complete fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant); or

[0701] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and an expression cassette encoding RNA targeting the target gene site; the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), and a gene encoding fragment 2 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant); fragment 1 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) constitute the complete fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant);

[0702] 3) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), a gene encoding reverse transcriptase (RT), and a gene encoding fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein); fragment 1 of the first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein) and fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein) constitute the complete first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0703] 4) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein or a truncated form of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0704] In this invention, by fusing fragment 1 and fragment 2 of fluorescent protein, which can form a complete fluorescent protein, with Cas9 nickase (nCas9) or reverse transcriptase (RT) respectively, samples containing the complex of Cas9 nickase (nCas9) and reverse transcriptase (RT) can be screened by fluorescent protein, thereby improving the efficiency of sample editing.

[0705] In some embodiments, the carrier includes: a first carrier, a second carrier, and a third carrier;

[0706] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0707] The second vector contains (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT) and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0708] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein (preferably a mutant of the third fluorescent protein);

[0709] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths);

[0710] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0711] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT) and a gene encoding segment 2 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0712] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0713] The first fluorescent protein or fragment 1 of the first fluorescent protein mutant and fragment 2 of the first fluorescent protein or the first fluorescent protein mutant constitute the complete first fluorescent protein or the first fluorescent protein mutant; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein and the second fluorescent protein are different (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0714] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0715] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0716] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0717] In some embodiments, the carrier comprises: a first carrier, a second carrier, and a third carrier; (the corresponding pre-editing system is named TRU-PE6e).

[0718] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein;

[0719] The second vector contains (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT) and a gene encoding a second fluorescent protein;

[0720] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein;

[0721] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0722] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0723] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT) and a gene encoding segment 2 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0724] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0725] Fragment 1 of the first fluorescent protein or a mutant of the first fluorescent protein and fragment 2 of the first fluorescent protein or a mutant of the first fluorescent protein constitute the complete first fluorescent protein or a mutant of the first fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); and

[0726] The expression cassette of the gene encoding RNA targeting the target gene site includes: a gene encoding guide editing RNA (pegRNA) targeting the target gene site, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA.

[0727] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a first fluorescent protein.

[0728] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): a second promoter, a gene encoding reverse transcriptase (RT), and a gene encoding a second fluorescent protein.

[0729] In some embodiments, the third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein.

[0730] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, and a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site.

[0731] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (ttttttt).

[0732] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): EF-1α promoter (SEQ ID NO:297), tRNA, gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (when the vector is a viral vector (e.g., a lentiviral vector)).

[0733] In some embodiments, the 5' end of the expression frame of the gene encoding RNA targeting the target gene site further includes: an EF-1α promoter ((SEQ ID NO:297) when the vector is a viral vector (e.g., a lentiviral vector)).

[0734] In some embodiments, the first promoter and the second promoter are each independently selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; furthermore, the first promoter and the second promoter are both EF-1α promoter (SEQ ID NO:297) or CMV promoter; even further, the first promoter and the second promoter are both CMV promoters.

[0735] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0736] b1)SEQ ID NO:244; or

[0737] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0738] In some embodiments, the first fluorescent protein is BFP.

[0739] In some embodiments, the nucleotide sequence of the BFP comprises:

[0740] c0) The coding sequence of BFP, wherein the amino acid sequence of BFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:262; or

[0741] c1)SEQ ID NO:262; or

[0742] c2) A nucleotide sequence of SEQ ID NO:262 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:262.

[0743] In some embodiments, the second fluorescent protein is EGFP.

[0744] In some embodiments, the nucleotide sequence of the EGFP comprises:

[0745] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[0746] c1)SEQ ID NO:256; or

[0747] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[0748] In some embodiments, the third fluorescent protein is RFP.

[0749] In some embodiments, the nucleotide sequence of the RFP comprises:

[0750] The encoding sequence of m0)RFP, wherein the amino acid sequence of the RFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:260; or

[0751] m1)SEQ ID NO:260; or

[0752] m2) A nucleotide sequence of SEQ ID NO:260 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:260.

[0753] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0754] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:264; or

[0755] d1)SEQ ID NO:264; or

[0756] d2) A nucleotide sequence of SEQ ID NO:264 with one or more nucleotide substitutions, deletions and / or additions, and having the same function as the nucleic acid molecule shown in SEQ ID NO:264.

[0757] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0758] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:265; or

[0759] e1)SEQ ID NO:265; or

[0760] e2) A nucleotide sequence of SEQ ID NO:265 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:265.

[0761] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0762] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0763] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0764] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0765] j1)SEQ ID NO:245; or

[0766] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0767] In some embodiments, the gene encoding the Cas9 nickase (nCas9) and the gene encoding the first fluorescent protein further include (preferably from the 5' end to the 3' end): a second NLS and a first 2A peptide.

[0768] In some implementations, the second NLS is a second SV40 NLS.

[0769] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0770] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:261; or

[0771] l1)SEQ ID NO:261; or

[0772] l2) A nucleotide sequence of SEQ ID NO:261 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:261.

[0773] In some embodiments, a third NLS is also included between the second promoter and the gene encoding reverse transcriptase (RT).

[0774] In some implementations, the third NLS (Nuclear localization sequence) is the third SV40 NLS.

[0775] In some embodiments, the nucleotide sequence of the third SV40 NLS comprises:

[0776] j0) The coding sequence of the third SV40 NLS, wherein the amino acid sequence of the third SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0777] j1)SEQ ID NO:245; or

[0778] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0779] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding the second fluorescent protein further include (preferably from the 5' end to the 3' end): a fourth NLS, a fifth NLS, and a second 2A peptide.

[0780] In some implementations, the fourth NLS is a fourth SV40 NLS.

[0781] In some embodiments, the nucleotide sequence of the fourth SV40 NLS comprises:

[0782] l0) The coding sequence of the fourth SV40 NLS, wherein the amino acid sequence of the fourth SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0783] l1)SEQ ID NO:249; or

[0784] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0785] In some implementations, the fifth NLS is a C-Myc NLS.

[0786] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0787] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0788] n1)SEQ ID NO:250; or

[0789] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0790] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the third fluorescent protein further include (preferably from the 5' end to the 3' end): a third promoter, an resistance gene, and a third 2A peptide.

[0791] In some embodiments, the third promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0792] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0793] g1)SEQ ID NO:254; or

[0794] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0795] In some embodiments, the resistance gene is Puro.

[0796] In some embodiments, the nucleotide sequence of the Puro comprises:

[0797] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0798] h1)SEQ ID NO:255; or

[0799] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0800] In some embodiments, the first 2A peptide, the second 2A peptide, and the third 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, and the third 2A peptide are all P2A peptides.

[0801] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0802] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0803] i1)SEQ ID NO:251; or

[0804] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0805] In some implementations, the target gene loci are one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0806] In some embodiments, when there are multiple target gene loci, the expression frame of the gene encoding RNA targeting the target gene locus includes N expression frames of genes encoding RNA targeting the target gene locus, where N is the number of target gene loci; for example, the expression frame of the gene encoding RNA targeting the target gene locus includes: an expression frame of a gene encoding RNA targeting a first target gene locus, an expression frame of a gene encoding RNA targeting a second target gene locus, ..., an expression frame of a gene encoding RNA targeting the Nth target gene locus.

[0807] In some embodiments, the target gene site is CDKL5, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:224, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:241.

[0808] In some embodiments, the target gene site is CXCR4, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:225, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:242.

[0809] In some implementations, the pilot editing system is PE7.

[0810] In some embodiments, the vector further comprises a gene encoding the La protein (when the lead editing system includes PE7).

[0811] In some embodiments, the vector comprises: an expression cassette encoding a gene for Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, a gene encoding a La protein, and a gene encoding a fluorescent protein or a mutant or truncated form thereof; (the corresponding lead editing system is named TRU-PE7).

[0812] The expression cassette of the gene encoding RNA targeting the target gene site includes a gene encoding a guide editing RNA (pegRNA) targeting the target gene site, a gene encoding a nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA;

[0813] 1) An expression cassette containing a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding a La protein, located in a vector, said vector containing one or more fluorescent proteins or mutants thereof or truncated versions thereof; or

[0814] 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), the gene encoding RNA targeting the target gene site, and the gene encoding La protein are located in two vectors.

[0815] 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the vector encoding La protein further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and the vector containing an expression cassette encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein or a truncated form of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0816] 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or

[0817] 3) The expression cassettes of the genes encoding Cas9 nickase (nCas9), reverse transcriptase (RT), RNA targeting the target gene site, and La protein are located in three vectors;

[0818] 31) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) and the gene encoding La protein further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the vector containing the expression cassette containing the gene encoding RNA targeting the target gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein; the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins; the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0819] 32) The vector containing the gene encoding Cas9 nickase (nCas9) further contains a gene encoding fragment 1 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the gene encoding reverse transcriptase (RT) and the vector encoding La protein further contains a gene encoding fragment 2 of a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing the expression cassette of the gene encoding RNA targeting the target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein, wherein the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0820] In some embodiments, the carrier comprises: a first carrier;

[0821] 1) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding La protein, a gene encoding RNA targeting the target gene site, and a gene encoding a fluorescent protein or a mutant or a truncated form of it (preferably a fluorescent protein mutant).

[0822] 2) The first vector comprises (preferably from the 5' end to the 3' end) an expression cassette of: a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant), a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant), a gene encoding La protein, and a gene encoding RNA targeting the target gene site; fragment 1 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant) and fragment 2 of fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant) constitute the complete fluorescent protein or its mutant or its truncated form (preferably fluorescent protein mutant).

[0823] In some embodiments, the carrier comprises: a first carrier and a second carrier;

[0824] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant); the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant), a gene encoding La protein, and an expression cassette encoding RNA targeting the target gene site; fragment 1 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein; or

[0825] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding fragment 1 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and an expression cassette encoding RNA targeting the target gene site; the second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 of a fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant), and a gene encoding La protein; fragment 1 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) and fragment 2 of the fluorescent protein or its mutant or its truncated form (preferably a fluorescent protein mutant) constitute a complete fluorescent protein;

[0826] 3) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding a first fluorescent protein or a first fluorescent protein mutant (preferably a first fluorescent protein mutant) fragment 1, a gene encoding reverse transcriptase (RT), a gene encoding a first fluorescent protein or a first fluorescent protein mutant (preferably a first fluorescent protein mutant) fragment 2, and a gene encoding La protein; the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, a gene encoding a second fluorescent protein or a first fluorescent protein, and a gene encoding a second fluorescent protein. The gene of a second fluorescent protein mutant (preferably a second fluorescent protein mutant); fragment 1 of the first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant) and fragment 2 of the first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant) constitute the complete first fluorescent protein or its mutant or its truncated form (preferably a first fluorescent protein mutant); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); or

[0827] 4) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding La protein, and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein); the second vector comprises (preferably from the 5' end to the 3' end): an expression cassette encoding RNA targeting the target gene site, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein); the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0828] In some embodiments, the carrier includes: a first carrier, a second carrier, and a third carrier;

[0829] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0830] The second vector contains (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding La protein, and a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein (preferably a mutant of the second fluorescent protein);

[0831] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein (preferably a mutant of the third fluorescent protein);

[0832] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths);

[0833] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0834] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and a gene encoding La protein;

[0835] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein or a mutant or truncated form of it (preferably a mutant of the second fluorescent protein);

[0836] The first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths).

[0837] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a pegRNA targeting the fluorescent protein mutant, which, after being edited by the pegRNA targeting the fluorescent protein mutant, can express the fluorescent protein.

[0838] In some embodiments, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: a promoter (preferably the tRNA described above) and a pegRNA targeting the fluorescent protein mutant.

[0839] In some embodiments, the promoter and the pegRNA targeting the fluorescent protein mutant are located at the 3' end of the expression frame of the gene encoding the RNA of the target gene site.

[0840] In some embodiments, the carrier includes: a first carrier, a second carrier, and a third carrier;

[0841] 1) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein;

[0842] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding La protein, and a gene encoding a second fluorescent protein;

[0843] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting the target gene site, and a gene encoding a third fluorescent protein;

[0844] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths); or

[0845] 2) The first vector comprises (preferably from the 5' end to the 3' end): a gene encoding Cas9 nickase (nCas9) and a gene encoding fragment 1 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein);

[0846] The second vector comprises (preferably from the 5' end to the 3' end): a gene encoding reverse transcriptase (RT), a gene encoding fragment 2 encoding a first fluorescent protein or a mutant of the first fluorescent protein (preferably a mutant of the first fluorescent protein), and a gene encoding La protein;

[0847] The third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a second fluorescent protein or a mutant or truncated form of it (preferably a mutant of the second fluorescent protein);

[0848] The first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein and the second fluorescent protein have different excitation wavelengths); and

[0849] The expression cassette of the gene encoding RNA targeting the target gene site includes: a gene encoding guide editing RNA (pegRNA) targeting the target gene site, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA.

[0850] This invention effectively solves the problem of low delivery efficiency in different types of cell lines due to the large size of the vector, which makes it difficult to enrich gene-edited cells, by placing the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) (which may or may not contain the gene encoding MLH1dn protein and the gene encoding La protein) in two vectors.

[0851] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a first fluorescent protein.

[0852] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): a second promoter, a gene encoding reverse transcriptase (RT), a gene encoding the La protein, and a gene encoding a second fluorescent protein.

[0853] In some embodiments, the third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein.

[0854] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, and a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site.

[0855] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (ttttttt).

[0856] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): EF-1α promoter (SEQ ID NO:297), tRNA, gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (when the vector is a viral vector (e.g., a lentiviral vector)).

[0857] In some embodiments, the 5' end of the expression frame of the gene encoding RNA targeting the target gene site further includes: an EF-1α promoter ((SEQ ID NO:297) when the vector is a viral vector (e.g., a lentiviral vector)).

[0858] In some embodiments, the first promoter and the second promoter are each independently selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; furthermore, the first promoter and the second promoter are both CMV promoters or EF-1α promoters (SEQ ID NO:297); even further, the first promoter and the second promoter are both CMV promoters.

[0859] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0860] b1)SEQ ID NO:244; or

[0861] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[0862] In some embodiments, the first fluorescent protein is BFP.

[0863] In some embodiments, the nucleotide sequence of the BFP comprises:

[0864] c0) The coding sequence of BFP, wherein the amino acid sequence of BFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:262; or

[0865] c1)SEQ ID NO:262; or

[0866] c2) A nucleotide sequence of SEQ ID NO:262 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:262.

[0867] In some embodiments, the second fluorescent protein is EGFP.

[0868] In some embodiments, the nucleotide sequence of the EGFP comprises:

[0869] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[0870] c1)SEQ ID NO:256; or

[0871] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[0872] In some embodiments, the third fluorescent protein is RFP.

[0873] In some embodiments, the nucleotide sequence of the RFP comprises:

[0874] The encoding sequence of m0)RFP, wherein the amino acid sequence of the RFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:260; or

[0875] m1)SEQ ID NO:260; or

[0876] m2) A nucleotide sequence of SEQ ID NO:260 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:260.

[0877] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[0878] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[0879] d1)SEQ ID NO:246; or

[0880] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[0881] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[0882] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[0883] e1)SEQ ID NO:248; or

[0884] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[0885] In some embodiments, the nucleotide sequence of the gene encoding the La protein comprises:

[0886] p0) The coding sequence of the La protein, wherein the amino acid sequence of the La protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:266; or

[0887] p1)SEQ ID NO:266; or

[0888] p2) A nucleotide sequence of SEQ ID NO:266 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:266.

[0889] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[0890] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[0891] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[0892] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0893] j1)SEQ ID NO:245; or

[0894] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0895] In some embodiments, the gene encoding the Cas9 nickase (nCas9) and the gene encoding the first fluorescent protein further include (preferably from the 5' end to the 3' end): a second NLS and a first 2A peptide.

[0896] In some implementations, the second NLS is a second SV40 NLS.

[0897] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[0898] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:261; or

[0899] l1)SEQ ID NO:261; or

[0900] l2) A nucleotide sequence of SEQ ID NO:261 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:261.

[0901] In some embodiments, a third NLS is also included between the second promoter and the gene encoding reverse transcriptase (RT).

[0902] In some implementations, the third NLS (Nuclear localization sequence) is the third SV40 NLS.

[0903] In some embodiments, the nucleotide sequence of the third SV40 NLS comprises:

[0904] j0) The coding sequence of the third SV40 NLS, wherein the amino acid sequence of the third SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[0905] j1)SEQ ID NO:245; or

[0906] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[0907] In some embodiments, the gene encoding the La protein and the gene encoding the second fluorescent protein further include (preferably from the 5' end to the 3' end): a fourth NLS, a fifth NLS, and a second 2A peptide.

[0908] In some implementations, the fourth NLS is a fourth SV40 NLS.

[0909] In some embodiments, the nucleotide sequence of the fourth SV40 NLS comprises:

[0910] l0) The coding sequence of the fourth SV40 NLS, wherein the amino acid sequence of the fourth SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[0911] l1)SEQ ID NO:249; or

[0912] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[0913] In some implementations, the fifth NLS is a C-Myc NLS.

[0914] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[0915] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[0916] n1)SEQ ID NO:250; or

[0917] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[0918] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the third fluorescent protein further include (preferably from the 5' end to the 3' end): a third promoter, an resistance gene, and a third 2A peptide.

[0919] In some embodiments, the third promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[0920] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[0921] g1)SEQ ID NO:254; or

[0922] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[0923] In some embodiments, the resistance gene is Puro.

[0924] In some embodiments, the nucleotide sequence of the Puro comprises:

[0925] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[0926] h1)SEQ ID NO:255; or

[0927] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[0928] In some embodiments, the first 2A peptide, the second 2A peptide, and the third 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, and the third 2A peptide are all P2A peptides.

[0929] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[0930] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[0931] i1)SEQ ID NO:251; or

[0932] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[0933] In some implementations, the target gene loci are one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0934] In some embodiments, when there are multiple target gene loci, the expression frame of the gene encoding RNA targeting the target gene locus includes N expression frames of genes encoding RNA targeting the target gene locus, where N is the number of target gene loci; for example, the expression frame of the gene encoding RNA targeting the target gene locus includes: an expression frame of a gene encoding RNA targeting a first target gene locus, an expression frame of a gene encoding RNA targeting a second target gene locus, ..., an expression frame of a gene encoding RNA targeting the Nth target gene locus.

[0935] In some embodiments, the target gene site is CDKL5, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:224, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:241.

[0936] In some embodiments, the target gene site is CXCR4, the sequence of the guide editing RNA (pegRNA) targeting the target gene site is shown in SEQ ID NO:225, and the sequence of the nicking sgRNA targeting the target gene site is shown in SEQ ID NO:242.

[0937] In some embodiments, the vector is a plasmid or a viral vector.

[0938] In some embodiments, the viral vector is an adeno-associated virus vector, an adenovirus vector, a retroviral vector, a lentiviral vector, or a herpes simplex virus vector.

[0939] A third aspect of the invention provides a delivery system comprising the gene editing system of the second aspect of the invention.

[0940] In some embodiments, the delivery system comprises at least one of liposomes, nanoparticles, and exosomes.

[0941] A fourth aspect of the present invention provides a cell comprising: the gene editing system of the second aspect of the present invention, the vector of the second aspect of the present invention, or the delivery system of the third aspect of the present invention.

[0942] In some embodiments, the cells do not contain reproductive material.

[0943] In some embodiments, the cells are selected from prokaryotic cells and eukaryotic cells.

[0944] In some embodiments, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0945] In some embodiments, the eukaryotic cells include yeast cells, plant cells, mammalian cells, insect cells, etc.

[0946] In some embodiments, the mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats.

[0947] In some embodiments, the cells comprise at least one of HEK293T cells, HeLa cells, MCF7 cells, iPSC cells, and U2OS cells.

[0948] A fifth aspect of the invention provides the use of the gene editing system of the second aspect of the invention, the delivery system of the third aspect of the invention, or the cell of the fourth aspect of the invention in any one of a1)-a5):

[0949] a1) Editing nucleic acid molecules;

[0950] a2) Prepare products for editing nucleic acid molecules;

[0951] a3) Construct plant, animal, or cell models;

[0952] a4) Prepare products for constructing plant, animal, or cell models;

[0953] a5) Prepare products for treating gene-related diseases.

[0954] In some implementations, the editing described in a1) and a2) includes at least one of replacement, deletion, and insertion.

[0955] In some implementations, the substitution is a conversion or transposition.

[0956] In some embodiments, the nucleic acid molecules described in a1) and a2) are derived from an organism or a biological cell.

[0957] In some implementations, the products described in a2) and a4) are reagents or kits.

[0958] In some embodiments, the product described in a5) is a drug or a drug composition.

[0959] In some implementations, the gene-related diseases described in a5) include genetic hematologic disorders, including but not limited to bone marrow failure syndrome, cancer susceptibility syndrome, and hemoglobinopathies.

[0960] In some embodiments, the gene-related diseases described in a5) include at least one of the following: megakaryocyte thrombocytopenic purpura, Deutsche-Bruce anemia, congenital dyskeratosis, Fanconi anemia, Pearson syndrome, severe congenital neutropenia, SDS, thrombocytopenia-radial agenesis syndrome, IVIC syndrome, Cockayne syndrome, radioulnar bony junction, ataxia, and various types of cytopenia.

[0961] In some embodiments, the gene-related disease described in a5) includes mucopolysaccharidosis type I (MPS I), which includes at least one of Holler syndrome (MPS IH), Holler-Sch'e syndrome (MPS IH / S), or Sch'e syndrome (MPS IS).

[0962] Preferably, the gene-related disease described in a5) includes at least one of GATA2 deficiency syndrome, germline DDX41 mutation-related myeloid tumors, and germline SAMD9 / SAMD9L mutation-related phantom syndrome.

[0963] In some embodiments, the gene-related diseases described in a5) include at least one of adenosine deaminase (ADA) deficiency, α-1 antitrypsin deficiency, cystic fibrosis, Duchenne muscular dystrophy, galactosemia, hemochromatosis, Huntington's disease, maple syrup diabetes, Marfan syndrome, neurofibromatosis type 1, congenital pachyonychia, phenylketonuria, severe combined immunodeficiency, sickle cell anemia, Sleigh-O. syndrome, trinucleotide duplication syndrome, prions, Tay-Sachs disease, heart disease, hypertension, Alzheimer's disease, arthritis, diabetes, cancer, and obesity.

[0964] A sixth aspect of the present invention provides a product comprising: a gene editing system of the second aspect of the present invention, a delivery system of the third aspect of the present invention, or a cell of the fourth aspect of the present invention.

[0965] In some embodiments, the product is used for any one of b1)-b3):

[0966] b1) Editing nucleic acid molecules;

[0967] b2) Construct plant, animal, or cell models;

[0968] b3) Treatment of gene-related diseases.

[0969] In some embodiments, the editing is the editing described in the fifth aspect of the present invention.

[0970] In some embodiments, the nucleic acid molecule is the nucleic acid molecule of the fifth aspect of the present invention.

[0971] In some embodiments, the gene-related disease is the gene-related disease of the fifth aspect of the present invention.

[0972] In some embodiments, the product is a reagent combination or kit (preferably the product is used for b1)-b2).

[0973] In some embodiments, the product is a drug or a drug composition (preferably the product is used for b3).

[0974] In some embodiments, the product is a drug or a drug composition, and the product further comprises a pharmaceutically acceptable carrier.

[0975] In some implementations, the animal is a non-human animal.

[0976] A seventh aspect of the present invention provides a method for editing nucleic acid molecules, wherein the nucleic acid molecules are brought into contact with a gene editing system of a second aspect of the present invention, a delivery system of a third aspect of the present invention, or a cell of a fourth aspect of the present invention.

[0977] In some embodiments, the editing is the editing described in the fifth aspect of the present invention.

[0978] In some embodiments, the nucleic acid molecule is the nucleic acid molecule of the fifth aspect of the present invention.

[0979] An eighth aspect of the invention provides a method for constructing a plant, animal, or cell model by contacting the plant, animal, or cell with a gene editing system of the second aspect of the invention, a delivery system of the third aspect of the invention, or a cell of the fourth aspect of the invention.

[0980] In some implementations, the animal is a non-human animal.

[0981] In some embodiments, a method for constructing a GATA2 deficiency syndrome (G2DS) model involves contacting an animal or cell with a gene editing system (TRU-PE5max-N) of the second aspect of the invention, wherein the target gene sites are GATA2, SETBP1, and ASXL1.

[0982] The gene editing system provided by this invention significantly facilitates in-depth research on complex polygenic genetic diseases. For example, when researchers need to establish cell models with different combinations of gene mutations in vitro while maintaining the same genetic background and the same potential off-target effects to explore the association between these gene mutations and diseases, this lead editing system can effectively support this need and can be used to construct various plant, animal, or cell models. At the same time, compared with other versions of PE editing systems, this lead editing system can not only be delivered in a variety of different ways, but is also highly compatible with other versions of the system.

[0983] In some embodiments, the gene editing system includes a vector, the vector comprising: a first vector, a second vector, and a third vector;

[0984] The first vector contains: a gene encoding Cas9 nickase (nCas9) and a gene encoding a first fluorescent protein;

[0985] The second vector contains: a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein;

[0986] The third vector comprises: an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein;

[0987] The expression cassette of the gene encoding RNA targeting the target gene site includes: a gene encoding guide editing RNA (pegRNA) targeting the target gene site, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA;

[0988] The first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different (the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein have different excitation wavelengths).

[0989] In some embodiments, the first vector comprises (preferably from the 5' end to the 3' end): a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a first fluorescent protein.

[0990] In some embodiments, the second vector comprises (preferably from the 5' end to the 3' end): a second promoter, a gene encoding reverse transcriptase (RT), a gene encoding MLH1dn protein, and a gene encoding a second fluorescent protein.

[0991] In some embodiments, the third vector comprises (preferably from the 5' end to the 3' end): an expression cassette of a gene encoding RNA targeting a specific gene site, and a gene encoding a third fluorescent protein.

[0992] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, and a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site.

[0993] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): tRNA, a gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, a gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (ttttttt).

[0994] In some embodiments, the first promoter and the second promoter are each independently selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; furthermore, the first promoter and the second promoter are both EF-1α promoters or CMV promoters; even further, the first promoter and the second promoter are both CMV promoters.

[0995] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site includes (preferably from the 5' end to the 3' end): EF-1α promoter, tRNA, gene encoding guide editing RNA (pegRNA) targeting the target gene site, tRNA, gene encoding nicking sgRNA (nicking RNA) targeting the target gene site, and polyT (when the vector is a viral vector (e.g., a lentiviral vector)).

[0996] In some embodiments, the 5' end of the expression frame of the gene encoding RNA targeting the target gene site further includes an EF-1α promoter (when the vector is a viral vector (e.g., a lentiviral vector)).

[0997] In some embodiments, the nucleotide sequence of the CMV promoter comprises:

[0998] b1)SEQ ID NO:244; or

[0999] b2) A nucleotide sequence of SEQ ID NO:244 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:244.

[1000] In some embodiments, the first fluorescent protein is BFP.

[1001] In some embodiments, the nucleotide sequence of the BFP comprises:

[1002] c0) The coding sequence of BFP, wherein the amino acid sequence of BFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:262; or

[1003] c1)SEQ ID NO:262; or

[1004] c2) A nucleotide sequence of SEQ ID NO:262 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:262.

[1005] In some embodiments, the second fluorescent protein is EGFP.

[1006] In some embodiments, the nucleotide sequence of the EGFP comprises:

[1007] c0) The coding sequence of EGFP, wherein the amino acid sequence of EGFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:256; or

[1008] c1)SEQ ID NO:256; or

[1009] c2) A nucleotide sequence of SEQ ID NO:256 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:256.

[1010] In some embodiments, the third fluorescent protein is RFP.

[1011] In some embodiments, the nucleotide sequence of the RFP comprises:

[1012] The encoding sequence of m0)RFP, wherein the amino acid sequence of the RFP is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:260; or

[1013] m1)SEQ ID NO:260; or

[1014] m2) A nucleotide sequence of SEQ ID NO:260 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:260.

[1015] In some embodiments, the nucleotide sequence of the gene encoding the Cas9 nickase (nCas9) comprises:

[1016] d0) The coding sequence of the Cas9 nickase, wherein the amino acid sequence of the Cas9 nickase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:246; or

[1017] d1)SEQ ID NO:246; or

[1018] d2) A nucleotide sequence of SEQ ID NO:246 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:246.

[1019] In some embodiments, the nucleotide sequence of the gene encoding reverse transcriptase (RT) comprises:

[1020] e0) The coding sequence of the reverse transcriptase, wherein the amino acid sequence of the reverse transcriptase is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:248; or

[1021] e1)SEQ ID NO:248; or

[1022] e2) A nucleotide sequence of SEQ ID NO:248 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:248.

[1023] In some embodiments, the nucleotide sequence of the gene encoding the MLH1dn protein comprises:

[1024] f0) The coding sequence of the MLH1dn protein, wherein the amino acid sequence of the MLH1dn protein is the amino acid sequence encoded by the nucleotides shown in SEQ ID NO:259; or

[1025] f1)SEQ ID NO:259; or

[1026] f2) A nucleotide sequence of SEQ ID NO:259 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:259.

[1027] In some embodiments, a first NLS is also included between the first promoter and the gene encoding the Cas9 nickase (nCas9).

[1028] In some implementations, the first NLS (Nuclear localization sequence) is the first SV40 NLS.

[1029] In some embodiments, the nucleotide sequence of the first SV40 NLS comprises:

[1030] j0) The coding sequence of the first SV40 NLS, wherein the amino acid sequence of the first SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[1031] j1)SEQ ID NO:245; or

[1032] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[1033] In some embodiments, the gene encoding the Cas9 nickase (nCas9) and the gene encoding the first fluorescent protein further include (preferably from the 5' end to the 3' end): a second NLS and a first 2A peptide.

[1034] In some implementations, the second NLS is a second SV40 NLS.

[1035] In some embodiments, the nucleotide sequence of the second SV40 NLS comprises:

[1036] l0) The coding sequence of the second SV40 NLS, wherein the amino acid sequence of the second SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:261; or

[1037] l1)SEQ ID NO:261; or

[1038] l2) A nucleotide sequence of SEQ ID NO:261 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:261.

[1039] In some embodiments, a third NLS is also included between the second promoter and the gene encoding reverse transcriptase (RT).

[1040] In some implementations, the third NLS (Nuclear localization sequence) is the third SV40 NLS.

[1041] In some embodiments, the nucleotide sequence of the third SV40 NLS comprises:

[1042] j0) The coding sequence of the third SV40 NLS, wherein the amino acid sequence of the third SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:245; or

[1043] j1)SEQ ID NO:245; or

[1044] j2) A nucleotide sequence of SEQ ID NO:245 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:245.

[1045] In some embodiments, the gene encoding reverse transcriptase (RT) and the gene encoding MLH1dn protein further include (preferably from the 5' end to the 3' end): a fourth NLS, a fifth NLS, and a second 2A peptide.

[1046] In some implementations, the fourth NLS is a fourth SV40 NLS.

[1047] In some embodiments, the nucleotide sequence of the fourth SV40 NLS comprises:

[1048] l0) The coding sequence of the fourth SV40 NLS, wherein the amino acid sequence of the fourth SV40 NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:249; or

[1049] l1)SEQ ID NO:249; or

[1050] l2) A nucleotide sequence of SEQ ID NO:249 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:249.

[1051] In some implementations, the fifth NLS is a C-Myc NLS.

[1052] In some embodiments, the nucleotide sequence of the C-Myc NLS comprises:

[1053] The coding sequence of n0)C-Myc NLS, wherein the amino acid sequence of C-Myc NLS is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:250; or

[1054] n1)SEQ ID NO:250; or

[1055] n2) A nucleotide sequence of SEQ ID NO:250 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:250.

[1056] In some embodiments, a third 2A peptide is further included between the gene encoding the MLH1dn protein and the gene encoding the second fluorescent protein.

[1057] In some embodiments, the expression cassette of the gene encoding RNA targeting the target gene site and the gene encoding the third fluorescent protein further include (preferably from the 5' end to the 3' end): a third promoter, an resistance gene, and a fourth 2A peptide.

[1058] In some embodiments, the third promoter is selected from at least one of the following: CBh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, β-glucuronidase (GUSB) promoter, chicken β-actin (CBA) promoter, CAG promoter, CB promoter, ubiquitin C (UBC) promoter, and PGK promoter; further, it is a PGK promoter; and even further, it is an hPGK promoter.

[1059] In some embodiments, the nucleotide sequence of the hPGK promoter comprises:

[1060] g1)SEQ ID NO:254; or

[1061] g2) A nucleotide sequence of SEQ ID NO:254 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:254.

[1062] In some embodiments, the resistance gene is Puro.

[1063] In some embodiments, the nucleotide sequence of the Puro comprises:

[1064] h0) The coding sequence of Puro, wherein the amino acid sequence of Puro is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:255; or

[1065] h1)SEQ ID NO:255; or

[1066] h2) A nucleotide sequence of SEQ ID NO:255 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:255.

[1067] In some embodiments, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are each independently selected from at least one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide; furthermore, the first 2A peptide, the second 2A peptide, the third 2A peptide, and the fourth 2A peptide are all P2A peptides.

[1068] In some embodiments, the nucleotide sequence of the P2A peptide comprises:

[1069] i0) The coding sequence of the P2A peptide, wherein the amino acid sequence of the P2A peptide is the amino acid sequence encoded by the nucleotide shown in SEQ ID NO:251; or

[1070] i1)SEQ ID NO:251; or

[1071] i2) A nucleotide sequence of SEQ ID NO:251 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:251.

[1072] A ninth aspect of the present invention provides a method for treating gene-related diseases by administering an effective amount of the drug or pharmaceutical composition of the sixth aspect of the present invention to a subject.

[1073] In some embodiments, the gene-related disease is the gene-related disease of the fifth aspect of the present invention.

[1074] In some embodiments, the subject may include mammals, such as humans or non-human mammals. In some embodiments, the non-human mammals may include, but are not limited to, non-human primates (e.g., monkeys, orangutans), mice, rats, hamsters, gerbils, cats, dogs, guinea pigs, rabbits, horses, sheep, cattle, pigs, etc. Attached Figure Description

[1075] Figure 1 shows the design scheme diagrams of the PE5max, PE5max-GR and uPE5max-G systems.

[1076] Figure 2 shows the differences in editing efficiency of the PE5max, PE5max-GR, and uPE5max-G systems at four different gene loci in HEK293T and HeLa cells.

[1077] Figure 3 shows the design scheme diagrams of PE5max and TRU-PE5max systems and a comparison of their experimental schemes.

[1078] Figure 4 shows the editing efficiency of the PE5max and TRU-PE5max systems in HEK293T cells.

[1079] Figure 5 shows the editing efficiency of the PE5max and TRU-PE5max systems in HeLa cells.

[1080] Figure 6 shows the editing efficiency of the PE5max and TRU-PE5max systems in MCF7 cells.

[1081] Figure 7 shows the editing efficiency of the PE5max and TRU-PE5max systems in iPSC cells.

[1082] Figure 8 shows a schematic diagram of the TRU-MPE5max-N multisite gene editing system, where N represents the number of target gene sites.

[1083] Figure 9 shows the editing efficiency of the TRU-MPE5max-10 system in HEK293T cells.

[1084] Figure 10 shows the gene editing results of the MPE5max-6 and TRU-MPE5max-6 systems in HEK293T cells.

[1085] Figure 11 shows the editing efficiency of the TRU-MPE5max-10 system in HEK293T cells.

[1086] Figure 12 shows the gene editing results of the TRU-MPE5max-10 system in HEK293T cells.

[1087] Figure 13 shows the editing efficiency of the TRU-MPE5max-3 and TRU-MPE5max-6 systems in HeLa cells.

[1088] Figure 14 shows the editing efficiency of the TRU-MPE5max-3 and TRU-MPE5max-6 systems in iPSC cells.

[1089] Figure 15 shows the editing efficiency of the TRU-MPE5max-10 system in HeLa cells and iPSC cells.

[1090] Figure 16 shows the editing efficiency of lentivirus-mediated MPE5max-3 and TRU-MPE5max-3 systems in HEK293T cells.

[1091] Figure 17 shows the editing efficiency of lentivirus-mediated MPE5max-3 and TRU-MPE5max-3 systems in Jurkat cells.

[1092] Figure 18 shows the design scheme diagrams for the TRU-PE6e and TRU-PE7 systems.

[1093] Figure 19 shows the editing efficiency of TRU-PE6e and TRU-PE7 in HEK293T, HeLa and U2OS cells.

[1094] Figure 20 shows the experimental workflow diagram of the TRU-MPEmax-3 system for in vitro iPSC editing.

[1095] Figure 21 shows the editing efficiency of the TRU-MPE5max-3 system at the GATA2, SETBP1, and ASXL1 gene loci in iPSCs.

[1096] Figure 22 shows the single-clone sequencing results after iPSC editing in vitro using the TRU-MPEmax-3 system.

[1097] Figure 23 shows the experimental procedure diagram of Example 7.

[1098] Figure 24 shows the editing efficiency of the system in Example 7 in HEK293T cells.

[1099] Figure 25 shows the editing efficiency of the system in Example 7 at different sites in HEK293T cells.

[1100] Figure 26 shows the experimental procedure diagram of Example 8.

[1101] Figure 27 shows the editing efficiency of the system in Example 8 in HEK293T cells.

[1102] In the above figures, Statistical Results Figure 7 shows three biological replicates, with two technical replicates per replicate. All other Statistical Results Figures show three biological replicates, with three technical replicates per replicate. Detailed Implementation

[1103] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[1104] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[1105] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[1106] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[1107] The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[1108] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[1109] In the description of this invention, the terms "first," "second," "third," and "fourth" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first," "second," "third," or "fourth" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[1110] Table 1 shows the sequences involved in the embodiments.

[1111] Table 2 shows the pegRNA and nicking sgRNA for different gene loci.

[1112] Table 3 shows the sequence of some components / fragments involved in the pilot editing system in the following embodiments.

[1113] In the following embodiments:

[1114] 1) The total PCR volume for constructing different vectors was 50 μL, including 25 μL of 2×PhantaFlash Master Mix (Dye Plus) high-fidelity DNA polymerase (Novizan), 2 μL each of 10 μM forward and reverse primers, 1 ng of template plasmid, and ddH2O to bring the reaction volume to 50 μL. The amplification program was as follows: pre-denaturation 98℃ for 30 s, denaturation 98℃ for 10 s, annealing 60℃ for 5 s, extension 72℃ for 5 s / kb, cycle number 35, and final extension for 1 min.

[1115] 2) All enzyme digestion systems are as follows: 1 μL restriction endonuclease, 5 μL 10X rCutSmart Buffer, appropriate amount of enzyme digestion vector template, and ddH2O to make up to 50 μL. The reaction conditions are: incubation at 37℃ for 30 min.

[1116] 3) All Gibson ligation reaction systems were in the form of 5 μL. HiFi DNA Assembly Master Mix, add an appropriate amount of DNA ligation fragment, and bring the reaction volume to 10 μL with ddH2O. Incubate at 50°C for 15-30 min.

[1117] 4) All T4 DNA ligation reaction systems were: 1 μL T4 DNA polymerase, 2 μL 10X T4 DNA ligase buffer, an appropriate amount of DNA ligation fragment, and ddH2O to bring the reaction system to 20 μL.

[1118] 5) The Goldengate reaction system is as follows: Appropriate amount of DNA reaction fragments, 1.25 μL BsaI (10 U / μL), 1.25 μL T4 DNA Ligase, 2 μL rCutsmart buffer, 2 μL T4 DNA ligase buffer, and water to a final volume of 20 μL. When the number of DNA reaction fragments in the above Goldengate reaction system is less than or equal to 6, incubate under the following conditions: 37℃, 1 min; 16℃, 1 min, 30 cycles; 60℃, 5 min. When the number of DNA reaction fragments in the above Goldengate reaction system is greater than 6, incubate under the following conditions: 37℃, 5 min; 16℃, 5 min, 30 cycles; 60℃, 5 min.

[1119] Example

[1120] Example 1: Construction of pilot editing vectors PE5max-GR and uPE5max-G and analysis of gene editing efficiency

[1121] Using the plasmid (Addgene: 51133) from Qingke Biotechnology, the stop codon (tga) after the expression fragment of the Puro gene in the plasmid was removed, and the synthesized P2A-EGFP fragment was directly inserted (P2A and EGFP are directly linked, and their nucleotide sequences are shown in SEQ ID NO: 251 and 256, respectively). The resulting positive clone was named LL078 (a vector that can express nicking sgRNA and contains both Puro and EGFP dual selection tags).

[1122] Using primers OL385 / OL386 and plasmid (Addgene:174828) as a template, PCR was performed under the conditions described in 1) above to obtain a fragment containing the CMV-nCas9-RT-MLH1dn element. Using primers OL397 / OL398 and LL078 as a template, PCR was performed under the conditions described in 1) above. After gel purification, the two PCR fragments were subjected to Gibson reaction under the conditions described in 3) above, and the resulting positive clone was named LL108 (a vector that can express the CMV-nCas9-RT-MLH1dn element, contains the Puro and EGFP dual selection tags, and can express nicking sgRNA).

[1123] Using primers OL485 / OL486 and the LL108 plasmid as a template, PCR amplification was performed as described in step 1) above. A positive clone containing only two BsaI restriction sites was obtained by mutating one of the BsaI restriction sites in the LL108 plasmid and named Mu-LL108.

[1124] After annealing and ligation using primers OL551 / OL552, the resulting positive clone was subjected to a Gibson reaction with Mu-LL108 digested with BsaI restriction endonuclease as described in 2) above, and the positive clone was named LL209 (with HindIII and XmaI restriction sites introduced between the U6 promoter and gRNA scaffold, and the remaining characteristics were consistent with LL108).

[1125] Using the plasmid (Addgene:132777) from Qingke Biotechnology as a base template vector, the vector was digested with BsaI, and the resulting fragment contained pegRNA, nicking RNA, and gRNA scaffold targeting the FANCF gene (its sequence is: GGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGGAAAAGCGATCAAGGTGCTGCAGAAGGGAtttttttgaacgctgacgtcatcaacccgctccaaggaatcgcgggcccagtgtcactaggcgggaacacccagcgcgcgtgcgccctgg caggaagatggctgtgagggacaggggagtggcgccctgcaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatgtctttggatttgggaatcttataagttctgtatgaggaccacGGGGTCCCAGGTGCTGACGTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc,SEQ ID NO:263) was inserted into the above-mentioned enzyme-digested plasmid to obtain the plasmid vector numbered LL109.

[1126] Using primers OL610 / OL574, and with LL109 as a template, a fragment containing pegRNA, nicking RNA, and gRNA scaffold targeting the FANCF gene was amplified as described in step 1). The LL209 vector was then digested with HindIII and XmaI restriction endonucleases as described in step 2). The digested LL209 vector and the amplified fragment were subjected to a Gibson reaction as described in step 3). The resulting positive clone was named LL241 (possessing both Puro and GFP dual selection tags; and integrating the pegRNA, nicking sgRNA, and CMV-nCas9-RT-MLH1dn expression elements targeting the FANCF gene into this plasmid vector).

[1127] Using primers OL899 / OL900 and LL241 as the PCR template, PCR amplification was performed as described in step 1) above. The DNA fragment was then purified and recovered via gel chromatography. Following step 2) above, the PE5max plasmid vector (Addgene ID: #174828) was digested with AgeI restriction endonuclease. The digested plasmid fragment and DNA fragment were recovered via gel chromatography and subjected to a Gibson reaction as described in step 3) above. The ligation product was transformed into DH5α *E. coli* (Kangti Life, KTSM101L), and the resulting positive clone was named LL368_PE5max_G (PE5max plasmid vector containing a GFP selection tag).

[1128] Using primers OL965 / OL966 and LL078 as the PCR template, PCR amplification was performed as described in step 1) above. Similarly, using primers OL902 / OL903 and the plasmid (Addgene:132777) as the PCR template, PCR amplification was performed as described in step 1) above. The DNA fragments were purified and recovered using gel electrophoresis, and Gibson reaction was performed as described in step 3) above. The ligation product was transformed into DH5α *E. coli* (Kangti Life, KTSM101L), and the resulting positive clone was named LL369 (an empty plasmid containing sgRNA expressing both RFP and puromycin dual selection tags).

[1129] Primers OL458 / OL459 containing BsaI restriction sites were used, and the basic sgRNA expression plasmid vector LL037 (Addgene ID: #51133) was used as the PCR template. PCR amplification was performed as described in step 1) above to obtain DNA fragments containing BsaI restriction sites at both ends. The resulting DNA fragments were then digested with BsaI restriction endonuclease and purified as described in step 2) above.

[1130] The purified DNA fragment was ligated with annealed primers OL441 and OL442, which contain two BsaI restriction sites, and a T4 DNA ligation reaction was performed as described in step 4 above. The ligation product was transformed into DH5α Escherichia coli (Kangti Life, KTSM101L), and the resulting positive clone was named LL169 as an empty plasmid vector (characterized by containing two BsaI restriction sites).

[1131] The synthesized hCtRNA-gRNA scaffold fragment (Table 3) was inserted into the BsaI-digested LL169 plasmid vector to construct the PCR amplification template plasmid LL170. The synthesized gRNA scaffold-hCtRNA fragment (Table 3) was inserted into the BsaI-digested LL169 plasmid vector to construct the PCR amplification template plasmid LL173.

[1132] Using primers OL305 / 306, the DNA fragment was amplified and purified using the LL169 basic vector as a template. Simultaneously, using primers OL901 and OL903, a plasmid vector containing the P2A-RFP fragment was amplified and purified using the LL369 basic vector as a template. The two purified DNAs were ligated using the Gibson reaction as described in step 3 above. The positive clones selected after transformation were named LL470 (characterized by containing two BsaI restriction sites and a DNA fragment capable of expressing RFP).

[1133] Using Addgene (132777) as the base plasmid, the plasmid was digested with BsaI by Qingke Biotechnology. Then, pegRNA fragments targeting different genes were synthesized and inserted into the digested plasmid vectors. The resulting vectors were LL018 (pegRNA vector targeting the FANCF gene site), LL019 (pegRNA vector targeting the DNMT1 gene site), LL022 (pegRNA vector targeting the EMX1 gene site), and LL070 (pegRNA vector targeting the ERCC6 gene site). The full sequences of the pegRNAs targeting different gene sites are shown in Table 2.

[1134] Annealing with primers OL212 / 213, and performing T4 DNA ligation reaction with the LL037 vector digested with BsaI as described in 2) above, according to 4) above. The positive clones selected after transformation were named LL028 (nicking sgRNA vector targeting the FANCF gene site).

[1135] Annealing was performed using primers OL210 / 211, and the LL037 vector digested with BsaI as described in step 2) was ligated to T4 DNA according to step 4) above. The positive clones selected after transformation were named LL026 nicking sgRNA vector targeting the DNMT1 gene locus.

[1136] Annealing with primers OL214 / 215, and performing T4 DNA ligation reaction with the LL037 vector digested with BsaI as described in 2) above, according to 4) above. The positive clones selected after transformation were named LL024 (nickingsgRNA vector targeting the EMX1 gene site).

[1137] Annealing with primers OL363 / 364, and performing T4 DNA ligation reaction with the LL037 vector digested with BsaI as described in 2) above, according to 4) above. The positive clones selected after transformation were named LL068 (nicking sgRNA vector targeting the ERCC6 gene site).

[1138] The nicking sgRNA sequences are shown in Table 2.

[1139] Primers OL535 / 546 and OL547 / 548 were used, both with LL173 as a template; primers OL549 / OL550 were used, with LL018 as a template, and PCR was performed as described in step 1) above. Using the three PCR products as templates, amplification was performed using primers OL535 / 550, and the amplified fragments and the digested LL169 were digested with BsaI. The digested fragments were ligated to the vector using T4 DNA ligase, and the positive clone was named LL206 (a pegRNA expression vector targeting the FANCF gene site without RFP fragments).

[1140] Using primers OL1451 / OL550, and with laboratory-synthesized LL206 as a template, amplification was performed. Following the enzyme digestion system described in section 2), BsaI restriction endonuclease was used to digest the PCR product and the LL470 plasmid vector. 1 μL of T4 DNA polymerase and 2 μL of 10X T4 DNA ligase buffer were added, and the reaction volume was brought to 20 μL with ddH2O. The mixture was incubated at room temperature for 2 hours before ligation. The ligation product was transformed into DH5α *E. coli* (Kangti Life, KTSM101L), and the resulting positive clone was named LL1216 (a pegRNA expression vector with an RFP fragment targeting the FANCF gene locus).

[1141] Amplification was performed using primers OL2721 / OL2641 with LL170 as a template; amplification was performed using primers OL2722 / OL2643 with LL173 as a template; and amplification was performed using primers OL2644 / OL2723 with LL019 as a template. Following the restriction enzyme digestion system described in 2) above, the LL470 plasmid vector was digested with BsaI restriction endonuclease. The DNA fragments and the digested plasmid vector were ligated using the Gibson reaction ligation system described in 3) above. The ligation product was transformed into DH5α *E. coli*, and the positive clone was named LL1209 (a pegRNA expression vector with an RFP fragment targeting the DNMT1 gene site with a 5G / T mutation).

[1142] Amplification was performed using primers OL2721 / OL2646 with LL170 as a template; primers OL2724 / OL2648 with LL173 as a template; and primers OL2649 / OL2725 with LL022 as a template. Following the restriction enzyme digestion system described in 2) above, the LL470 plasmid vector was digested with BsaI restriction endonuclease. The DNA fragments and the digested plasmid vector were ligated using the Gibson reaction ligation system described in 3) above. The ligation product was transformed into DH5α *E. coli*, and the positive clone was named LL1210 (a pegRNA expression vector with an RFP fragment targeting the EMX1 gene locus).

[1143] Amplification was performed using primers OL2721 / OL2651 with LL170 as a template; amplification was performed using primers OL2726 / OL2653 with LL173 as a template; and amplification was performed using primers OL2654 / OL2727 with LL070 as a template. Following the restriction enzyme digestion system described in 2) above, the LL470 plasmid vector was digested with BsaI restriction endonuclease. The DNA fragments and the digested plasmid vector were ligated using the Gibson reaction ligation system described in 3) above. The ligation product was transformed into DH5α *E. coli*, and the positive clone was named LL1211 (a pegRNA expression vector with an RFP fragment targeting the ERCC6 gene locus).

[1144] Amplification was performed using primers OL1044 / OL1048 with LL206 as a template; amplification was performed using primers OL1047 / OL1049 with LL241 as a template; the LL241 plasmid vector was digested with AgeI / SpeI restriction endonucleases according to the enzyme digestion system described in 2) above. The above DNA fragments and the digested plasmid vectors were ligated using the Gibson reaction ligation system described in 3) above. The ligation product was transformed into DH10β Escherichia coli, and the positive clone was named LL410 (a pegRNA expression vector without RFP fragment targeting the FANCF gene site).

[1145] Using primers OL2721 / OL2641 with LL170 as a template; using primers OL2722 / OL2643 with LL173 as a template; and using primers OL2644 / OL2723 with LL019 as a template, PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL169 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α *E. coli*, and the resulting positive clone was named LL1179 (a pegRNA expression vector targeting the DNMT1 gene locus without RFP fragments).

[1146] Using primers OL2721 / OL2646 with LL170 as a template; primers OL2724 / OL2648 with LL173 as a template; and primers OL2649 / OL2725 with LL022 as a template, PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL169 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α *E. coli*, and the resulting positive clone was named LL1180 (a pegRNA expression vector targeting the EMX1 gene locus without RFP fragments).

[1147] Using primers OL2721 / OL2651 with LL170 as a template; using primers OL2726 / OL2653 with LL173 as a template; and using primers OL2654 / OL2727 with LL070 as a template, PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL169 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α *E. coli*, and the resulting positive clone was named LL1181 (a pegRNA expression vector targeting the ERCC6 gene locus without RFP fragments).

[1148] Using primers OL1042 / 1043 with LL241 as a template; using primers OL1044 / OL1045 with LL206 as a template; and using primers OL1046 / 1047 with LL241 as a template, PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of AgeI / SpeI-digested LL003 vector via a Gibson reaction as described in step 3) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL409.

[1149] Amplification was performed using primers OL2782 / OL2784 with LL1179 as a template. Following the restriction enzyme digestion system described in 2) above, the LL409 plasmid vector was digested with XmaI / HindIII restriction endonucleases. The DNA fragment and the digested plasmid vector were ligated using the Gibson reaction ligation system described in 3) above. The ligation product was transformed into DH5α *E. coli*, and the positive clone was named LL1230 (uPE5max_G vector targeting the DNMT1 gene locus).

[1150] Amplification was performed using primers OL2782 / OL2784 with LL1180 as a template. Following the restriction enzyme digestion system described in 2) above, the LL409 plasmid vector was digested with XmaI / HindIII restriction endonucleases. The DNA fragment and the digested plasmid vector were ligated using the Gibson reaction ligation system described in 3) above. The ligation product was transformed into DH5α *E. coli*, and the positive clone was named LL1231 (uPE5max_G vector targeting the EMX1 gene site).

[1151] Amplification was performed using primers OL2782 / OL2784 with LL1181 as a template. Following the restriction enzyme digestion system described in 2) above, the LL409 plasmid vector was digested with XmaI / HindIII restriction endonucleases. The DNA fragment and the digested plasmid vector were ligated using the Gibson reaction ligation system described in 3) above. The ligation product was transformed into DH5α *E. coli*, and the positive clone was named LL1232 (uPE5max_G vector targeting the ERCC6 gene locus).

[1152] LL003 (Addgene: 174828), LL018, and LL028 together constitute the PE5max system targeting the FANCF gene locus; LL003 (Addgene: 174828), LL022, and LL024 together constitute the PE5max system targeting the EMX1 gene locus; LL003 (Addgene: 174828), LL019, and LL026 together constitute the PE5max system targeting the DNMT1 gene locus; LL003 (Addgene: 174828), LL070, and LL068 together constitute the PE5max system targeting the ERCC6 gene locus (illustrations of each plasmid are shown in Figure 1).

[1153] LL368 and LL1216 together constitute the PE5max_GR system targeting the FANCF gene site; LL368 and LL1209 together constitute the PE5max_GR system targeting the DNMT1 gene site; LL368 and LL1210 together constitute the PE5max_GR system targeting the EMX1 gene site; LL368 and LL1211 together constitute the PE5max_GR system targeting the ERCC6 gene site (the schematic diagram of each plasmid is shown in Figure 1, and the sequences of some elements / fragments are shown in Table 3).

[1154] Four different gene loci (FANCF, DNMT1, ERCC6, and EMX1) were tested in 24-well cell culture plates. The corresponding plasmid vector combinations were delivered to HEK293T or HeLa cells using PEI40000 transfection reagent. For the conventional PE5max system, the Prime Editor:pegRNA:nicking sgRNA vector ratio was 900:300:100 ng, 2.6 μL PEI40000. After 24 h of transfection, the medium was replaced with medium containing 4 μg / mL puromycin and cultured for 48 h. The cell pellet was then collected and transferred to QuickExtract DNA Extraction Solution (Lucigen, QE09050). For the PE5max-GR and uPE5max-G systems, the corresponding vectors were transfected at 900:300 ng (2.4 μL PEI40000) and 900 ng (1.8 μL PEI40000), respectively. Forty-eight hours post-transfection, 6000 cells expressing GFP and RFP (double-positive) or GFP (single-positive) were sorted using flow cytometry and transferred to QuickExtract DNA Extraction Solution (Lucigen, QE09050). DNA was extracted from cell populations collected using different strategies at 65℃ for 6 min and 98℃ for 2 min. The target sites were amplified using the corresponding primers (FANCF: OL633 / 634; DNMT1: OL643 / 644; ERCC6: OL639 / 640; EMX1: OL641 / 642), and NGS sequencing analysis was performed to compare the differences in editing efficiency. The editing efficiency of different vector combinations in HEK293T and HeLa cells is shown in Figure 2: Compared to the control group's Puromycin selection strategy, the fluorescence selection strategy better enriched gene-editing cells in HEK293T and HeLa cells.

[1155] Example 2: Construction and gene editing efficiency analysis of the TRU-PE5max gene editing system

[1156] Using primers OL1035 / OL1036 and OL1037 / OL1038, respectively, and the LL368_PE5max_G constructed in Example 1 as the PCR template, amplification was performed according to the PCR reaction system described in 1) above. The two different PCR fragments were purified by gel extraction and ligated using the Gibson reaction described in 3) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL392 (characterized by CMV promoter-driven expression of the RT-P2A-MLH1dn-P2A-EGFP element).

[1157] Primers OL1035 / OL1036, OL1039 / 1040, and the LL368_PE5max_G constructed in Example 1 were used as PCR templates, and amplification was performed according to the PCR reaction system described in 1) above. The two different PCR fragments were purified by gel extraction, and the two DNA fragments were ligated using the Gibson reaction. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL393 (characterized by CMV promoter-driven nCas9 element expression).

[1158] Using primers OL1313 and OL1314, the plasmid vector containing the P2A-BFP fragment from Example 1 was used as a PCR template, and amplification was performed according to the PCR reaction system described in 1) above. The PCR fragment was purified by gel extraction. Using the enzyme digestion reaction system described in 2) above, the LL393 vector digested with AgeI was ligated with the two DNA fragments via the Gibson reaction. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL622 (characterized by CMV promoter-driven nCas9-P2A-BFP element expression).

[1159] Using Addgene (132777) as the base plasmid, the plasmid was digested with BsaI, and pegRNA fragments targeting different genes were synthesized and inserted into the digested plasmid vector. The final synthesized vectors were LL055 (characterized by a U6 promoter-driven pegRNA vector targeting the BAG3 gene site) and LL057 (characterized by a U6 promoter-driven pegRNA vector targeting the NMB gene site). The full sequences of the pegRNAs targeting different gene sites are shown in Table 2.

[1160] Using primers OL535 / 536 and OL537 / 538, both with LL173 as a template; and primers OL539 / OL540, with LL055 as a template, PCR was performed as described in step 1) above. Using the three PCR products as templates, amplification was performed using primers OL535 / 540, and the amplified fragments and the digested LL169 were digested with BsaI. The digested fragments were ligated to the vector using T4 DNA ligase, and the positive clone was named LL204 (characterized by: hCtRNA driving a vector containing pegRNA and nicking sgRNA targeting the BAG3 gene).

[1161] Primers OL535 / 541 and OL542 / 543 were used, both with LL173 as a template; primers OL544 / OL545 were used, with LL057 as a template, and PCR was performed as described in step 1) above. Using the three PCR products as templates, amplification was performed using primers OL535 / 545, and the amplified fragments and the digested LL169 were digested with BsaI. The digested fragments were ligated to the vector using T4 DNA ligase, and the positive clone was named LL205 (characterized by: hCtRNA driving a vector containing pegRNA and nicking sgRNA targeting the NMB gene).

[1162] Using primers OL1451 / OL540 and the previously synthesized LL204 as a template, PCR amplification was performed as described in step 1) above. Following the enzyme digestion reaction system described in step 2), the DNA fragment and LL470 vector were digested with BsaI. T4 DNA ligation was performed as described in step 4), and the ligation product was transformed into DH5α E. coli. The resulting positive clone was named LL1214 (characterized by: hCtRNA-driven vector containing pegRNA and nicking sgRNA targeting the BAG3 gene site, capable of expressing RFP signals).

[1163] Using primers OL1451 / OL545 and the previously synthesized LL205 as a template, PCR amplification was performed as described in step 1) above. Following the enzyme digestion reaction system described in step 2), the DNA fragment and LL470 vector were digested with BsaI. T4 DNA ligation was performed as described in step 4), and the ligation product was transformed into DH5α E. coli. The resulting positive clone was named LL1215 (characterized by: hCtRNA driving a vector containing pegRNA and nicking sgRNA targeting the NMB gene, and capable of expressing RFP signals).

[1164] Using primers OL2745 / OL3444, with LL173 from Example 1 as a template; using primers OL3445 / OL3446, with LL173 from Example 1 as a template; and using primers OL3447 / OL3448, with LL037 from Example 1 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1415 (characterized by: an hCtRNA-driven vector containing pegRNA and nicking sgRNA targeting the TP53 gene, and capable of expressing RFP signals).

[1165] Using primers OL2745 / OL3436, with LL173 from Example 1 as a template; using primers OL3437 / OL3438, with LL173 from Example 1 as a template; and using primers OL3439 / OL3440, with LL037 from Example 1 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1413 (characterized by: hCtRNA-driven vector containing pegRNA and nicking sgRNA targeting the KRAS gene, and capable of expressing RFP signals).

[1166] Using primers OL2745 / OL3431, with LL173 from Example 1 as a template; using primers OL3432 / OL3433, with LL173 from Example 1 as a template; and using primers OL3434 / OL3435, with LL037 from Example 1 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1412 (characterized by: hCtRNA-driven vector containing pegRNA and nicking sgRNA targeting the LRRK2 gene, and capable of expressing RFP signals).

[1167] Using primers OL2745 / OL3426, with LL173 from Example 1 as a template; using primers OL3427 / OL3428, with LL173 from Example 1 as a template; and using primers OL3429 / OL3430, with LL037 from Example 1 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1411 (characterized by: an hCtRNA-driven vector containing pegRNA and nicking sgRNA targeting the GBA1 gene, and capable of expressing RFP signals).

[1168] Using primers OL1080 / OL4225, with LL173 from Example 1 as a template; using primers OL4226 / OL4227, with LL173 from Example 1 as a template; and using primers OL4228 / OL4317, with LL037 from Example 1 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1563 (characterized by: hCtRNA driving pegRNA and nicking sgRNA containing the DNMT1 gene deletion mutation del1-15, and capable of expressing RFP).

[1169] Using primers OL1080 / OL4232, with LL173 from Example 1 as a template; using primers OL4233 / OL4234, with LL173 from Example 1 as a template; and using primers OL4235 / OL4236, with LL037 from Example 1 as a template; PCR amplification was performed as described in step 1) above. Annealing was performed using primers OL4237 / OL4238. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1564 (characterized by: hCtRNA-driven vector containing pegRNA and nicking sgRNA targeting the RNF2 gene, and capable of expressing RFP signals).

[1170] For HEK293T, HeLa, and MCF7 cells, the LL368 vector in Example 1, together with LL1216, LL1209, LL1210, and LL1211 in Example 1, as well as the pegRNA expression vectors targeting different gene sites in this example (LL1214, LL1215, LL1415, LL1413, LL1412, LL1411, LL1563, and LL1564), constitute the PE5max system shown in Figure 3. The LL392 and LL622 vectors mentioned above, together with LL1216, LL1209, LL1210, and LL1211 in Example 1, and the pegRNA expression vectors targeting different gene sites in this example (LL1214, LL1215, LL1415, LL1413, LL1412, LL1411, LL1563, and LL1564), constitute the TRU-PE5max system shown in Figure 3 (the sequences of some elements / fragments are shown in Table 3).

[1171] To prevent the CMV promoter from being silenced in iPSC cells, plasmid vectors containing the EF-1α promoter were amplified using 2 μL of OL2675 / 2676 primers, and the PCR fragments were purified by gel recovery. These fragments were then Gibson-ligated with LL392 and LL622 vectors digested with NotI and SpeI, respectively. This yielded iPSC-specific gene editing vectors LL1184 (characterized by EF-1α promoter-driven expression of the RT-P2A-MLH1dn-P2A-EGFP element) and LL1185 (characterized by EF-1α promoter-driven expression of the nCas9-P2A-BFP element).

[1172] For the control group of the iPSC transfection experiment, the CMV promoter of the LL368 vector was replaced with the EF-1α promoter (SEQ ID NO:297): primers OL4012 / 4011 amplified the LL1184 vector, primers OL4013 / 2720 amplified the LL368 vector, primers OL2719 / 1034 amplified the LL368 vector, and primers OL1033 / 4014 amplified the aforementioned LL1184 vector. All amplification reactions used 25 μL of 2×PhantaFlash Master Mix (Dye Plus) high-fidelity DNA polymerase (Novizan) under the following conditions: pre-denaturation 98℃ for 30 s, denaturation 98℃ for 10 s, annealing 60℃ for 5 s, extension 72℃ for 5 s / kb, cycle number 35, and final extension for 1 min. The PCR fragments were purified by gel extraction. The recovered fragments were Gibson ligated, and the transformed positive clone was named LL1539 (characterized by: EF-1α promoter-driven nCas9-RT-P2A-MLH1dn-P2A-EGFP element expression).

[1173] For iPSC cells, the LL1539 vector, together with LL1216, LL1209, LL1210, and LL1211 from Example 1, and the pegRNA expression vectors targeting different gene loci in this example (LL1214, LL1215, LL1415, LL1413, LL1412, LL1411, LL1563, and LL1564), constitute the PE5max system shown in Figure 3. The LL1184 and LL1185 vectors, together with LL1216, LL1209, LL1210, and LL1211 from Example 1, and the pegRNA expression vectors targeting different gene loci in this example (LL1214, LL1215, LL1415, LL1413, LL1412, LL1411, LL1563, and LL1564), constitute the TRU-PE5max system shown in Figure 3.

[1174] In four different cell lines—HEK293T, Hela, MCF7, and iPSC—tests were performed on 12 different gene loci in 24-well plates.

[1175] For HEK293T, HeLa and MCF7 cell lines, the corresponding plasmid vector combinations were delivered to the target cells using PEI40000 transfection reagent. The vector ratio in the PE5max system was 900:300 ng (Prime Editor: pegRNA) (2.4 μL PEI40000), and the ratio of the three vectors in the TRU-PE5max system was 400:400:400 ng (2.4 μL PEI40000).

[1176] iPSC uses 2 μL / well of Lipofectamine TM Stem Transfection Reagent(Invitrogen TM The STEM00001 transfection plasmid vector was used for delivery. In the PE5max control group, cells were screened for 48 hours after transfection using medium containing 4 μg / mL puromycin (i.e., three days after transfection), and then collected into QuickExtract DNA Extraction Solution. For the TRU-PE5max gene editing system, 6000 positive cells co-expressing GFP, BFP, and RFP trifluorescein were sorted into lysis buffer using flow cytometry 48 hours after transfection.

[1177] The cells in the lysis buffer were subjected to DNA extraction at 65℃ for 6 min and 98℃ for 2 min. Target sites were amplified using primers, and NGS sequencing analysis was performed to compare differences in editing efficiency. The editing efficiency of different vector combinations in HEK293T cells is shown in Figure 4, in HeLa cells in Figure 5, in MCF7 cells in Figure 6, and in iPSC cells in Figure 7. Compared to the PE5max system in the control group, the TRU-PE5max experimental group showed an average 2-8 fold increase in editing efficiency across different cell lines, demonstrating the feasibility of the TRU-PE5max system design.

[1178] Example 3: Construction and Editing Efficiency Analysis of the TRU-MPE Multisite Gene Editing System

[1179] Based on the TRU-PE5max system in Example 2, corresponding pegRNA and nickRNA sequences were designed for 3, 6, and 10 different genes, respectively, and all of them were integrated with the pegRNA-nickRNA-RFP in the TRU-PE5max system to construct a TRU-MPE5max-3 / 6 / 10 system targeting multiple different gene loci, as shown in Figure 8. The specific operation is as follows:

[1180] Using primers OL1451 / OL1148, with LL1214 from Example 2 as a template; using primers OL1149 / OL1148, with LL1215 from Example 2 as a template; and using primers OL1149 / OL2756, with LL1216 from Example 2 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1217 (a pegRNA expression vector simultaneously targeting three different gene loci: FANCF, BAG3, and NMB).

[1181] Using primers OL1451 / OL1148, with LL1179 from Example 2 as a template; using primers OL1149 / OL1148, with LL1180 from Example 2 as a template; and using primers OL1149 / OL2756, with LL1181 from Example 2 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1212 (a pegRNA expression vector simultaneously targeting three different gene loci: DNMT1, EMX1, and ERCC6).

[1182] Using primers OL1451 / OL2989, with LL1209 from Example 2 as a template; using primers OL2990 / OL2991, with LL1216 from Example 2 as a template; using primers OL2992 / OL2993, with LL1215 from Example 2 as a template; using primers OL2994 / OL2995, with LL1214 from Example 2 as a template; using primers OL2996 / OL2997, with LL1211 from Example 2 as a template; and using primers OL2998 / OL2999, with LL1210 from Example 2 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were then ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1213 (a pegRNA expression vector that simultaneously targets six different gene loci: DNMT1, FANCF, NMB, BAG3, ERCC6, and EMX1).

[1183] Primers OL1451 / OL2989 were used, with LL1210 from Example 2 as a template; primers OL2990 / OL2991 were used, with LL1415 from Example 2 as a template; primers OL2992 / OL2993 were used, with LL1411 from Example 2 as a template; primers OL2994 / OL2995 were used, with LL1211 from Example 2 as a template; primers OL2996 / OL2997 were used, with LL1214 from Example 2 as a template; primers OL2... 998 / OL3574, using LL1215 from Example 2 as a template; using primers OL3575 / OL3576, using LL1216 from Example 2 as a template; using primers OL3577 / OL3578, using LL1209 from Example 2 as a template; using primers OL3579 / OL3580, using LL1412 from Example 2 as a template; using primers OL3581 / OL3582, using LL1413 from Example 2 as a template; PCR amplification was performed as described in step 1) above. The purified DNA fragments were ligated with an appropriate amount of the LL470 vector using a Goldengate reaction as described in step 5) above. The ligation product was transformed into DH5α Escherichia coli, and the resulting positive clone was named LL1450 (a pegRNA expression vector simultaneously targeting ten different gene loci: EMX1, TP53, GBA1, ERCC6, BAG3, NMB, FANCF, DNMT1, LRRK2, and KRAS).

[1184] For HEK293T and HeLa cells, LL001 (Addgene: 132775) and LL1213 constitute MPE3-6, LL003 (Addgene: 174828) and LL1213 constitute MPE5max-6, LL392, LL622 and LL1217 in Example 2 constitute TRU-MPE5max-3a, LL392, LL622 and LL1212 in Example 2 constitute TRU-MPE5max-3b, LL392, LL622 and LL1213 in Example 2 constitute TRU-MPE5max-6, and LL392, LL622 and LL1450 in Example 2 constitute TRU-MPE5max-10.

[1185] For iPSC cells, LL001 (Addgene: 132775) and LL1213 constitute MPE3-6, LL1184, LL1185 and LL1217 in Example 2 constitute TRU-MPE5max-3a, LL1184, LL1185 and LL1212 in Example 2 constitute TRU-MPE5max-3b, LL1184, LL1185 and LL1213 in Example 2 constitute TRU-MPE5max-6, and LL1184, LL1185 and LL1450 in Example 2 constitute TRU-MPE5max-10.

[1186] The experiment was conducted in three different cell types: HEK293T, HeLa, and iPSC. The plasmid vector ratio was 400:400:400 ng. 48 h post-transfection, 6000 cells co-expressing GFP, BFP, and RFP (tri-fluorescence positive) were sorted using flow cytometry and added to cell lysis buffer. DNA was extracted by lysis at 65°C for 6 min and 98°C for 2 min. The corresponding target gene loci were amplified using the primer sequences described in Example 2, and NGS sequencing analysis was performed to compare the differences in editing efficiency. The results in HEK293T are shown in Figure 9: Compared to the traditional MPE3-6 and MPE5max-6 multi-site gene editing systems, the editing efficiency of TRU-MPE5max-6 was significantly improved by 1.1-2.5 times.

[1187] To further validate the performance of the TRU-MPE5max multi-site gene editing system in terms of gene editing completion, the MPE5max-6 and TRU-MPE5max-6 systems were delivered to HEK293T cells using Lipo3000 transfection reagent. For the MPE5max-6 system, single cells were sorted and cultured in 96-well plates after 48 hours. For the TRU-MPE5max-6 system, single cells co-expressing the three fluorescent markers were sorted and cultured in 96-well plates after 48 hours. After 10 days of culture, approximately 30 single-clonal cell clusters were collected and lysed in cell lysate. Different gene loci were amplified using primers, and Sanger sequencing was used to analyze the genotypes of different gene loci. The results are shown in Figure 10: Compared to the MPE5max-6 system (left), where only 7 / 30 cells achieved co-editing of 6 gene loci, the TRU-MPE5max-6 system achieved co-editing of 6 gene loci in 14 / 28 cells. Furthermore, after gene editing, the number of homozygotes (red circles) in the TRU-MPE5max-6 system is about three times that of the MPE5max-6 system.

[1188] Simultaneously, three different plasmids, LL392, LL622, and LL1450, were co-transfected into HEK293T cells at a ratio of 400:400:400 ng using PEI40000. This allowed them to jointly target and edit 10 different gene loci. 48 hours post-transfection, 6000 cells co-expressing GFP, BFP, and RFP (tri-fluorescence positive) were sorted using flow cytometry and added to cell lysis buffer. DNA was extracted by lysing at 65°C for 6 min and then at 98°C for 2 min. The corresponding target gene loci were amplified using the primer sequences described in Example 2, and NGS sequencing analysis was performed to compare the differences in editing efficiency. The results are shown in Figure 11: In HEK293T cells, the TRU-MPE5max-10 system successfully achieved efficient gene editing at all 10 different gene loci.

[1189] Furthermore, to verify whether co-editing of 10 gene sites occurred in cells edited using the TRU-MPE5max-10 system, the inventors performed NGS analysis on single cells. The corresponding experimental results are shown in Figure 12: the TRU-MPE5max-10 system achieved co-editing of 10 gene sites in 2 / 32 HEK293T single cells, and 8 / 32 and 7 / 32 clones achieved co-editing of up to 6-7 gene sites in HEK293T cells.

[1190] Figure 13 shows the test results of multi-site editing efficiency in HeLa cells: the TRU-MPE5max-3 system not only exhibits high editing efficiency when simultaneously targeting 3 sites, but also, compared to the traditional MPE3-6 gene editing system, shows a maximum 7-fold improvement in editing efficiency when simultaneously targeting 6 gene sites. Figure 14 shows the test results of multi-site editing efficiency in iPSC cells: the TRU-MPE5max-3 system not only exhibits high editing efficiency when simultaneously targeting 3 sites, but also, compared to the traditional MPE3-6 gene editing system, shows a maximum 21-fold improvement in editing efficiency when simultaneously targeting 6 gene sites. Furthermore, the inventors also tested the TRU-MPE5max-10 system in HeLa and iPSC cells, and the results are shown in Figure 15: the TRU-MPE5max-10 system not only achieved co-editing of up to 10 sites in HeLa and iPSC cells, but also demonstrated relatively high editing efficiency across 10 different gene sites.

[1191] The above experimental results show that the optimized TRU-MPE5max not only possesses the ability to perform multi-site gene editing in various cell lines, but also achieves co-editing of up to 10 gene sites in cell lines including HEK293T, HeLa, and iPSC. This fully demonstrates the multi-site gene editing capability of the TRU-MPE5max system.

[1192] Example 4: The TRU-MPE5max system can be delivered using a lentivirus system.

[1193] Using primers OL3078 / OL3079 and plasmid (Addgene:52961) as a template, PCR amplification was performed as described in step 1) above to obtain the plasmid backbone expressing nCas9 in the lentiviral system. Using primers OL3080 / OL3081 and LL392 as a template, PCR amplification was performed as described in step 1) above to obtain the GFP fragment. The purified DNA fragments were subjected to Gibson reaction as described in step 3) above, and the resulting positive clone was named LL1294 (characterized by: a lentiviral system containing a GFP fluorescent selection tag and a plasmid vector expressing Cas9).

[1194] Using primers OL5080 / OL5081 and LL1294 as a template, PCR amplification was performed as described in step 1) above. Using primers OL5082 / OL5083 and LL1184 as a template, PCR amplification was performed as described in step 1) above. The purified DNA fragment was subjected to a Gibson reaction as described in step 3) above. The resulting positive clone was named LL1820 (characterized by: EF1α promoter (SEQ ID NO:297) driving the expression of the RT-P2A-MLH1dn-P2A-EGFP element).

[1195] Using primers OL5080 / OL5081 and LL1294 as a template, PCR amplification was performed as described in step 1) above. Using primers OL5082 / OL5084 and LL1185 as a template, PCR amplification was performed as described in step 1) above. The purified DNA fragments were subjected to Gibson reaction as described in step 3) above. The resulting positive clone was named LL1821 (characterized by the expression of the nCas9-P2A-BFP element driven by the EF1α promoter).

[1196] Using primers OL5080 / OL5085 and LL1294 as a template, amplification was performed as described in step 1) above. Using primers OL5086 / 5087 and LL1214 as a template, amplification was performed as described in step 1) above, resulting in an element containing hCtRNA driving the expression of BAG3 gene pegRNA and nicking sgRNA. Using primers OL5088 / OL5089 and LL1184 as a template, amplification was performed as described in step 1) above. Using primers OL5090 / OL5091 and LL1214 as a template, amplification was performed as described in step 1) above. The purified DNA fragments were subjected to a Gibson reaction as described in step 3) above, and the resulting positive clone was named LL1822 (characterized by: EF1α promoter driving the expression of BAG3 gene hCtRNA-nicking sgRNA-hCtRNA pegRNA elements).

[1197] Using primers OL5080 / OL5085 and LL1294 as a template, amplification was performed as described in step 1) above. Using primers OL5092 / 5087 and LL1215 as a template, amplification was performed as described in step 1) above, resulting in elements containing hCtRNA driving the expression of NMB gene pegRNA and nicking sgRNA. Using primers OL5088 / OL5089 and LL1184 as a template, amplification was performed as described in step 1) above. Using primers OL5090 / OL5091 and LL1214 as a template, amplification was performed as described in step 1) above. The purified DNA fragments were subjected to a Gibson reaction as described in step 3) above, and the resulting positive clone was named LL1823 (characterized by: EF1α promoter driving the expression of NMB gene hCtRNA-nicking sgRNA-hCtRNA pegRNA elements).

[1198] Using primers OL5080 / OL5085 and LL1294 as a template, amplification was performed as described in step 1) above. Using primers OL5093 / 5087 and LL1216 as a template, amplification was performed as described in step 1) above, resulting in an element containing hCtRNA driving the expression of FANCF gene pegRNA and nicking sgRNA. Using primers OL5088 / OL5089 and LL1184 as a template, amplification was performed as described in step 1) above. Using primers OL5090 / OL5091 and LL1214 as a template, amplification was performed as described in step 1) above. The purified DNA fragments were subjected to a Gibson reaction as described in step 3) above, and the resulting positive clone was named LL1824 (characterized by: EF1α promoter driving the expression of FANCF gene hCtRNA-nicking sgRNA-hCtRNA pegRNA elements). Using primers OL5080 / OL5085 and LL1294 as a template, amplification was performed as described in step 1) above. Using primers OL5114 / 5115 and LL1217 as a template, amplification was performed as described in step 1) above, resulting in an element containing hCtRNA driving the expression of pegRNA and nicking sgRNA of the three genes BAG3 / NMB / FANCF. Using primers OL5088 / OL5089 and LL1184 as a template, amplification was performed as described in step 1) above. Using primers OL5090 / OL5091 and LL1214 as a template, amplification was performed as described in step 1) above. The purified DNA fragments were subjected to a Gibson reaction as described in step 3) above, and the resulting positive clone was named LL1825 (characterized by: EF1α promoter driving the expression of hCtRNA-nicking sgRNA-hCtRNA pegRNA elements targeting the three genes BAG3 / NMB / FANCF).

[1199] To prepare lentiviral particles, HEK293T cells were cultured at approximately 1.2 × 10⁻⁶ cells per 15 cm culture dish. 7 Cells were seeded at a density of 10 μg / mL in 20 mL of complete culture medium. After 16 hours, each culture dish was transiently transfected with a transfection mixture consisting of 22.5 μg of the target gene plasmid, 19.1 μg of the packaging plasmid psPAX2, and 5.6 μg of the envelope plasmid pMD2.G. Transfection was performed using PEI 40000 (working concentration 1 mg / mL) strictly according to the manufacturer's instructions. After 6 hours, the medium was replaced with fresh complete culture medium. 72 hours post-transfection, the virus-containing supernatant was collected, first centrifuged at 400 rcf for 10 minutes at 4°C to remove cell debris, and then filtered through a 0.45 μm PES filter (Biosharp, BP-PES-45). The lentiviral particles were then precipitated by centrifugation at 15000 rcf for 2 hours at 4°C. The lentiviral pellet obtained from each culture dish was resuspended in 200 μL of RPMI 1640 medium, and then aliquoted into 50 μL tubes into 1.5 mL centrifuge tubes and stored at -80°C for later use. 50 μL of resuspended lentivirus containing LL1820+LL1821+LL1822 / LL1823 / LL1824 / LL1825 was added to HEK293T or Jurkat cells cultured in 24-well plates, designated as D0. Starting from D2, the same batch of cells was treated with 1 μg / mL puromycin as the lentivir-mediated MPE5max experimental group. After 6 days of culture, half of the lentivir-infected HEK293T and Jurkat cells were collected for flow cytometry sorting. Cells co-expressing GFP / BFP / RFP trifluorescein were collected and added to lysis buffer, while the other half continued normal culture. On day 13, half of the lentivirally infected HEK293T and Jurkat cells were collected for flow cytometry sorting. Cells co-expressing GFP / BFP / RFP trifluorescence-positive cells were collected and added to lysis buffer. The other half continued normal culture. On day 20, all HEK293T and Jurkat cells were collected for flow cytometry sorting. Cells co-expressing GFP / BFP / RFP trifluorescence-positive cells were added to lysis buffer. DNA was extracted from the cells in lysis buffer after 65℃ for 6 min and 98℃ for 2 min. Target sites were amplified using different primers, and NGS sequencing analysis was performed.

[1200] Figure 16 shows the lentiviral editing efficiency in HEK293T cells. Analysis reveals that after day 13 of culture, the lentivir-mediated TRU-MPE5max-3 system showed a gene editing efficiency approximately 22-70 times higher than the lentivir-mediated MPE5max-3 system. Figure 17 shows the lentiviral editing efficiency in iPSC cells. Similarly, after day 13 of culture, the lentivir-mediated TRU-MPE5max-3 system showed a gene editing efficiency approximately 9-17 times higher than the lentivir-mediated MPE5max-3 system. In summary, the above experimental results indicate that the lentivir-mediated TRU-MPE5max gene editing system has high editing efficiency and can achieve co-editing of up to three gene loci.

[1201] Example 5: The TRU-PE system has excellent compatibility with other PE versions.

[1202] Based on the TRU-MPE5max design scheme in Example 2, the same engineered vector was modified for two different gene editing versions, PE6e and PE7, to construct the TRU-PE6e and TRU-PE7 gene editing systems shown in Figure 18. The specific operations are as follows:

[1203] Using primers OL2766 / 2768, OL2767 / 1033, and OL2769 / 1034, and with the LL622 vector from Example 2 as a template, PCR amplification was performed as described in step 1) above. The purified and recovered fragment was ligated using the Gibson reaction as described in step 3) above, and the transformed positive clone was named LL1218 (a vector expressing nCas9-BFP in the TRU-PE6e system).

[1204] Using primers OL2670 / 2674, OL2673 / 2669, and OL2671 / 2672, and with the LL392 vector from Example 2 as a template, PCR amplification was performed as described in step 1) above. The purified and recovered fragment was ligated using a Gibson reaction as described in step 3) above, and the transformed positive clone was named LL1186 (a vector expressing RT-GFP in the TRU-PE6e system).

[1205] Using primers OL4255 / 4256 with LL003 as a template; using primers OL4257 / 4258 with the La-SZBL library plasmid (horizon-OHS5898) as a template; using primers OL4259 / 4203 with LL003 as a template; and using primers OL4282 / 4283 with OL4260 as a template, PCR amplification was performed as described in step 1) above. Using LL003 as a template, restriction endonucleases BamHI and AgeI were used for digestion as described in step 2) above. The purified digested plasmid backbone was ligated with the purified DNA fragment template using a Gibson reaction as described in step 3) above. The ligation product was transformed into DH5α Escherichia coli, and the positive clone was named LL1449(PE7).

[1206] Using primers OL1335 / 4201 with LL1218 as a template; using primers OL4202 / 4203 with LL1186 as a template; and using primers OL1207 / 400 with LL1186 as a template, PCR amplification was performed as described in step 1) above. The purified and recovered fragments were ligated using the Gibson reaction as described in step 3) above, and the transformed positive clone was named LL1467(...

Claims

A gene editing system comprising a vector; The vector contains: a gene encoding a guide RNA (gRNA) targeting a specific gene site, a tRNA (transfer RNA), and a gene encoding a selection marker. The gene editing system according to claim 1, wherein The selection marker does not contain resistance genes. The gene editing system according to any one of claims 1-2 is characterized in that, The gene editing system includes a CRISPR / Cas9 system; further includes at least one of a cytosine base editing system, an adenine base editing system, a guanine base editing system, and a lead editing system; and even further, is a lead editing system; Preferably, the pilot editing system includes at least one of PE1, PE2, PE3, PEmax, PE4, PE5, PE6 (e.g., PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, PE6g), and PE7; Optionally, the vector comprises: a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding guide RNA (pegRNA) targeting a target gene site, tRNA, and a gene encoding a selection marker. The gene editing system according to any one of claims 1-3, characterized in that The screening marker is a fluorescent protein or a mutant thereof or a truncated thereof; Preferably, the fluorescent protein comprises green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, AzamiGreen, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent protein (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent protein (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent protein (e.g., CFP, eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), and red fluorescent protein (e.g., RFP, At least one of the following: eRFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, monomer Kusabira-Orange, mTangerine, tdTomato), and luciferase. The gene editing system according to any one of claims 1-4, characterized in that The vector contains: an expression cassette encoding a gene for Cas9 nickase (nCas9), a gene for reverse transcriptase (RT), a gene for RNA targeting a specific gene site, and a gene encoding a fluorescent protein or a mutant or truncated form thereof. The expression cassette of the gene encoding RNA targeting the target gene site includes: tRNA (Transfer RNA) and a gene encoding guide RNA (pegRNA) targeting the target gene site; Preferably, when the vector contains a fluorescent protein mutant, the expression cassette of the gene encoding RNA targeting the target gene site further includes: pegRNA targeting the fluorescent protein mutant; Preferably, the vector comprises: an expression cassette encoding a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding a fluorescent protein or a mutant or truncated form thereof; The expression cassette of the gene encoding RNA targeting the target gene site includes: tRNA (Transfer RNA) and a gene encoding guide RNA (pegRNA) targeting the target gene site; 1) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in a vector, said vector containing genes encoding one or more fluorescent proteins or their mutants or truncated forms; or 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in two vectors; 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; or 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or 3) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in three vectors; 31) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; a vector containing a gene encoding a reverse transcriptase (RT) further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; a vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein, wherein the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different; or 32) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a fragment 1 of a first fluorescent protein mutant; a vector containing a gene encoding a reverse transcriptase (RT) further contains a gene encoding a first fluorescent protein or a fragment 2 of a first fluorescent protein mutant; a vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a second fluorescent protein or a second fluorescent protein mutant, wherein the first fluorescent protein or fragment 1 of a first fluorescent protein mutant and fragment 2 of a first fluorescent protein mutant constitute the complete first fluorescent protein or a first fluorescent protein mutant; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; Preferably, the vector further comprises: a gene encoding the MLH1dn protein; Preferably, the vector comprises: an expression cassette encoding a gene for Cas9 nickase, a gene for reverse transcriptase, a gene for RNA targeting a specific gene site, a gene for MLH1dn protein, and a gene for a fluorescent protein or a mutant or truncated form thereof. The expression cassette of the gene encoding RNA targeting the target gene site includes a gene encoding guide editing RNA (pegRNA) and tRNA targeting the target gene site; 1) An expression cassette containing a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding MLH1dn protein, wherein the vector contains genes encoding one or more fluorescent proteins, mutants thereof, or truncated forms thereof; or 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), the gene encoding RNA targeting the target gene site, and the gene encoding MLH1dn protein are located in two vectors. 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the vector encoding MLH1dn protein further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein or a truncated form of the second fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; or 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or 3) The expression cassettes of the genes encoding Cas9 nickase (nCas9), reverse transcriptase (RT), RNA targeting the target gene site, and MLH1dn protein are located in three vectors. 31) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; a vector containing a gene encoding a reverse transcriptase (RT) or an MLH1dn protein further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; a vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein, wherein the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different; or 32) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein fragment 1; a vector containing a gene encoding a reverse transcriptase (RT) or a vector encoding an MLH1dn protein further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein fragment 2; a vector containing an expression cassette encoding RNA targeting a target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein, wherein the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; Preferably, the vector further comprises: a gene encoding a nicking sgRNA targeting a specific gene site; Preferably, the vector comprises: an expression cassette encoding a gene for Cas9 nickase, a gene for reverse transcriptase, a gene for RNA targeting a specific gene site, a gene for MLH1dn protein, and a gene for fluorescent protein or a mutant or truncated form thereof. The expression cassette of the gene encoding RNA targeting the target gene site includes a gene encoding a guide editing RNA (pegRNA) targeting the target gene site, a gene encoding a nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA; 1) An expression cassette containing a gene encoding Cas9 nickase (nCas9), a gene encoding reverse transcriptase (RT), a gene encoding RNA targeting a specific gene site, and a gene encoding MLH1dn protein, wherein the vector contains genes encoding one or more fluorescent proteins, mutants thereof, or truncated forms thereof; or 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), the gene encoding RNA targeting the target gene site, and the gene encoding MLH1dn protein are located in two vectors. 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the vector encoding MLH1dn protein further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein or a truncated form of the second fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; or 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or 3) The expression cassettes of the genes encoding Cas9 nickase (nCas9), reverse transcriptase (RT), RNA targeting the target gene site, and MLH1dn protein are located in three vectors. 31) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; a vector containing a gene encoding a reverse transcriptase (RT) or an MLH1dn protein further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; a vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a third fluorescent protein or a mutant of the third fluorescent protein, wherein the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different; or 32) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein fragment 1; a vector containing a gene encoding a reverse transcriptase (RT) or a vector encoding an MLH1dn protein further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein fragment 2; a vector containing an expression cassette encoding RNA targeting a target gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein, wherein the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; Preferably, the vector comprises: an expression cassette encoding a gene encoding a Cas9 nickase, a gene encoding a reverse transcriptase, a gene encoding RNA targeting a specific gene site, and a gene encoding a fluorescent protein or a mutant or truncated form thereof; The expression cassette of the gene encoding RNA targeting the target gene site includes a gene encoding a guide editing RNA (pegRNA) targeting the target gene site, a gene encoding a nicking sgRNA (nicking RNA) targeting the target gene site, and tRNA; 1) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in a vector, said vector containing genes encoding one or more fluorescent proteins or their mutants or truncated forms; or 2) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in two vectors; 21) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in the same vector, the vector containing the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; the vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; or 22) When the gene encoding Cas9 nickase (nCas9) and the gene encoding reverse transcriptase (RT) are located in different vectors, wherein one vector contains a gene encoding fragment 1 of fluorescent protein or its mutant or truncated form, and the other vector contains a gene encoding fragment 2 of fluorescent protein or its mutant or truncated form, and fragment 1 of fluorescent protein or its mutant or truncated form and fragment 2 of fluorescent protein or its mutant or truncated form constitute the complete fluorescent protein or its mutant or truncated form; or 3) The expression cassettes of the gene encoding Cas9 nickase (nCas9), the gene encoding reverse transcriptase (RT), and the gene encoding RNA targeting the target gene site are located in three vectors; 31) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a mutant of the first fluorescent protein; a vector containing a gene encoding a reverse transcriptase (RT) further contains a gene encoding a second fluorescent protein or a mutant of the second fluorescent protein; a vector containing an expression cassette encoding RNA targeting a specific gene site further contains a third fluorescent protein or a mutant of the third fluorescent protein, wherein the first fluorescent protein, the second fluorescent protein, the third fluorescent protein, or their mutants or truncated forms are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different; or 32) A vector containing a gene encoding a Cas9 nickase (nCas9) further contains a gene encoding a first fluorescent protein or a fragment 1 of a first fluorescent protein mutant; a vector containing a gene encoding a reverse transcriptase (RT) further contains a gene encoding a first fluorescent protein or a fragment 2 of a first fluorescent protein mutant; a vector containing an expression cassette encoding RNA targeting a specific gene site further contains a gene encoding a second fluorescent protein or a second fluorescent protein mutant, wherein the first fluorescent protein or fragment 1 of a first fluorescent protein mutant and fragment 2 of a first fluorescent protein mutant constitute the complete first fluorescent protein or a first fluorescent protein mutant; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; Preferably, the vector further comprises: a gene encoding the La protein; Preferably, the vector comprises: a gene encoding a Cas9 nickase, a gene encoding a reverse transcriptase, an expression cassette of a gene encoding an RNA targeting a target gene site, a gene encoding a La protein, and a gene encoding a fluorescent protein or a mutant thereof or a truncation thereof; the expression cassette of the gene encoding the RNA targeting a target gene site comprises a gene encoding a prime editing guide RNA (pegRNA) targeting a target gene site, a gene encoding a nicking sgRNA (nicking RNA) targeting a target gene site, a tRNA; 1) the gene encoding a Cas9 nickase (nCas9), the gene encoding a reverse transcriptase (RT), the expression cassette of the gene encoding an RNA targeting a target gene site, and the gene encoding a La protein are located in one vector, and the vector comprises a gene encoding one or more fluorescent proteins or a mutant thereof or a truncation thereof; or 2) the gene encoding a Cas9 nickase (nCas9), the gene encoding a reverse transcriptase (RT), the expression cassette of the gene encoding an RNA targeting a target gene site, and the gene encoding a La protein are located in two vectors; 21) when the gene encoding a Cas9 nickase (nCas9) and the gene encoding a reverse transcriptase (RT) are located in the same vector, the vector comprising the gene encoding a Cas9 nickase (nCas9), the gene encoding a reverse transcriptase (RT), and the gene encoding a La protein further comprises a gene encoding a first fluorescent protein or a mutant thereof, and the vector comprising the expression cassette of the gene encoding an RNA targeting a target gene site further comprises a gene encoding a second fluorescent protein or a mutant thereof or a truncation thereof; the first fluorescent protein and the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; or 22) when the gene encoding a Cas9 nickase (nCas9) and the gene encoding a reverse transcriptase (RT) are located in different vectors, wherein one vector comprises a gene encoding a fluorescent protein or a mutant thereof or a truncation thereof in fragment 1, and the other vector comprises a gene encoding a fluorescent protein or a mutant thereof or a truncation thereof in fragment 2, and the fluorescent protein or the mutant thereof or the truncation thereof in fragment 1 and the fluorescent protein or the mutant thereof or the truncation thereof in fragment 2 constitute a complete fluorescent protein or a mutant thereof or a truncation thereof; or 3) the gene encoding a Cas9 nickase (nCas9), the gene encoding a reverse transcriptase (RT), the expression cassette of the gene encoding an RNA targeting a target gene site, and the gene encoding a La protein are located in three vectors; 31) the vector comprising a gene encoding a Cas9 nickase (nCas9) further comprises a gene encoding a first fluorescent protein or a first fluorescent protein mutant, the vector comprising a gene encoding a reverse transcriptase (RT) further comprises a gene encoding a second fluorescent protein or a second fluorescent protein mutant, the vector comprising an expression cassette of a gene encoding an RNA targeting a target genetic locus further comprises a gene encoding a third fluorescent protein or a third fluorescent protein mutant, each of the first fluorescent protein, the second fluorescent protein, the third fluorescent protein or a mutant or a truncation thereof is independently selected from the fluorescent proteins, the first fluorescent protein, the second fluorescent protein, the third fluorescent protein are different; or 32) the vector comprising a gene encoding a Cas9 nickase (nCas9) further comprises a gene encoding a first fluorescent protein or a first fluorescent protein mutant, fragment 1, the vector comprising a gene encoding a reverse transcriptase (RT) further comprises a gene encoding a first fluorescent protein or a first fluorescent protein mutant, fragment 2, the vector comprising an expression cassette of a gene encoding an RNA targeting a target genetic locus further comprises a gene encoding a second fluorescent protein or a second fluorescent protein mutant, each of the first fluorescent protein, the second fluorescent protein is independently selected from the fluorescent proteins, the first fluorescent protein is different from the second fluorescent protein. The gene editing system according to any one of claims 1-5, wherein the vector comprises a first vector; the first vector comprises a gene encoding a Cas9 nickase, a gene encoding a reverse transcriptase, an expression cassette of a gene encoding an RNA targeting a target genetic locus, a gene encoding a MLH1dn protein, and a gene encoding a fluorescent protein or a mutant or a truncation thereof; the expression cassette of a gene encoding an RNA targeting a target genetic locus comprises a gene encoding a prime editing guide RNA targeting a target genetic locus, a gene encoding a nicking sgRNA targeting a target genetic locus, and a tRNA; preferably, the vector comprises a first vector; the first vector comprises a first promoter, a gene encoding a Cas9 nickase, a gene encoding a reverse transcriptase, a gene encoding a MLH1dn protein, an expression cassette of a gene encoding an RNA targeting a target genetic locus, and a gene encoding a fluorescent protein; preferably, the expression cassette of a gene encoding an RNA targeting a target genetic locus comprises a tRNA, a gene encoding a prime editing guide RNA targeting a target genetic locus, a tRNA, and a gene encoding a nicking sgRNA targeting a target genetic locus. The gene editing system according to any one of claims 1-5, wherein the vector comprises a first vector and a second vector; the first vector comprises a gene encoding a Cas9 nickase, a gene encoding a reverse transcriptase, a gene encoding a MLH1dn protein, and a gene encoding a first fluorescent protein or a first fluorescent protein mutant; The second vector comprises: an expression cassette of a gene encoding an RNA targeting a target gene site, and a gene encoding a second fluorescent protein or a second fluorescent protein mutant; The expression cassette of the gene encoding the RNA targeting the target gene site comprises: a gene encoding a prime editing guide RNA targeting the target gene site, a gene encoding a nicking sgRNA targeting the target gene site, and a tRNA; The first fluorescent protein, the second fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein is different from the second fluorescent protein; Preferably, the first vector comprises: a first promoter, a gene encoding a Cas9 nickase, a gene encoding a reverse transcriptase, a gene encoding a MLH1dn protein, and a gene encoding a first fluorescent protein; Preferably, the second vector comprises: an expression cassette of a gene encoding an RNA targeting a target gene site, and a gene encoding a second fluorescent protein; Preferably, the expression cassette of the gene encoding the RNA targeting the target gene site comprises: a tRNA, a gene encoding a prime editing guide RNA targeting the target gene site, a tRNA, and a gene encoding a nicking sgRNA targeting the target gene site. The gene editing system according to any one of claims 1-5, wherein The vector comprises: a first vector, a second vector, and a third vector; 1) the first vector comprises: a gene encoding a Cas9 nickase, and a gene encoding a first fluorescent protein or a first fluorescent protein mutant; The second vector comprises: a gene encoding a reverse transcriptase, a gene encoding a MLH1dn protein, and a gene encoding a second fluorescent protein or a second fluorescent protein mutant; The third vector comprises: an expression cassette of a gene encoding an RNA targeting a target gene site, and a gene encoding a third fluorescent protein or a third fluorescent protein mutant; The first fluorescent protein, the second fluorescent protein, the third fluorescent protein are each independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, the third fluorescent protein are different; or 2) the first vector comprises: a gene encoding a Cas9 nickase (nCas9), a gene encoding a first fluorescent protein or a first fluorescent protein mutant, fragment 1; The second vector comprises: a gene encoding a reverse transcriptase (RT), a gene encoding a first fluorescent protein or a first fluorescent protein mutant, fragment 2, a gene encoding a MLH1dn protein; The third vector comprises: an expression cassette of a gene encoding an RNA targeting a target gene, and a gene encoding a second fluorescent protein or a mutant thereof or a truncated thereof; The fragment 1 of the first fluorescent protein or the first fluorescent protein mutant and the fragment 2 of the first fluorescent protein or the first fluorescent protein mutant constitute a complete first fluorescent protein or a first fluorescent protein mutant; The first fluorescent protein, the second fluorescent protein are each independently selected from the fluorescent proteins, the first fluorescent protein is different from the second fluorescent protein (the first fluorescent protein, the second fluorescent protein have different excitation wavelengths); and The expression cassette of the gene encoding the RNA targeting the target gene site comprises: a gene encoding a prime editing guide RNA targeting the target gene site, a gene encoding a nicking sgRNA targeting the target gene site, and a tRNA; Preferably, the first vector comprises: a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a fragment 1 of a second fluorescent protein; Preferably, the second vector comprises: a second promoter, a gene encoding a reverse transcriptase (RT), a gene encoding a fragment 2 of a second fluorescent protein, and a gene encoding a MLH1dn protein; Preferably, the third vector comprises: an expression cassette of a gene encoding the RNA targeting the target gene site, and a gene encoding a third fluorescent protein or a mutant thereof or a truncation thereof; Preferably, the first vector comprises: a first promoter, a gene encoding a Cas9 nickase, and a gene encoding a first fluorescent protein; Preferably, the second vector comprises: a second promoter, a gene encoding a reverse transcriptase, a gene encoding a MLH1dn protein, and a gene encoding a second fluorescent protein; Preferably, the third vector comprises: an expression cassette of a gene encoding the RNA targeting the target gene site, and a gene encoding a third fluorescent protein; Preferably, the first vector comprises: a first promoter, a gene encoding a Cas9 nickase (nCas9), and a gene encoding a first fluorescent protein; Preferably, the second vector comprises: a second promoter, a gene encoding a reverse transcriptase (RT), a gene encoding a MLH1dn protein, and a gene encoding a second fluorescent protein; Preferably, the third vector comprises: an expression cassette of a gene encoding the RNA targeting the target gene site, and a gene encoding a third fluorescent protein mutant; Preferably, the expression cassette of the gene encoding the RNA targeting the target gene site further comprises: a pegRNA targeting the third fluorescent protein mutant; Preferably, the expression cassette of the gene encoding the RNA targeting the target gene site comprises: a tRNA, a gene encoding a prime editing guide RNA targeting the target gene site, a tRNA, and a gene encoding a nicking sgRNA targeting the target gene site. The gene editing system according to any one of claims 1-5, wherein, The vector comprises: a first vector, a second vector, and a third vector; The first vector comprises: a gene encoding a Cas9 nickase, and a gene encoding a first fluorescent protein or a first fluorescent protein mutant; The second vector comprises: a gene encoding a reverse transcriptase, and a gene encoding a second fluorescent protein or a second fluorescent protein mutant; The third vector comprises: an expression cassette of a gene encoding the RNA targeting the target gene site, a gene encoding a third fluorescent protein or a third fluorescent protein mutant; The expression cassette of the gene encoding the RNA targeting the target gene site comprises: a gene encoding the prime editing guide RNA targeting the target gene site, a gene encoding the nicking sgRNA targeting the target gene site, and a tRNA; each of the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein is independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different; Preferably, the first vector comprises: a first promoter, a gene encoding a Cas9 nickase, and a gene encoding a first fluorescent protein; Preferably, the second vector comprises: a second promoter, a gene encoding a reverse transcriptase, and a gene encoding a second fluorescent protein; Preferably, the third vector comprises: an expression cassette of a gene encoding an RNA targeting a target gene site, and a gene encoding a third fluorescent protein; Preferably, the expression cassette of the gene encoding the RNA targeting the target gene site comprises: a tRNA, a gene encoding a prime editing guide RNA targeting the target gene site, a tRNA, and a gene encoding a nicking sgRNA targeting the target gene site. The gene editing system according to any one of claims 1-5, wherein the vector comprises: a first vector, a second vector, and a third vector; the first vector comprises: a gene encoding a Cas9 nickase, and a gene encoding a first fluorescent protein or a first fluorescent protein mutant; the second vector comprises: a gene encoding a reverse transcriptase, a gene encoding a La protein, and a gene encoding a second fluorescent protein or a second fluorescent protein mutant; the third vector comprises: an expression cassette of a gene encoding an RNA targeting a target gene site, and a gene encoding a third fluorescent protein or a third fluorescent protein mutant; the expression cassette of the gene encoding the RNA targeting the target gene site comprises: a gene encoding a prime editing guide RNA targeting the target gene site, a gene encoding a nicking sgRNA targeting the target gene site, and a tRNA; each of the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein is independently selected from the fluorescent proteins, and the first fluorescent protein, the second fluorescent protein, and the third fluorescent protein are different; Preferably, the first vector comprises: a first promoter, a gene encoding a Cas9 nickase, and a gene encoding a first fluorescent protein; Preferably, the second vector comprises: a first promoter, a gene encoding a Cas9 nickase, and a gene coding a first fluorescent protein; Preferably, the third vector comprises: an expression cassette of a gene encoding a RNA targeting a target gene site, and a gene encoding a third fluorescent protein; Preferably the expression cassette of the gene encoding the RNA targeting the target gene site comprises: a tRNA; a gene encoding a prime editing guide RNA targeting the target gene site; a tRNA; and a gene encoding a nicking sgRNA targeting the target gene site. The gene editing system of any one of claims 1-10, wherein the target gene site is one or more; Preferably, when the target gene site is multiple, the expression cassette of the gene encoding the RNA targeting the target gene site comprises N expression cassettes of the gene encoding the RNA targeting the target gene site, N being the number of target gene sites. Preferably, the target gene site is FANCF, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 210, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 227; Preferably, the target gene site is DNMT1, the sequence of the prime editing guide RNA (pegRNA) targeting the site-directed mutation of the target gene site is shown as SEQ ID NO: 215, and the sequence of the nicking sgRNA targeting the site-directed mutation of the target gene site is shown as SEQ ID NO: 232; Preferably, the target gene site is DNMT1, the sequence of the prime editing guide RNA (pegRNA) targeting the deletion mutation of the target gene site is shown as SEQ ID NO: 226, and the sequence of the nicking sgRNA targeting the deletion mutation of the target gene site is shown as SEQ ID NO: 243; Preferably, the target gene site is ERCC6, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 211, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 228; Preferably, the target gene site is EMX1, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 212, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 229; Preferably, the target gene site is KRAS, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 219, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 236; Preferably, the target gene site is LRRK2, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 218, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 235; Preferably, the target gene site is NMB, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 214, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 231; Preferably, the target gene site is BAG3, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 213, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 230; Preferably, the target gene site is GBA1, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 217, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 234; Preferably, the target gene site is TP53, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 216, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 233; Preferably, the target gene site is RNF2, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is shown as SEQ ID NO: 220, and the sequence of the nicking sgRNA targeting the target gene site is shown as SEQ ID NO: 237; Preferably, the target gene sites are BAG3, NMB and FANCF; wherein the sequence of the prime editing guide RNA (pegRNA) targeting BAG3 is shown as SEQ ID NO: 213, the sequence of the nicking sgRNA targeting BAG3 is shown as SEQ ID NO: 230, the sequence of the prime editing guide RNA (pegRNA) targeting NMB is shown as SEQ ID NO: 214, the sequence of the nicking sgRNA targeting NMB is shown as SEQ ID NO: 231, the sequence of the prime editing guide RNA (pegRNA) targeting FANCF is shown as SEQ ID NO: 210, and the sequence of the nicking sgRNA targeting FANCF is shown as SEQ ID NO: 227; Preferably, the target gene sites are DNMT1, EMX1 and ERCC6; wherein the sequence of the prime editing guide RNA (pegRNA) targeting a site-directed mutation of DNMT1 is shown as SEQ ID NO: 215, the sequence of the nicking sgRNA targeting a site-directed mutation of DNMT1 is shown as SEQ ID NO: 232, the sequence of the prime editing guide RNA (pegRNA) targeting EMX1 is shown as SEQ ID NO: 212, the sequence of the nicking sgRNA targeting EMX1 is shown as SEQ ID NO: 229, the sequence of the prime editing guide RNA (pegRNA) targeting ERCC6 is shown as SEQ ID NO: 211, the sequence of the nicking sgRNA targeting ERCC6 is shown as SEQ ID NO: 228; Preferably, the target gene sites are GATA2, SETBP1 and ASXL1; wherein the sequence of the prime editing guide RNA (pegRNA) targeting GATA2 is shown as SEQ ID NO: 221, the sequence of the nicking sgRNA targeting GATA2 is shown as SEQ ID NO: 238, the sequence of the prime editing guide RNA (pegRNA) targeting SETBP1 is shown as SEQ ID NO: 222, the sequence of the nicking sgRNA targeting SETBP1 is shown as SEQ ID NO: 239, the sequence of the prime editing guide RNA (pegRNA) targeting ASXL1 is shown as SEQ ID NO: 223, the sequence of the nicking sgRNA targeting ASXL1 is shown as SEQ ID NO: 240; Preferably, the target gene sites are BAG3, NMB, FANCF, DNMT1, EMX1 and ERCC6; wherein the sequence of the prime editing guide RNA (pegRNA) targeting BAG3 is shown as SEQ ID NO:213, the sequence of the nicking sgRNA targeting BAG3 is shown as SEQ ID NO:230, the sequence of the prime editing guide RNA (pegRNA) targeting NMB is shown as SEQ ID NO:214, the sequence of the nicking sgRNA targeting NMB is shown as SEQ ID NO:231, the sequence of the prime editing guide RNA (pegRNA) targeting FANCF is shown as SEQ ID NO:210, the sequence of the nicking sgRNA targeting FANCF is shown as SEQ ID NO:227, the sequence of the prime editing guide RNA (pegRNA) targeting DNMT1 site-directed mutation is shown as SEQ ID NO:215, the sequence of the nicking sgRNA targeting DNMT1 site-directed mutation is shown as SEQ ID NO:232, the sequence of the prime editing guide RNA (pegRNA) targeting EMX1 is shown as SEQ ID NO:212, the sequence of the nicking sgRNA targeting EMX1 is shown as SEQ ID NO:229, the sequence of the prime editing guide RNA (pegRNA) targeting ERCC6 is shown as SEQ ID NO:211, the sequence of the nicking sgRNA targeting ERCC6 is shown as SEQ ID NO:228; Preferably, the target gene sites are BAG3, NMB, FANCF, DNMT1, EMX1 and ERCC6; wherein the sequence of the prime editing guide RNA (pegRNA) targeting BAG3 is shown as SEQ ID NO:213, the sequence of the nicking sgRNA targeting BAG3 is shown as SEQ ID NO:230, the sequence of the prime editing guide RNA (pegRNA) targeting NMB is shown as SEQ ID NO:214, the sequence of the nicking sgRNA targeting NMB is shown as SEQ ID NO:231, the sequence of the prime editing guide RNA (pegRNA) targeting FANCF is shown as SEQ ID NO:210, the sequence of the nicking sgRNA targeting FANCF is shown as SEQ ID NO:227, the sequence of the prime editing guide RNA (pegRNA) targeting DNMT1 site-directed mutation is shown as SEQ ID NO:215, the sequence of the nicking sgRNA targeting DNMT1 site-directed mutation is shown as SEQ ID NO:232, the sequence of the prime editing guide RNA (pegRNA) targeting EMX1 is shown as SEQ ID NO:212, the sequence of the nicking sgRNA targeting EMX1 is shown as SEQ ID NO:229, the sequence of the prime editing guide RNA (pegRNA) targeting ERCC6 is shown as SEQ ID NO:211, the sequence of the nicking sgRNA targeting ERCC6 is shown as SEQ ID NO:228; Preferably, the target gene site is BAG3, NMB, FANCF, DNMT1, EMX1, ERCC6, TP53, GBA1, LRRK2 and KRAS; wherein the sequence of the prime editing guide RNA (pegRNA) targeting BAG3 is shown as SEQ ID NO: 213, the sequence of the nicking sgRNA targeting BAG3 is shown as SEQ ID NO: 230, the sequence of the prime editing guide RNA (pegRNA) targeting NMB is shown as SEQ ID NO: 214, the sequence of the nicking sgRNA targeting NMB is shown as SEQ ID NO: 231, the sequence of the prime editing guide RNA (pegRNA) targeting FANCF is shown as SEQ ID NO: 210, the sequence of the nicking sgRNA targeting FANCF is shown as SEQ ID NO: 227, the sequence of the prime editing guide RNA (pegRNA) targeting DNMT1 site-directed mutation is shown as SEQ ID NO: 215, the sequence of the nicking sgRNA targeting DNMT1 site-directed mutation is shown as SEQ ID NO: 232, the sequence of the prime editing guide RNA (pegRNA) targeting EMX1 is shown as SEQ ID NO: 212, the sequence of the nicking sgRNA targeting EMX1 is shown as SEQ ID NO: 229, the sequence of the prime editing guide RNA (pegRNA) targeting ERCC6 is shown as SEQ ID NO: 211, the sequence of the nicking sgRNA targeting ERCC6 is shown as SEQ ID NO: 228, the sequence of the prime editing guide RNA (pegRNA) targeting TP53 is shown as SEQ ID NO: 216, the sequence of the nicking sgRNA targeting TP53 is shown as SEQ ID NO: 233, the sequence of the prime editing guide RNA (pegRNA) targeting GBA1 is shown as SEQ ID NO: 217, the sequence of the nicking sgRNA targeting GBA1 is shown as SEQ ID NO: 234, the sequence of the prime editing guide RNA (pegRNA) targeting LRRK2 is shown as SEQ ID NO: 218, the sequence of the nicking sgRNA targeting LRRK2 is shown as SEQ ID NO: 235, the sequence of the prime editing guide RNA (pegRNA) targeting KRAS is shown as SEQ ID NO: 219, the sequence of the nicking sgRNA targeting KRAS is shown as SEQ ID NO: 236; Preferably, the target gene site is CDKL5, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is set forth in SEQ ID NO: 224, and the sequence of the nicking sgRNA targeting the target gene site is set forth in SEQ ID NO:

241. Preferably, the target gene site is CXCR4, the sequence of the prime editing guide RNA (pegRNA) targeting the target gene site is set forth in SEQ ID NO: 225, and the sequence of the nicking sgRNA targeting the target gene site is set forth in SEQ ID NO:

242. Preferably, the vector is a plasmid or a viral vector. A delivery system comprising the gene editing system of any one of claims 1-11. The delivery system of claim 12, wherein, The delivery system comprises at least one of a liposome, a nanoparticle, an exosome. A cell comprising: the gene editing system of any one of claims 1-11, the vector of any one of claims 1-11, or the delivery system of any one of claims 12-13. The cell of claim 14, wherein, The cell does not comprise reproductive material; Preferably, the cell is selected from a prokaryotic cell and a eukaryotic cell. The gene editing system of any one of claims 1-11, the delivery system of any one of claims 12-13, or the cell of any one of claims 14-15 for use in any one of a1)-a5): a1) editing a nucleic acid molecule; a2) preparing a product for editing a nucleic acid molecule; a3) constructing a plant, an animal, or a cell model; a4) preparing a product for constructing a plant, an animal, or a cell model; a5) preparing a product for treating a gene-related disease. The use of claim 16, wherein, a1) and a2) the editing comprises at least one of substitution, deletion, insertion; the substitution is a transition or a transversion; Preferably, a1) and a2) the nucleic acid molecule is from an organism, or a biological cell; Preferably, a2) and a4) the product is a reagent, or a kit; Preferably, a5) the product is a drug or a pharmaceutical composition; Preferably, a5) the gene-related disease comprises a genetic hematological disease such as bone marrow failure syndromes, cancer predisposition syndromes, hemoglobinopathies, Preferably, a5) the gene-related disease comprises at least one of amegakaryocytic thrombocytopenia, Diamond-Blackfan anemia, dyskeratosis congenita, Fanconi anemia, Pearson syndrome, severe congenital neutropenia, Shwachman-Diamond syndrome (SDS), thrombocytopenia-absent radius syndrome, IVIC syndrome, WT syndrome, radioulnar synostosis, ataxia, and various types of cytopenia, Preferably, a5) the gene-related disease comprises at least one of amegacaryocytic thrombocytopenia, Diamond-Blackfan anemia, dyskeratosi congenita, Fanconi anemia, Pearson syndrome, severe congenital neutropenia, Shvachman-Diamond syndrome (SDS), thrombocytopenia-absent radius syndrome (TAR), IVIC syndrome, WT syndrome, radioulnar synostosis, ataxia, various types of cytopenia, Preferably, the genetic related disease of a5) comprises at least one of mucopolysaccharidosis type I (MPS I), including Hurler syndrome (MPS I-H), Hurler-Scheie syndrome (MPS I-H / S), or Scheie syndrome (MPS I-S), Preferably, the genetic related disease of a5) comprises at least one of GATA2 deficiency syndrome, germline DDX41 mutation associated myeloid neoplasms, germline SAMD9 / SAMD9L mutation such as phantom syndrome, Preferably, the genetic related disease of a5) comprises at least one of adenosine deaminase deficiency, alpha-1 antitrypsin deficiency, cystic fibrosis, Duchenne muscular dystrophy, galactosemia, hemochromatosis, Huntington's disease, maple syrup urine disease, Marfan syndrome, neurofibromatosis type 1, pachyonychia congenita, phenylketonuria, severe combined immunodeficiency, sickle cell anemia, Steele-Richardson-Olszewski syndrome, trinucleotide repeat disorders, prion disease, Tay-Sachs disease, heart disease, hypertension, Alzheimer's disease, arthritis, diabetes, cancer, and obesity. A product comprising: the gene editing system of any one of claims 1-11, the delivery system of any one of claims 12-13, or the cell of any one of claims 14-15. The product of claim 18, wherein, The product is for any one of b1)-b3): b1) editing a nucleic acid molecule; b2) constructing a plant, animal, or cell model; b3) treating a genetic related disease; The editing is the editing of claim 17; Preferably, the nucleic acid molecule is the nucleic acid molecule of claim 17; Preferably, the genetic related disease is the genetic related disease of claim 17; Preferably, the product is a reagent combination or a kit; Preferably, the product is a medicament or a pharmaceutical composition; Preferably, the product is a medicament or a pharmaceutical composition, further comprising: a pharmaceutically acceptable carrier; Preferably, the animal is a non-human animal. A method of editing a nucleic acid molecule, contacting the nucleic acid molecule with the gene editing system of any one of claims 1-11, the delivery system of any one of claims 12-13, or the cell of any one of claims 14-15. A method of constructing a model of a plant, animal or cell, characterized in that, The method comprises contacting the plant, animal, or cell with the gene editing system of any one of claims 1-11, the delivery system of any one of claims 12-13, or the cell of any one of claims 14-15. A method of constructing a GATA2 deficiency syndrome model, contacting an animal or a cell with the gene editing system of any one of claims 1-11. The method of claim 20, wherein The target genetic locus is GATA2, SETBP1, and / or ASXL1. The method of claim 22, wherein The gene editing system is the gene editing system of claim 8. Use of a fluorescent protein as a selection marker in the gene editing system of any one of claims 1-11, Preferably, the fluorescent protein comprises at least one of a green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, AzamiGreen, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), a yellow fluorescent protein (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), a blue fluorescent protein (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), a cyan fluorescent protein (e.g., CFP, eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), a red fluorescent protein (e.g., RFP, eRFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), an orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato), a luciferase; or a variant thereof or a truncation thereof. Preferably, the fluorescent protein comprises at least one of a green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, AzamiGreen, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), a yellow fluorescent protein (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), a blue fluorescent protein (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), a cyan fluorescent protein (e.g., CFP, eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), a red fluorescent protein (e.g., RFP, eRFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), an orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato), a luciferase; or a variant thereof or a truncation thereof.

Images