Reporter plasmid system for multi-gene fixed-point knock-in cells and screening method

Abstract

The invention provides a reporter plasmid system for multi-gene fixed-point knock-in cells and a screening method. Specifically, the invention provides a set of multi-gene knock-in reporter plasmid system which comprises sgRNA plasmids of a plurality of target genes, a plurality of genome donor plasmids, a target gene knock-in reporter plasmid, a reporter plasmid donor plasmid and a Cas9 plasmid. Wherein the reporter plasmid comprises a truncated eGFP (enhanced green fluorescent protein) gene, a target site sequence is introduced, and a gene knock-in event is accurately reported. The invention further discloses a screening method which is a method for screening simultaneous knock-in of multiple genes only through one target gene knock-in reporter plasmid during multi-gene knock-in editing, and the efficiency of sorting gene homozygous knock-in cells is improved. The method can be efficiently used for gene knock-in through HDR repair, including accurate point mutation and large fragment insertion or deletion, and a clean gene knock-in cell line without residues in a report system is obtained.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology and relates to a reporter plasmid system and screening method for multi-gene knock-in cells. Background Technology

[0002] Precise manipulation of the genome, particularly through in situ gene knock-in (KI) technology, has revolutionized biomedical research, enabling scientists to study gene function, build complex disease models, and develop advanced therapeutic strategies. In recent years, the need for gene knock-in systems has become increasingly urgent, as mounting evidence suggests that many biological processes and diseases are determined by the expression and regulation of specific genes. For example, accurately inserting target genes into primary cells is crucial for constructing sophisticated disease models that can mimic complex human pathologies such as cancer and neurodegenerative diseases. Furthermore, this technology shows great potential in optimizing genetically modified animal models for xenotransplantation and advancing gene and cell therapies. As research progresses, single-gene manipulation is insufficient to unravel the networks of multi-gene synergistic regulation; a multi-site knock-in strategy capable of simultaneously introducing multiple genes or regulatory elements into the same cell is urgently needed to realistically reproduce the complex molecular landscape of diseases.

[0003] However, despite significant advancements in gene editing technologies (especially CRISPR-Cas9 and related systems), achieving efficient and precise gene knock-in in primary cells remains a formidable challenge, with the difficulty increasing exponentially when the target extends to multiple genes and multiple sites. Existing methods, such as CRISPR-mediated homologous recombination repair (HDR) insertion, while achieving precise integration, are inefficient, especially in primary cells where transfection and genetic manipulation are difficult. Furthermore, the HDR method requires homologous arms, limiting its application. Another approach—CRISPR-mediated non-homologous end joining (NHEJ) insertion—is more efficient but lacks precision and cannot control the insertion direction. Other emerging technologies, such as Cpf1-mediated insertion and PE-based systems (e.g., PAINT, eePASSIGE), have shown potential but are still limited by low insertion efficiency, residual adapter sequences, and the inability to achieve seamless integration.

[0004] Researchers have attempted to improve HDR efficiency through various strategies, including using cell cycle regulators, inhibiting the NHEJ and microhomology-mediated end-joining (MMEJ) pathways, and optimizing donor DNA templates, but these efforts have yielded only limited improvements. These advances have not fully resolved the problem of low gene knock-in efficiency, especially in primary cells. To overcome these obstacles, researchers have developed reporter plasmids to improve gene knock-in recovery rates by attaching reporter genes or simultaneously editing harmless target sites. While these methods are effective, they often leave residual reporter genes or editing scars, limiting their application. Although the HDR-USR system and methods such as PEAR have shown potential, highly efficient gene knock-in has not yet been achieved in primary cells.

[0005] Therefore, there is an urgent need in the field to develop efficient methods for simultaneous, targeted, and scarless knock-in of multiple genes at multiple sites in primary cells. Summary of the Invention

[0006] In view of the deficiencies in the prior art, this invention proposes a reporter plasmid system and screening method for multi-gene knock-in cells.

[0007] This invention provides a reporter plasmid system for multi-gene knock-in cells. The reporter plasmid system is a multi-component plasmid system, comprising: a gene editing plasmid, a target gene knock-in reporter plasmid, and a reporter plasmid donor plasmid.

[0008] The gene editing plasmids include: target gene sgRNA plasmid, Cas9 plasmid, and genome donor plasmid.

[0009] Furthermore, the target gene knock-in reporter plasmid (pKISR) uses a truncated eGFP gene as the reporter gene, which can avoid the false positives caused by the Indel generated by NHEJ repair in the reporter plasmid frameshift mutation caused by the conventional introduction of universal target sites.

[0010] In the reporter plasmid system, the insertion of the target site sequence into the reporter plasmid is achieved by linking it into the reporter plasmid via a linker, reducing the plasmid construction process.

[0011] The genome donor plasmid in the gene editing plasmid is knocked into the gene via HDR repair, which can introduce precise mutations, including point mutations, large fragment insertions or deletions.

[0012] Intracellularly, Cas9 targets the target gene's target site sequence (TS), while simultaneously targeting the same target gene knock-in reporter plasmid (TS). This linearizes the reporter plasmid and mediates repair in the donor plasmid (pDonor), thereby restoring complete eGFP gene expression. Simultaneously, the location of the target site sequence (TS) in the genome also mediates repair in the donor plasmid (pDonor-TS), achieving precise gene knock-in.

[0013] In target gene knock-in reporter plasmids (pKISRs), the target site recognized by the reporter plasmid sgRNA is not universal; it is designed specifically for the gene knock-in target site. Therefore, the reporter vector can only be repaired and thus report the gene knock-in event if the target site sgRNA is effective. Universal sgRNAs, on the other hand, may be repaired and the reporter gene expressed regardless of whether the target site sgRNA is effective, potentially leading to false positives.

[0014] Furthermore, for multi-gene knock-in editing, only one target gene knock-in reporter plasmid (pKISR-TS) is constructed, which introduces only one specific target site sequence (TS). This specific target site sequence (TS) is the target site sequence corresponding to the sgRNA plasmid with the lowest editing efficiency among multiple target gene sgRNA plasmids (pSG-TS). Similar to the "barrel effect," the reporter plasmid selects the target site with the lowest editing efficiency for gene knock-in, thereby screening for cells with simultaneous knock-in of multiple genes.

[0015] Preferably, the genome donor plasmid (pDonor-TS) and the reporter plasmid donor plasmid (pDonor) have the same backbone. Both have a universal specific target site TS-A and the corresponding sgRNA (pSG-A) expressed by the hU6 promoter designed and added downstream of the right homologous arm. After transfection into the cell, TS-A is cleaved by pSG-A via Cas9, which linearizes the donor plasmid in the cell and improves the efficiency of gene knock-in.

[0016] This invention discloses a screening method using the reporter plasmid system, comprising the following steps: (1) Construct a reporter plasmid system targeting multiple target genes; (2) The constructed reporter plasmid system was mixed and transfected into cells, and M3814 small molecules were added to inhibit the NEHJ repair pathway. (3) After transfection, high-positive cell populations (H population) were sorted by flow cytometry. (4) After sorting, the cells are centrifuged and lysed, and then identified by PCR.

[0017] It can efficiently obtain cells with precise gene knock-in, and the reporter plasmid is non-integrative, making it easy to obtain clean gene knock-in cell lines without any residue in the reporter system.

[0018] Compared with existing technologies, this invention has the following advantages: This invention provides a multi-gene knock-in reporter plasmid system with precise reporter gene knock-in events, and discloses a screening method. When performing multi-gene knock-in editing, this method uses only one target gene knock-in reporter plasmid to screen for simultaneous knock-in of multiple genes, improving the efficiency of sorting homozygous knock-in cells. This method can be efficiently applied to gene knock-in via HDR repair, including precise point mutations, large fragment insertions or deletions, to obtain clean gene knock-in cell lines with no residue from the reporter system. Attached Figure Description

[0019] Figure 1 Map of sgRNA plasmid (pSG-TS).

[0020] Figure 2 Map of genome donor plasmid (pDonor-TS).

[0021] Figure 3 A. Target gene knock-in reporter plasmid (pKISR) B. Reporter plasmid donor plasmid (pDonor) plasmid map.

[0022] Figure 4 Map of knock-in reporter plasmid (pKISR-TS).

[0023] Figure 5 Map of universal reporter plasmid donor plasmids (pDonor).

[0024] Figure 6 Gene knock-in reporter plasmid and conventional flow cytometry cell sorting strategy.

[0025] Figure 7 Flow cytometry results of GGTA1 gene knock-in report plasmids.

[0026] Figure 8 Comparison of gene knock-in reporter plasmid efficiency and conventional flow cytometry sorting efficiency of cells.

[0027] Figure 9 Efficiency of simultaneous knock-in of long fragments of two genes.

[0028] Figure 10 The precision of long-fragment gene knock-in. Detailed Implementation

[0029] The technical solutions of the embodiments of this application will be further described clearly and completely below with reference to the accompanying drawings. It should be noted that the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0030] This invention proposes a reporter plasmid system and screening method for multi-gene knock-in cells.

[0031] Example 1: This example involves the precise deletion of 40 bp, 47 bp, and 29 bp of the GGTA1, CMAH, and B4GALNT2 genes, respectively, in porcine embryonic fibroblasts (PFFs), comparing the gene knock-in reporter plasmid and screening method provided by this invention with conventional methods in terms of gene knock-in efficiency.

[0032] This includes the construction of target gene sgRNA plasmids (pSG-GGTA1, pSG-CMAH, pSG-B4GALNT2), genome donor plasmids (pDonor-GGTA1, pDonor-CMAH, pDonor-B4GALNT2), gene knock-in reporter plasmids (pKISR-GGTA1, pKISR-CMAH, pKISR-B4GALNT2), reporter plasmid repair plasmids (pDonor), and red fluorescent control plasmids (pETP); and a technical scheme for cell electroporation, flow cytometry sorting of reporter plasmids with different fluorescence intensities, and control cells with high fluorescence intensity for genotyping. Unless otherwise specified, the experimental or detection methods involved in the following examples are conventional experimental or detection methods already existing in the art.

[0033] (1) Construction of the target gene sgRNA plasmid The gene-editing plasmids are sgRNA-expressing plasmids, including pSG-GGTA1, pSG-CMAH, and pSG-B4GALNT2. The target site for GGTA1 is the sequence shown in SEQ ID NO.1. The synthesized forward primer is shown in SEQ ID NO.2, and the synthesized reverse primer is shown in SEQ ID NO.3. The target site for CMAH is the sequence shown in SEQ ID NO.4. The synthesized forward primer is shown in SEQ ID NO.5, and the synthesized reverse primer is shown in SEQ ID NO.6. The target site for B4GALNT2 is the sequence shown in SEQ ID NO.7. The synthesized forward primer is shown in SEQ ID NO.8, and the synthesized reverse primer is shown in SEQ ID NO.9. The primers were annealed, and the T4 enzyme was used to ligate the pSG backbone recovered from BbsI digestion (see plasmid map). Figure 1 ).

[0034] (2) Construction of genome donor plasmid The genomic donor plasmid contains 500 bp upstream and downstream of the target site as left and right homologous arms, and introduces the target mutation. In addition, there is a universal and specific sgA target site on the outer side of the right homologous arm, as well as sgA expressed by U6, for Donor linearization after Cas9 activation in cells. To construct pDonor-GGTA1, the nucleotide sequence of the PCR forward primer for the left homologous arm is shown in SEQ ID NO.10, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.11. The nucleotide sequence of the PCR forward primer for the right homologous arm is shown in SEQ ID NO.12, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.13. To construct pDonor-CMAH, the nucleotide sequences of the PCR primers for the left homologous arm are shown in SEQ ID NO.14 and SEQ ID NO.15. The nucleotide sequence of the forward primer for the PCR of the right homologous arm is shown in SEQ ID NO.16, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.17. For constructing pDonor-B4GALNT2, PCR primers for the left homologous arm were synthesized; the nucleotide sequence of the forward primer is shown in SEQ ID NO.18, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.19. For the PCR primers of the right homologous arm, the nucleotide sequence of the forward primer is shown in SEQ ID NO.20, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.21. PCR was performed using the genome of the target cells (PFFs) as a template. The ligation was performed via Gibson ligation into the pDonor backbone recovered from PacI and AscI digestion (see plasmid map). Figure 2 ).

[0035] (3) Construction of target gene knock-in reporter plasmid The target gene knock-in reporter plasmid contains eGFP gene expression initiated by CMV, but 17 nt of the 5' end of the eGFP gene is deleted to disrupt eGFP expression, and a 36 nt "BamHI-Target Site-XhoI" sequence is inserted to be identified and linearized as the target site. Figure 3A). To construct pKISR-GGTA1, upstream and downstream primers containing the target site were synthesized, and their nucleotide sequences are shown in SEQ ID NO. 22 and SEQ ID NO. 23. To construct pKISR-CMAH, upstream and downstream primers containing the target site were synthesized, and their nucleotide sequences are shown in SEQ ID NO. 24 and SEQ ID NO. 25. To construct pKISR-B4GALNT2, upstream and downstream primers containing the target site were synthesized, and their nucleotide sequences are shown in SEQ ID NO. 26 and SEQ ID NO. 27. The primers were annealed, and T4 enzyme was used to ligate them into the pKISR backbone recovered from gel digestion with BamHI and XhoI (see plasmid map). Figure 4 ).

[0036] (4) Construction of the report plasmid donor plasmid (pDonor) The reporter plasmid donor plasmid contains a truncated CMV promoter and a truncated eGFP gene. Downstream of the eGFP gene is a universal and specific sgA target site, as well as sgA expressed by U6, used for Donor linearization after Cas9 activation in cells, mediating homologous recombination repair in the knock-in reporter plasmid. Figure 3 B). pDonor is a universal plasmid and does not need to be reconstructed depending on changes in the editing site (see plasmid map). Figure 5 ).

[0037] (5) Cell electroporation The target gene sgRNA plasmid (pSG-GGTA1 or pSG-CMAH or pSG-B4GALNT2), genome donor plasmid (pDonor-GGTA1 or pDonor-CMAH or pDonor-B4GALNT2), target gene knock-in reporter plasmid (pKISR-GGTA1 or pKISR-CMAH or pKISR-B4GALNT2), reporter plasmid donor plasmid (pDonor), and Cas9 plasmid (pM3-Cas9, CMV-initiated Cas9 expression alone) constructed above were mixed at mass values ​​of 1 μg, 3 μg, 3 μg, 3 μg, and 6 μg, and electroporated into 2 × 10⁻⁶ cells using a Lonza 2B electroporator. 6 In the PFFs, 2 μM of M3814 was added to the culture medium.

[0038] (6) Flow cytometry sorting of gene knock-in positive cells and genotype identification Forty-eight hours after electroporation, different intensities of green fluorescent cell populations (GH, GL, GN) and different intensities of red fluorescent cell populations (RH, R, RN) were sorted using a BD FACSAria™ III flow cytometer (see [link to BD FACSAria™ III flow cytometer]). Figure 6 , Figure 7 1000 cells were centrifuged and lysed before direct PCR identification. The nucleotide sequence of the forward primer for PCR identification of the GGTA1 gene knock-in is shown in SEQ ID NO.28, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.29. The nucleotide sequence of the forward primer for PCR identification of the CMAH gene knock-in is shown in SEQ ID NO.30, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.31. The nucleotide sequence of the forward primer for PCR identification of the B4GALNT2 gene knock-in is shown in SEQ ID NO.32, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.33. After Sanger sequencing, the gene knock-in efficiency was analyzed using ICE. The results showed that, at all three sites, the gene knock-in efficiency of the green fluorescent high-intensity cell population (GH, i.e., the screening method using gene knock-in reporter plasmids) was significantly higher than that of the red fluorescent high-intensity cell population (RH, i.e., the conventional method for screening gene editing) (see [link to relevant documentation]). Figure 8 ).

[0039] Example 2 provides another example of precise insertion of two long segments in primary cells (porcine embryonic fibroblasts, PFFs). One is the precise replacement of the CDS of the THBD gene with the CDS sequence of the human hTHBD gene. The other is the precise insertion of the CDS expression sequence of the human hGP1BB gene before the ATG start codon of the GP1BA gene.

[0040] This includes the construction of sgRNA plasmids for the THBD gene (pSG-THBD-1, pSG-THBD-2), sgRNA plasmids for the GP1BA gene (pSG-GP1BA-1, pSG-GP1BA-2), THBD gene knock-in donor plasmid (pDonor-THBD), GP1BA gene knock-in donor plasmid (pDonor-hGP1BA), gene knock-in reporter plasmid (pKISR-THBD-2, preliminary experiments show that pSG-THBD-2 has relatively low editing efficiency), and the repair plasmid for the reporter plasmid (pDonor); cell electroporation, flow cytometry sorting, and genotyping techniques. Unless otherwise specified, the experimental or detection methods described in the following examples are conventional experimental or detection methods already existing in the art.

[0041] (1) Construction of the target gene sgRNA plasmid The gene editing plasmids include sgRNA plasmids for the THBD gene (pSG-THBD-1, pSG-THBD-2) and GP1BA gene (pSG-GP1BA-1, pSG-GP1BA-2). These plasmids express sgRNA alone. The nucleotide sequence of the target site for THBD-1 is shown in SEQ ID NO. 34, the nucleotide sequence of the forward primer is shown in SEQ ID NO. 35, and the nucleotide sequence of its reverse primer is shown in SEQ ID NO. 36. The nucleotide sequence of the target site for THBD-2 is shown in SEQ ID NO. 37, the nucleotide sequence of the forward primer is shown in SEQ ID NO. 38, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 39. The nucleotide sequence of the target site for GP1BA-1 is shown in SEQ ID NO. 40, the nucleotide sequence of the forward primer is shown in SEQ ID NO. 41, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 42. The nucleotide sequence of the target site of GP1BA-2 is shown in SEQ ID NO.43, the nucleotide sequence of the forward primer of the synthesized primer is shown in SEQ ID NO.44, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.45.

[0042] Primer annealing and T4 enzyme ligation were performed into the pSG backbone recovered from BbsI digestion (see plasmid map). Figure 1 ).

[0043] (2) Construction of genome donor plasmids (pDonor-hTHBD, pDonor-hGP1BA) The THBD gene knock-in donor plasmid (pDonor-THBD) contains 1000 bp upstream and downstream of the THBD target site as left and right homologous arms, and introduces the CDS sequence of the human THBD gene. In addition, a universal and specific sgA target site and U6-expressed sgA are located on the outer side of the right homologous arm for Donor linearization after Cas9 activation in cells. To construct pDonor-hTHBD, PCR primers for the left homologous arm were synthesized; the nucleotide sequence of the forward primer is shown in SEQ ID NO.46, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.47. The nucleotide sequence of the PCR forward primer for the right homologous arm is shown in SEQ ID NO.48, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.49. PCR was performed using the genome of the target cells (PFFs) as a template, and the ligation was performed via Gibson into the pDonor backbone recovered from PacI and AscI digestion, serving as an intermediate vector. PCR primers for the CDS sequence of the human THBD gene were synthesized. The nucleotide sequence of the forward primer is shown in SEQ ID NO. 50, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 51. PCR was performed using human cDNA as a template, and the fragment was ligated into the intermediate vector backbone recovered from NotI / NheI digestion via Gibson assay to obtain pDonor-hTHBD. The GP1BA gene was knocked into the donor plasmid (pDonor-hGP1BA) using the same Donor as the backbone. The LA-hGP1BB-RA fragment was synthesized and ligated into the pDonor backbone recovered from PacI and AsciI digestion to obtain pDonor-hGP1BA (see plasmid map). Figure 2 ).

[0044] (3) Construction of the target gene knock-in reporter plasmid (pKISR-THBD) The target gene knock-in reporter plasmid contains eGFP gene expression initiated by CMV, but 17 nt of the 5' end of the eGFP gene is deleted to disrupt eGFP expression, and a 36 nt "BamHI-Target Site-XhoI" sequence is inserted to be identified and linearized as the target site. Figure 3 A). To construct pKISR-THBD, upstream and downstream primers containing the target site were synthesized, and their nucleotide sequences are shown in SEQ ID NO. 52 and SEQ ID NO. 53. The primers were annealed, and the T4 enzyme was used to ligate them into the pKISR backbone recovered from gel digestion with BamHI and XhoI (see plasmid map). Figure 4 ).

[0045] (4) Construction of the report plasmid donor plasmid (pDonor) The reporter plasmid donor plasmid contains a truncated CMV promoter and a truncated eGFP gene. Downstream of the eGFP gene is a universal and specific sgA target site, as well as sgA expressed by U6, used for Donor linearization after Cas9 activation in cells, mediating homologous recombination repair in the knock-in reporter plasmid. Figure 3 B). pDonor is a universal plasmid and does not need to be reconstructed depending on changes in the editing site (see plasmid map). Figure 5 ).

[0046] (5) Cell electroporation The target gene sgRNA plasmids (pSG-THBD-1, pSG-THBD-2, pSG-GP1BA-1, pSG-GP1BA-2), genome donor plasmids (pDonor-hTHBD, pDonor-hGP1BA), target gene knock-in reporter plasmids (pKISR-THBD), reporter plasmid donor plasmids (pDonor), and Cas9 plasmids (pM3-Cas9, CMV-initiated Cas9 expression) constructed above were mixed at mass values ​​of 0.5 μg, 1 μg, 0.5 μg, 0.5 μg, 3 μg, 3 μg, 3 μg, 3 μg, and 6 μg, and electroporated into 2*10^6 PFFs using a Lonza2B electroporator. 2 μM of M3814 was added to the culture medium.

[0047] (6) Flow cytometry sorting of gene knock-in positive cells and genotype identification Forty-eight hours after electroporation, high-positive cell populations (H population) were sorted using a BD FACSAria™ III flow cytometer. 1000 cells were centrifuged and lysed, followed by direct PCR identification. The remaining cells were plated for further culture, and single clones were picked. The nucleotide sequences of the forward primer for PCR detecting THBD gene insertion across the left homologous arm are shown in SEQ ID NO. 54, and the reverse primer is shown in SEQ ID NO. 55. The nucleotide sequences of the forward primer for PCR detecting THBD gene insertion across the right homologous arm are shown in SEQ ID NO. 56, and the reverse primer is shown in SEQ ID NO. 57. The nucleotide sequences of the forward primer for PCR detecting GP1BA gene insertion across the left homologous arm are shown in SEQ ID NO. 58, and the reverse primer is shown in SEQ ID NO. 59. The nucleotide sequences of the forward primer for PCR detecting GP1BA gene insertion across the right homologous arm are shown in SEQ ID NO. 60, and the reverse primer is shown in SEQ ID NO. 61. A positive PCR result indicates successful gene knock-in. Gene knock-in mixed cells after electroporation served as a positive control, while wild-type genomes served as a negative control. Results showed that the gene knock-in efficiency for GP1BA was 11 / 26, for THBD it was 15 / 26, and the efficiency of simultaneous GP1BA and THBD gene knock-in was 7 / 26 (26.9%) (see [link to relevant documentation]). Figure 9 After the PCR products were sequenced by Sanger sequencing, the accuracy of the inserted sequences was checked by comparison. The results showed that all clones had accurate insertions, and the sequencing results of the clones participated in the sequencing process. Figure 10 .

[0048] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

Claims

1. A reporter plasmid system for multi-gene site-directed knock-in cells, characterized in that, The report plasmid system is a multi-plasmid system, comprising: a gene editing plasmid, a target gene knock-in report plasmid, and a report plasmid donor plasmid.

2. The reporter plasmid system of claim 1, wherein, The gene editing plasmid comprises: a target gene sgRNA plasmid, a Cas9 plasmid, and a genome donor plasmid.

3. The reporter plasmid system of claim 1, wherein, The truncated eGFP gene is used as a reporter gene in the target gene knock-in report plasmid, and the nucleotide sequence is shown as SEQ ID NO.

62.

4. The reporter plasmid system of claim 1, wherein, In the target gene knock-in report plasmid, the insertion of the target site sequence is connected to the report plasmid through a linker.

5. The reporter plasmid system of claim 2, wherein, The genome donor plasmid in the gene editing plasmid is subjected to gene knock-in through HDR repair.

6. The reporter plasmid system of claim 1, wherein, The target gene knock-in report plasmid only introduces one specific target site sequence, which is the target site sequence corresponding to the sgRNA plasmid with the lowest editing efficiency among the sgRNA plasmids of multiple target genes.

7. The reporter plasmid system of claim 3, wherein, The genome donor plasmid and the report plasmid donor plasmid are both designed to add a universal specific target site TS-A downstream of the right homologous arm, and the corresponding sgRNA expressed by the hU6 promoter.

8. A screening method using the reporter plasmid system of claim 1, characterized by, The method comprises the following steps: (1) constructing a report plasmid system for multiple gene knock-in editing; (2) mixing the constructed report plasmid system and transfecting it into cells, and adding M3814 small molecules to inhibit the NEHJ repair pathway; (3) sorting high-positive cell groups through a flow cytometer after transfection; (4) centrifugal lysis of the sorted cells, and then performing PCR identification.

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