CN114763559A

CN114763559A - Targeting gene capturing system independent of homologous recombination and application thereof

Info

Publication number: CN114763559A
Application number: CN202110057414.8A
Authority: CN
Inventors: 吴森; 杜旭光; 鲁恒星
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-01-15
Filing date: 2021-01-15
Publication date: 2022-07-19

Abstract

The invention relates to the technical field of biology, in particular to a targeting gene capturing system independent of homologous recombination and application thereof. The invention provides a targeting gene capturing system independent of homologous recombination, which comprises the following modules: a Cas9 protein recognition site module, a sgRNA expression module for recognizing a Cas9 protein recognition site, a gene capture module, a Cas9 protein expression module and a sgRNA expression module for recognizing a gene capture target site. The system can realize efficient targeted gene capture, has higher knockin efficiency in various mammalian cells, greatly simplifies the preparation of mutant alleles, and provides a new path for efficiently introducing targeted mutation into the mammalian cells.

Description

Targeting gene capturing system independent of homologous recombination and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a targeting gene capturing system independent of homologous recombination and application thereof.

Background

Gene mutation technology plays an important role in genetic engineering technology. On the one hand, gene mutation is the most direct method for studying gene function, and after a target gene is mutated, the phenotype of the mutant is analyzed, and the gene function can be determined. On the other hand, disease model animals prepared by using gene editing mutation disease-related genes are of great significance for the research of human diseases and the development of related therapeutic means. Therefore, the development of rapid and efficient gene mutation technology has been the focus of research.

Gene trapping (gene trapping), also known as gene trapping, and the like, is a high-throughput gene mutation technology established based on insertional mutagenesis of mouse ES cells and trapping vectors, and the basic principle is to integrate a gene trapping vector randomly into the genome of mouse ES cells, destroy the function of the inserted gene by using a transcription element on the vector, and provide a marker capable of indicating the expression of the gene. Research shows that the gene capturing technology combined with the targeting capturing technology generated by homologous recombination can realize efficient mutation on a specific gene, and the combination with the site-specific recombination technology can be used for preparing conditional knockout and researching embryonic lethal genes. To date, these gene mutation strategies based on gene capture technology have achieved a series of important results in the study of mouse functional genomics. However, most of these techniques are currently applied to mouse ES cells, and the efficiency is low in other animals that have not yet obtained ES cells, and the application still has some limitations.

The technique of knock-in to integrate foreign DNA precisely at a specific location on the genome is the basis of genome-directed insertional mutagenesis. In addition, the gene knock-in technology can be used for marking the target gene so as to conveniently research the function of the target gene. Moreover, stable transgene overexpression at a specific position can be realized by knocking in the gene, and the possible adverse effect caused by random integration is avoided. Therefore, the knock-in technique occupies an important position in basic and applied biology. However, traditional knock-in relies on homologous recombination, is inefficient, and is not applicable in many cell types and tissues. NHEJ repair dominates in mammalian cells, and random integration of exogenous DNA via NHEJ-mediated has been widely used in the generation of transgenic animals and cell lines with efficiencies greater than about 1000-fold over HDR-mediated DNA insertion. The potential of the NHEJ pathway for targeted integration of foreign DNA has been developed in recent years. Orlando et al first found that short oligonucleotides (<100bp) could be efficiently inserted into ZFN-induced genomic DSBs by means of NHEJ repair (Orlando, S.J., Santiago, Y., DeKelver, R.C., Freyvert, Y., Boydton, E.A., Moehle, E.A., Choi, V.M., Gopalan, S.M., Lou, J.F., Li, J., et al (2010) Zinc-finger nucleic acid with used chromosomal homology. In a further study, Cristea and Maresca et al can site-specifically integrate plasmid DNA into genomic DSB by NHEJ repair after cutting plasmid and genomic DNA simultaneously by introducing ZFN or TALEN target sequence In donor plasmid vector (Cristea, S., Freyvert, Y., Santiago, Y., Holmes, M.C., Urnov, F.D., Gregory, P.D. and Cost, G.J, (2013) In vivo restriction of gene vectors, biotechnol. en. 110, 880; escra, M.Lin, V.G., and G., N.Yang, Y. 2013) injection-modified gene, supplement, 23. gene expression, coding, protein, coding, and coding, cloning, coding. This design approach is also applicable to NHEJ repair generated by CRISPR/Cas9, Suzuki et al developed a homologously-independent targeted integration (HITI) technique using CRISPR/Cas9 and NHEJ, which can efficiently integrate foreign DNA to a target site In vivo and In vitro, even In non-dividing neuronal cells, while HDR repair was not active In such cells (Suzuki, k, Tsunekawa, y., Hernandez-Benitez, r., Wu, j., Zhu, j., Kim, e.j., hataraka, f., Yamamoto, m., Araoka, t., Li, z., al, (2016) In vivo gene/9 medium-encoded 149, nati, 144). In subsequent researches, a series of applications such as the marking of a reporter gene, the site-specific integration of a large-fragment vector, the destruction of a target gene and the like are respectively realized by people based on NHEJ-mediated DNA site-specific knock-in, so that the NHEJ-mediated DNA site-specific integration is a molecular biology tool with high efficiency, simplicity and rich functions and is paid attention to by people. Although significant progress has been made in NHEJ-mediated site-directed knock-in of DNA, targeted gene mutation technology has not been found for use in conjunction with gene trapping technology.

Disclosure of Invention

The invention aims to provide a targeting gene capturing system independent of homologous recombination, and the invention also aims to provide a targeting gene capturing system independent of homologous recombination and capable of realizing recoverability and conditional knockout.

In order to achieve the above objects, the present invention combines CRISPR/Cas 9-mediated DNA knock-in strategy relying on Non-homologous end-joining (NHEJ) and gene trapping technology to develop a new targeted gene mutation system named as a targeting-gene trapping system (HIT-trapping) independent of homologous recombination. Unlike traditional gene capture vectors, the system comprises a gene capture vector which contains a universal Cas9 recognition site, so that the gene capture vector can be cut simultaneously with a genome in a cell, and then the cell integrates the vector into a target site in a process of repairing the genome by using a NHEJ signal pathway, and further comprises a Cas9 protein expression module and a sgRNA expression module. When the system is used for gene mutation, the integration of the capture carrier does not need to use a site-specific homology arm, and the system is generally used for all target sites of mammals, and only sgRNA aiming at the target sites needs to be redesigned when the target sites are replaced. On the basis of the targeted gene capture system, the invention provides the targeted gene capture system which is independent of homologous recombination and can realize recoverability and conditional knockout. The system integrates a FlEx conversion system mediated by a site-specific recombinase technology into a carrier, so that a capture element can be inverted under the action of Cre enzyme after being inserted into a target site. The gene capturing carrier capable of inverting combines the characteristic of NHEJ knock-in, and can simultaneously obtain recoverable and conditional knock-out in one experiment.

Specifically, the invention provides the following technical scheme:

the invention provides a targeted gene capture system independent of homologous recombination, comprising the following modules:

a Cas9 protein recognition site module comprising a nucleotide sequence for Cas9 protein recognition;

a sgRNA expression module recognizing a Cas9 protein recognition site, comprising a promoter 1 and a sgRNA1 operably linked downstream of the promoter 1, the sgRNA1 being capable of recognizing a Cas9 protein recognition site for guiding a Cas9 protein to cleave a vector linearly;

a gene capture module comprising a splice acceptor SA, an IRES derived from an encephalomyocarditis virus, a marker gene, and a poly A-tailing sequence (polyA), and not comprising a homology arm of a gene capture target site;

a Cas9 protein expression module comprising promoter 2 and a Cas9 gene operably linked downstream of the promoter 2;

an sgRNA expression module that recognizes a gene capture target site, comprising a promoter 1 and an sgRNA2 operably linked downstream of the promoter 1, the sgRNA2 capable of recognizing the gene capture target site.

Further, to facilitate the screening of the cells into which the gene capture element is inserted, the gene capture system further comprises a gene capture screening module comprising promoter 3 and a drug screening gene for screening the cells for successful insertion of the gene capture module into the target site.

Preferably, the promoter 3 is a promoter that can be expressed continuously in a cell of interest. More preferably, the promoter 3 is an SV40 promoter, and the drug selection gene is a Puro resistance gene.

In the targeted gene capture system described above, the gene capture screening module is operably linked downstream of the gene capture module, and the sgRNA expression module that recognizes the Cas9 protein recognition site and the Cas9 protein recognition site module are sequentially and operably linked upstream of the gene capture module in the upstream-downstream direction.

The invention discovers that the targeted gene capture system consisting of the modules can realize targeted gene capture in cells. In order to further improve the knock-in efficiency of the targeted gene capture, the invention screens and optimizes the element selection of each module.

Specifically, the promoter 1 is a U6 promoter, and the promoter 2 is a CMV promoter.

Preferably, the nucleotide sequence for Cas9 protein recognition is shown as SEQ ID No.1, and the sequence of sgRNA1 is shown as SEQ ID No. 2.

Preferably, the sequence of the gene capture module (SA-ires-GFP-pA) is shown as SEQ ID NO. 3.

To further facilitate the improvement of the knock-in efficiency of targeted gene capture and at the same time facilitate the manipulation, the Cas9 protein recognition site module, the sgRNA expression module recognizing the Cas9 protein recognition site, and the gene capture module are preferably placed on a first vector.

The Cas9 protein expression module and the sgRNA expression module that recognizes the gene capture target site were placed on a second vector and a third vector, respectively.

Preferably, the backbone vector of the first vector is pX330, and the backbone vector of the second vector is pMax-gfp (lonza).

The backbone vector of the third vector may be any vector commonly used in the art for expressing sgRNA, including but not limited to pCRISPR-sg6(Xu C, Qi X, Du X, et al. piggyBac media effects in vivo CRISPR library screening for modeling in the microorganism [ J ] Proceedings of the National Academy of Sciences of the United States of America,2017,114(4): 201615735.).

As a preferred embodiment of the present invention, the targeted gene capture system described above comprises three vectors. The first vector is a gene capture vector and comprises a Cas9 protein recognition site module, a sgRNA expression module for recognizing a Cas9 protein recognition site and a gene capture module, and the sequence of the gene capture module is shown as SEQ ID No. 4. The 15 th to 233 th sites of the sequence shown in SEQ ID NO.4 are U6 promoters. The second vector is a Cas9 expression vector, comprises a CMV promoter and a Cas9 gene, and the sequence of the expression vector is shown as SEQ ID NO. 5. The 1 st to 798 th sites of the sequence shown in SEQ ID NO.5 are CMV promoters, and the 1077 th to 5201 th sites are Cas9 genes. The third vector is a target site sgRNA expression vector, and comprises a U6 promoter and sgRNA for recognizing a gene capture target site.

On the basis of the targeting gene capturing system, the invention also provides a recoverable and conditional knockout targeting gene capturing system, which comprises the targeting gene capturing system and a Cre enzyme mediated FlEx conversion module, wherein the FlEx conversion module comprises a loxP/lox2272 sequence and a Cre enzyme expression element, and the loxP/lox2272 sequence is respectively positioned between the gene capturing module and the Cas9 protein recognition site module and at the downstream of the gene capturing screening module.

Specifically, the Cre enzyme expression element is separately transferred into the cell when the targeted gene capture needs reversion or conditional knockout.

To avoid integration of the promoter of the sgRNA into the genome, the Cas9 protein recognition site module, the gene capture screening module are preferably placed on a first vector; the sgRNA expression module that recognizes the Cas9 protein recognition site is placed on a second vector.

The Cas9 protein expression module and the sgRNA expression module for recognizing the gene capturing target site are respectively positioned on a third vector and a fourth vector.

Preferably, the backbone vector of the second vector is pX330, and the backbone vector of the third vector is pMax-GFP.

The backbone vector of the fourth vector may be a vector that can express sgRNA, which is commonly used in the art, including but not limited to pCRISPR-sg 6.

Further preferably, when the first vector is constructed, the Cas9 protein recognition site module, the gene capture module and the gene capture screening module are firstly connected with a vector pUC57-simple, and then a vector framework of pUC57-simple is removed, so that the first vector is obtained and is used for transforming cells to target.

As a preferable scheme of the invention, pUC57-simple connecting the Cas9 protein recognition site module, the gene capture module and the gene capture screening module is treated by restriction endonuclease PvuI, the skeleton part of the vector is removed by electrophoretic separation, the insertion element is purified, and the vector is treated by T4 DNA ligase to be cyclized to obtain the first vector.

The invention also provides the application of the targeted gene capturing system independent of homologous recombination or the recoverable and conditional knockout targeted gene capturing system in targeted gene capturing, animal model preparation or drug screening.

The targeted gene capture systems of the invention that do not rely on homologous recombination and targeted gene capture systems that are recoverable and conditional knockout can be applied to animal cells (including but not limited to mammalian cells).

The invention has the beneficial effects that:

the targeted gene capturing system (HIT-trapping) independent of homologous recombination provided by the invention can realize efficient targeted gene capturing, has higher knockin efficiency in mouse ES cells and various mammalian cells, greatly simplifies the preparation of mutant alleles, and provides a new path for efficiently introducing targeted mutation into the mammalian cells.

The targeted gene capture system which is independent of homologous recombination and can realize the recoverability and the conditional knockout can realize the recoverability gene knockout and the conditional knockout of genes through a FlEx conversion system connected with Cre enzyme, and provides powerful molecular tools and a novel method for the recoverability and the conditional gene mutation.

Drawings

FIG. 1 is a schematic diagram of the design and action principle of the HIT-bridging system in example 1 of the present invention, wherein, a, the diagram shows the core part of the system, HIT-trap-1C is a gene capture vector, a U6-sgA expression element and sgA recognition site are arranged on the gene capture element SA-ires-GFP-pA for the Cas9 intracellular cleavage vector itself, the Puro resistance gene in the drug screening element is started by SV40 promoter, SA, splice acceptor; ATS, sgA target site (sgA target site); pM3-Cas9 is a Cas9 protein transient expression vector and is started by a CMV promoter; b, the figure shows that when the HIT-drawing technology is used for expressing a gene, a Cas9 RNP compound cuts a target site on a genome and a carrier HIT-trap-1C, the carrier is integrated on the target site by NHEJ repair, a gene capture element in the carrier has splicing action with an endogenous gene, the endogenous promoter is transcribed and translated into a truncated protein and GFP, and meanwhile, a drug screening element on the carrier independently expresses Puro resistance genes to provide drug resistance for cells.

FIG. 2 is a graph showing the examination of the knocking-in efficiency of HIT-trapping in mouse ES cells in example 2 of the present invention, wherein a, the state of clones of mouse ES cells after drug screening, some of the clones survived after the cells were drug screened and recovered from culture, the uppermost arrow indicates apoptotic cells after drug screening, the middle arrow indicates a clone that survived but does not express GFP, and the lowermost arrow indicates a clone that survived and expresses GFP. Scale bar 50 μm; b, statistics of the efficiency of vector knockin in mouse ES cells.

FIG. 3 is a diagram showing the results of knocking out a target gene by the HIT-mapping system in example 3 of the present invention, wherein a, sequencing confirmed that the vector was knocked into the target site correctly, Seamless is the sequence when the vector was seamlessly ligated to the genome and used as a reference, the genome sequence is indicated in green, the vector sequence is indicated in black, the PAM sequence is indicated in blue, the dotted line indicates a base deletion, the red portion indicates a base addition or mismatch, 5/3J, and a 5 '/3' linker (5 '/3' junction); b, c, detecting the mutagenicity of the Hprt gene by the HIT-trapping technology at the protein and RNA level. Th-1, Th2-4 and Th3-5 are monoclonal cell lines with HIT-trapping vector integrated in the intron of Hprt gene. Hk-2/8/12 is a monoclonal cell line with indels on the exon of the Hprt gene. Histograms are statistics of three technical replicates, mean ± standard deviation (. + -. P <0.01, Student's t-test); d, phenotypic analysis of Hprt gene knockout by the HIT-trapping technology, which is shown in the figure as a typical result of drug resistance test on the obtained clones in HIT-trapping experiments for the Hprt gene, only GFP clones positive by PCR (middle) will have 6-TG resistance, and clones positive by GFP but negative by PCR (right) and wild-type cells (left) are apoptotic after 6-TG treatment; scale bar 100 μ M.

FIG. 4 is a schematic diagram of the detection of the effectiveness of the HIT-trapping system in various mammalian cell lines in example 4 of the present invention, wherein, a, the detection process is shown; b, counting the efficiency of knocking-in the carrier in the mammalian cells by using an HIT-trapping technology, wherein each group of data comprises at least two repeated experiments, and the average value is +/-standard deviation; and c, counting the repair form of the connection part of the vector and the genome of each target site. 5/3J,5 '/3' linker, n ═ number of TA clones sequenced; d, statistics of base deletion conditions, including base deletion caused by indels and MMEJ.

FIG. 5 is a schematic diagram of a process for simultaneously obtaining recoverability and conditional knockout of a target gene by using a vector HIT-trap-FlEx in example 5 of the present invention, wherein, a, the vector optimization process is shown in the figure; b, an HIT-trap-FlEx vector schematic diagram, wherein SA-ires-pA is a promoter capture element, and SV40-Puro-pA is a drug sieve element; blue triangle, loxP site; red triangle, lox2272 site; magenta arrow, PvuI restriction site, ATS, sgA target site; c, carrying out enzyme digestion on the vector by PvuI, and then carrying out electrophoretic separation on the insertion element part and the vector framework part, wherein the uppermost strip is the insertion element part and has the size of 3399 bp; d, schematic representation of the mechanism of action of the HIT-trap-FlEx vector. Treating the plasmid with PvuI, separating and purifying the part of the inserted element in vitro, and obtaining a vector LigFV3 through self-concatemerisation; vector LigFV3 was integrated into the target site in a forward or reverse manner by the action of Cas 9-generated NHEJ in the cell to give an allele with a recoverable or conditional knockout. Under the action of Cre enzyme-mediated FlEx conversion, the gene capturing element is inverted and the drug screening element is deleted, so that phenotype reversion or conditional knockout is realized.

FIG. 6 shows the PCR identification of selected clones for the Nes and Hprt sites in mESC and the ACTB site in A375 cells, respectively, as determined by the knock-in efficiency of the optimized vector LigFV3 in mammalian cells of example 5 of the present invention, wherein for the Nes site, both forward and reverse insertions in each clone were determined. 5/3J,5 '/3' linker; d, statistics of the knock-in efficiency of the optimized vector LigFV3 at each target site.

FIG. 7 shows the change of GFP expression in cells caused by the inversion of the Cre enzyme-recombinantly produced vector in example 5 of the present invention, and the state of the cloned GFP with the vector integrated at the target site has been changed after the Cre enzyme is transferred into the cells, indicating that the vector elements in the cells have been inverted; the vectors in the monoclonal cell lines AG1 and H10 inserted forward to the target site, while the vectors in AN3 and H17 inserted backward to the target site; scale bar 50 μm.

FIG. 8 shows the sequencing in example 5 of the present invention to further confirm the effect of FlEx transduction, and Cre enzyme mediated FlEx transduction not only can invert the capture element, but also can remove the drug-sieve element from the carrier, to generate a new specific carrier-genome linker sequence.

FIG. 9 shows phenotypic reversion and conditional knock-out by FlEx transformation, survival of cells detected by methylene blue staining, and the results of H10 and H17 dividing into mESCs monoclonal cell lines with HIT-bridging vectors integrated in the forward and reverse directions of the intron of Hprt gene in example 5 of the present invention, and transformation of resistance of the clones to 6-TG and HAT before and after Cre enzyme treatment, which confirmed the phenotypic change caused by vector inversion.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 design and construction of a Targeted Gene trapping System (HIT-trapping) independent of homologous recombination

The design concept of the targeting gene capturing system independent of homologous recombination of the invention is as follows: due to the fact that NHEJ repair generated by the CRISPR/Cas9 can integrate exogenous DNA into DSB of a genome efficiently, the CRISPR/Cas 9-mediated NHEJ knock-in technology and the gene capture technology are combined, a gene capture carrier is knocked into an intron of a target gene without depending on homologous recombination, and therefore targeted capture of the gene is achieved.

For this reason, this example constructs a gene targeting gene capture system that can realize knock-in using NHEJ repair, and the system comprises three vectors: the gene trap vector HIT-trap-1C, Cas9 expresses vector pM3-Cas9 (a of figure 1) and target site sgRNA expression vector. Unlike traditional targeted gene capture vectors, in HIT-trap-1C, the promoter-free SA-ires-GFP-pA (promoter capture element, sequence shown in SEQ ID No. 3) is flanked by no homology arms, and upstream thereof is added a Cas9 recognition site (a sequence of zebrafish gene tia1l, shown in SEQ ID No. 1) that is nonhomologous in mammals, and a U6-sgA expression element (sequence of sgRNA shown in SEQ ID No. 2) that transcribes sgRNA (designated sgA) that recognizes this site, for Cas9 intracellular cleavage of the vector itself. Expression of the Cas9 protein was initiated by a CMV promoter in pM3-Cas 9. Target site sgRNA expression vector sgRNA recognizing the target site is initiated by a U6 promoter.

When the HIT-trapping vector, the Cas9 protein and the sgRNA expression vector aiming at the target site on the genome are co-transferred into a cell, the expressed and transcribed Cas9 protein is combined with the two sgRNAs to form an RNP complex aiming at the vector and the genome respectively, and the vector and the corresponding target site on the genome are cut, and then the cell can introduce the in-vivo linearized gene capture vector to the target site in a mode of end connection when repairing the DSB of the genome by using an NHEJ signal channel.

In addition, the carrier also comprises a drug screening element (SV40-Puro-pA) which is used for enriching cells successfully captured in a targeted mode. After the carrier is integrated into the intron of the expression gene, the splicing acceptor on the gene capturing element (SA-ires-GFP-pA) and the endogenous gene generate splicing action, and finally a section of truncated endogenous target gene protein and GFP signal protein are generated, so that the target gene is damaged and the expression condition of the target gene is indicated at the same time. The drug screening marker is initiated by the promoter SV40 which is expressed continuously on the vector, so that Puro can be used to screen for cells that have successfully inserted the capture vector into the target site regardless of whether the target gene is expressed or not (b of FIG. 1).

Through screening and optimization of all elements, the sequence of the finally determined gene capture vector HIT-trap-1C is shown in SEQ ID No.4 and comprises a Cas9 protein recognition site, a U6 promoter, sgRNA for recognizing the Cas9 protein recognition site and a gene capture element, wherein 15 th to 233 th sites of the sequence shown in the SEQ ID No.4 are U6 promoters. The sequence of the Cas9 expression vector pM3-Cas9 is shown in SEQ ID NO.5, and comprises a CMV promoter and a Cas9 gene, the 1 st to 798 th sites of the sequence shown in SEQ ID NO.5 are CMV promoters, and the 1077 th to 5201 th sites are Cas9 genes.

Example 2 detection of knock-in efficiency of HIT-trapping System in mouse ES cells

To examine the efficiency of vector knock-in to target sites in the HIT-trapping system, three target sites were first selected on the first intron of the mouse Hprt gene for the detection experiment. Co-transferring the HIT-trapping vector, Cas9 protein, and sgRNA expression vector for the target site into mouse ES cells, adding Puro for drug screening and resuming culture, it was found that the expression of partially surviving clone GFP was positive, which initially indicated successful integration of the vector to the target site, but also a small amount of clone GFP was not expressed, probably due to reverse integration of the vector to the target site or to other sites (a of fig. 2).

In addition, Sall4 and Tet1 were selected as target genes for the detection experiment, and the sgRNA sequences used for the target genes were as shown in Table 1.

The GFP positive clones surviving the drug screening were PCR-characterized to determine if the vector was knocked correctly into the target site. The results showed that successful insertion of the vector into the target site was observed in all of the clones selected for the three genes Hprt, Sall4, and Tet1, and the total efficiency reached 51.6% (16/31) (FIG. 2 b).

TABLE 1 sgRNA sequences of the respective target genes

Example 3 mutagenic detection of target genes by the HIT-trapping System

To examine whether the HIT-trapping system produced a gene knock-out effect at the target site, three monoclonal cell lines determined to have a gene trap vector integrated at the Hprt site were selected from the cell lines obtained in example 2: th1-1, Th2-4 and Th3-5 were used as experimental groups to detect target gene activity at protein and RNA levels.

The sequence of the vector-genome junction in these clones was sequenced and the occurrence of knock-in events was further confirmed, as a result of the imprecision of NHEJ repair, with a degree of indels present at the junction (fig. 3 a). At the same time, three monoclonal cell lines with Hprt gene knock-out by traditional Cas9 technology were also selected: hk-2, hk-8 and hk-12 (the second exon of hk-2 and hk-8 is deleted with 12bp and 5bp bases, respectively, and the second exon of hk-12 is added with 2bp bases) and wild type cell lines as controls.

Western blot results showed that knocking in the HIT-mapping vector in the intron and producing indels in the exon were both effective in knocking out the Hprt gene at the protein level (b of FIG. 3). While the q-PCR results indicate that both forms of knock-out degrade the mRNA of the target gene to some extent, but the gene capture results in more complete degradation (c in FIG. 3), which is consistent with previous studies (Reber, S., Mechtersheimer, J., Nasif, S., Benitez, J.A., Colombo, M., Domanski, M., Jutzi, D., Hedlund, E.and Ruepp, M.D. (2018) CRISPR-Trap: a clean approach for the generation of gene knockouts and gene reproductions in human cells, mol.biol.cell,29, 75-83.).

Cells that are knocked out by Hprt develop 6-TG resistance. Therefore, in order to phenotypically detect a knockout cell line prepared by the HIT-tapping technology, mouse ES cell clones targeting Hprt in the above experiment were treated with 6-TG, and as a result, it was revealed that some clones were not resistant to 6-TG, although GFP was positive, and no vector insertion was detected by PCR, and only those clones positive for PCR identification developed drug resistance (d in FIG. 3), demonstrating that cells produced by the HIT-tapping technology also phenotypically achieved the knockout criteria, and also demonstrating the reliability and stringency of the PCR detection method.

The above results show that the HIT-trapping technology has high targeting efficiency when applied to mouse ES cells, and although NHEJ repair introduces certain indels, the effect of knocking-in vector on gene mutagenesis is not affected.

Example 4HIT-trapping System is general for multiple mammalian cell lines

When the gene trap vector is inserted into a gene having transcriptional activity, the gene trap element hijacks the transcript of the endogenous gene and disrupts the gene, while the promoter of the trapped gene initiates GFP expression on the vector. Therefore, the efficiency of knocking-in of the vector to the target site can be detected directly by FACS technique at the overall level (a of fig. 4). To examine the efficiency and versatility of the HIT-trapping technique in mammalian cell lines, four cell lines, mouse ES cells, human 293T and a375 cells, porcine fetal fibroblasts (pFFs), were selected for experiments.

In the aspect of target selection, Hprt, Sall4 and Tet1 were selected as targets on the mouse genome, ACTB and Hprt1 were selected as targets on the human genome, ROSA26 and THY1 were selected as targets on the pig genome, these genes were all expressed in the corresponding cell lines, and the sgRNA sequences used for the respective target genes are shown in table 1. Co-transferring an HIT-bridging vector (HIT-trap-1C) and a Cas9 protein and a sgRNA expression vector into a corresponding cell line, and performing drug screening and enrichment to obtain a large number of cells containing capture vector integration for subsequent analysis, wherein the conditions in a control group are the same except that sgRNA aiming at a genome is absent.

The FACS results show (b of fig. 4) that a certain proportion of GFP positive cells are present in the cell population enriched by the drug screen. The proportion of GFP positive cells in the experimental group correlated with cell type and target site, with a minimum of 24.6 + -2.7% (pFF, THY1) and a maximum of 68.0 + -0.4% (A375, ACTB). Although random integration of the vector in the control group also resulted in a fraction of GFP positive cells (12.6 + -1.3% -17.9 + -2.0%), the control group was associated with a lower survival rate after drug screening, and therefore, in general, most of the GFP positive cells screened in the experimental group were generated by correct insertion of the vector into the target site.

Since NHEJ repair produces a degree of indels that may damage the capture elements on the vector, thereby rendering the capture vector non-functional, the precise degree of vector integration into the target site is also an important indicator for the evaluation of HIT-trapping technology. For this purpose, the Hprt site in mouse ES cells, the ACTB site in human A375 cells and the THY1 site in pFLs were selected, and the conditions of indels generated by NHEJ repair during integration of the HIT-trapping vector into these three sites were examined. And collecting the cells screened and enriched by the three targets in the previous step, extracting a genome, amplifying sequences at joints of two sides of the vector and the genome by PCR, and performing TA cloning sequencing and comparison analysis. The results show that there is a proportion of seamless junctions between vector and genome in each locus, with a higher proportion of seamless junctions in a375 cells versus pFFs relative to mouse ES cells, up to 92.4% (a375, 5' linker); the ratio of seamless junctions is related to the cell type, while the ratio of seamless junctions between 5 'and 3' ends on the same target is not very different. Furthermore, it was found that some of the ligation was caused by microhomology-terminated ligation (MMEJ) repair, which is more common in mouse ES cells. Statistical base deletion (c in FIG. 4) found that 91% of sequence deletions were less than 50bp bases, which did not destroy the functional elements in the vector, and the slight base deletion at this position had less effect on the genome because the target site was located in the intron. Therefore, indels produced by NHEJ do not have much influence on the experiment, and most of the capture vectors inserted into the target site can play a role. The above results indicate that the HIT-trapping technology can be efficiently generalized to a variety of mammalian cell lines.

Example 5 the HIT-trapping System can achieve both recoverability of a target gene and conditional knockdown

Since NHEJ-mediated knock-in of the vector is not directional, both forward and reverse knock-in of the vector to the target site alleles can be obtained in the experiment. Previous studies have shown that when a Gene capture vector is inserted forward in an intron, a loss-of-function mutation can be generated, which acts as a knock-out, whereas when inserted backward, the Gene capture element does not act, which has little effect on Gene function (Schn ü tgen, F., De-Zolt, S., Van Sloun, P., Hollatz, M., Floss, T., Hansen, J., Altschmied, J., Seisenger, C., Gyneselink, N.B., Ruiz, P., et al (2005) genomic vector of multiple applications for the amplification of the Gene product, Proc. Natl.Acad.U.S.A., 102, 7226, Xn. H7226, U.S.S.A., S.A., 3, J.S.C., D.C., D.S.S.S.S.S.A., K., P.S.E., S.S.S.E. Pat. No. 10, J., Gene, S.S.S.S.E.S.S.E.S.E.E., S.E., K., S. 1, S.E., S. 1, J., Gene, S. 1, U.S. 2, U.S. 1, U.S. 2, U.S. 1, S. 2, U.S. 2, S. 2, U.S. 1, U.S. 2, U.S. 1, S. 2, U.S. publication, U.S. 2, U.S. publication, m., Maddison, L.A., Boyd, K.L., Huskey, L., Ju, B., Hesselson, D., Zhong, T.P., Page-McCaw, P.S., et al (2012) Conditional control of gene function by an invertible gene trap in zebrafish.Proc.Natl.Acad.Sci.U.S.A.,109, 15389-15394.).

In the embodiment, an HIT-trapping system is combined with a Site-specific recombinase (SSR) system, a gene capturing element is designed into a structure capable of being inverted, and the forward and reverse knockins of a vector are realized to generate recoverable and conditional knockouts respectively.

To achieve inversion of the gene capture element, a Cre enzyme-mediated FlEx-extension (flip-inversion) switching system was chosen, which has been shown to be able to achieve inversion of the vector structure efficiently and stably in mammals. Furthermore, to avoid integration of the vector backbone moiety into the target site, a simple optimization strategy was developed, as shown in a of fig. 5: the insertion element part is separated from the vector backbone part by the same restriction enzyme, and the purified linear DNA is self-ligated into a loop by T4 DNA ligase to serve as a targeting vector.

Based on the factors, a new HIT-mapping system is designed: the system comprises a gene capture vector HIT-trap-FlEx vector (b in figure 5), an sgRNA expression vector pKin-sgA for recognizing a Cas9 recognition site, a Cas9 expression vector pM3-Cas9 and a target site sgRNA expression vector. In the HIT-trap-FlEx vector, the gene trapping and drug screening elements are the same as HIT-trap-1C, but a pair of anti-parallel loxP/lox2272 sequences are added on both sides of the two functional elements for FlEx conversion. To achieve NHEJ-mediated knock-in, the system also carries an sgA target site for Cas9 in vivo cleavage vector, while sgA that transcriptionally cleaves this site is expressed separately from another vector pKin-sgA, avoiding integration of the U6 promoter into the target site.

Furthermore, to facilitate removal of the vector backbone moiety in vitro, only one PvuI restriction enzyme was required in the design to achieve efficient separation of the insertion element from the vector backbone moiety (fig. 5 c). In the experiment, firstly treating the HIT-trap-FlEx vector with restriction enzyme PvuI, after the enzyme digestion is completed, carrying out electrophoretic separation to remove the skeleton part of the vector and purify an insertion element, and then treating with T4 DNA ligase for cyclization, thereby obtaining the optimized vector for the HIT-trapping experiment and being named as LigFV 3. After the targeting vector, Cas9 and the corresponding sgRNA expression vector are co-transferred into cells, the vector will be inserted into the target site in random direction under the repair effect of NHEJ generated by Cas 9. Due to the invertibility of the gene capturing element, after the gene capturing element is transferred into Cre enzyme again, the Cre enzyme mediated staged recombination can invert the gene capturing element and delete the drug screen element at the same time, so that the knockout event of the vector in the forward integration of the target site is converted into non-mutagenic insertion, and conversely, the reverse integration without mutagenesis is converted into a mutant type, thereby realizing the phenotype reversion and conditional knockout of the target gene respectively. Thus, the forward insertion of the vector at the target site is a revertive knockout, while the reverse insertion is a conditional knockout (d of FIG. 5).

In order to test the knock-in efficiency of the optimized HIT-trapping vector LigFV3 in mammalian cells, the Nes gene and Hprt gene in mouse ES cells and the ACTB gene in human A375 cells were selected as targets for detection, and the sgRNA sequences used for the target genes are shown in Table 1. After cell electroporation is completed, the monoclonal cell line is selected by drug screening and PCR identification is carried out. Since the Hprt and ACTB genes are both expressed in their respective cell types, the clones selected for these two sites can be used to preliminarily determine the knock-in of the vector based on the GFP expression and to detect it correspondingly, i.e. GFP-positive clones detect the forward knock-in event, whereas reverse knock-in events are detected. While the Nes gene is not expressed in mouse ES cells, it was observed that most of the selected cloned GFP was negative, and the knock-in of the vector could not be judged by GFP. Thus, for each clone selected, both forward and reverse insertion events were detected. PCR identification showed that all target clones were successful in both forward and reverse knock-in of the vector, and that most of the identifications were positive for the 5 '/3' linker PCR (a, b, c of FIG. 6). Summarizing the results of genotyping, it was found that both forward and reverse insertions of the capture vector could be obtained by identifying a small number of clones (no more than 24), especially with respect to GFP positive clones obtained when expressing the gene, the proportion of clones containing vector integration in the forward direction was higher (d in fig. 6). These results demonstrate the high efficiency of knock-in of the optimized vector ligafv 3 to the target site by the action of NHEJ.

To examine whether the capture element integrated into the target site could be inverted by FlEx conversion, some of the clones obtained in the above experiments that were determined to have carrier incorporation were selected for the experiments. Vector forward insertion selected a375 monoclonal cell line AG1 targeting the ACTB gene and mESCs monoclonal cell line H10 targeting the Hprt gene; vector reverse insertion selection A375 monoclonal cell line AN3 targeting the ACTB gene and mESCs monoclonal cell line H17 targeting the Hprt gene. Since the GFP signal in these clones is correlated with the direction of insertion of the vector, the direction of the vector at the target site can be intuitively determined from the condition of GFP expression. After transferring the Cre enzyme expression vector into these four monoclonal cell lines, it was found that the expression of GFP was stopped in the original GFP-expressing clones AG1 and H10, while the expression of GFP was activated in the GFP-non-expressing clones AN3 and H17 (FIG. 7). Therefore, it was preliminarily confirmed from the change in GFP fluorescence that the vector element at the target site in the cell was inverted.

In order to further confirm the inversion of the gene capture element at the target site caused by FlEx conversion, the DNA of the mES cell lines H10 and H17 transformed into Cre enzyme cells after recombination is extracted, and the sequence of the junction between the two ends of the vector and the genome is amplified by site-specific PCR and sequenced. The results show that the vector integrated into the target site is subjected to FlEx conversion under the action of Cre enzyme to generate a new specific vector and genome linker sequence, and prove that the gene capture element is inverted and the drug screen element is deleted (FIG. 8).

The loss of Hprt function in mES cells results in a 6-TG resistant and HAT sensitive phenotype, as opposed to a phenotype of cells containing wild-type Hprt (resistant to HAT but sensitive to 6-TG). Therefore, the monoclonal cell lines H10 and H17 obtained in experiments directed against the Hprt gene provide convenience for detecting phenotypic complementation and conditional knockout generated by Cre enzyme-mediated inversion of the vector element. After treating these two cell lines and the subcloned cell line (H10+ Cre, H17+ Cre) obtained by transferring Cre enzyme with 6-TG and HAT, respectively, methylene blue staining was performed to examine the survival of the cells. The results showed that the vector forward-knock-in monoclonal cell line H10 survived 6-TG treatment and no clones formed after HAT treatment, whereas the phenotype of the vector reverse-knock-in monoclonal cell line H17 was opposite thereto. After they have been subjected to the action of Cre enzyme, the survival of the cells is reversed compared to that before Cre enzyme treatment (FIG. 9). These observations suggest that the function of the Hprt gene in these cells is related to the orientation of the gene capture element integrated in its intron, with forward insertion resulting in loss of Hprt gene function, and reverse insertion having little effect on its function, allowing phenotypic reversion and conditional knock-out, respectively, under the action of Cre enzyme.

The above results show from the molecular level and phenotype that the HIT-mapping system is capable of efficiently generating both recoverable and conditional knockouts for target genes.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Sequence listing

<110> university of agriculture in China

<120> targeting gene capturing system independent of homologous recombination and application thereof

<130> KHP201120031.0

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggtatgtcgg gaacctctcc agg 23

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggtatgtcgg gaacctctcc 20

<210> 3

<211> 1713

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcgattaagg gatctgtagg gcgcagtagt ccagggtttc cttgatgatg tcatacttat 60

cctgtccctt ttttttccac agctcgcggt tgaggacaaa ctcttcgcgg tctttccagt 120

ggggatcgac ggtatcgtag agtcgaggcc gctctagcgg atctgcccct ctccctcccc 180

cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat 240

gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt 300

cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt 360

gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc 420

gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc 480

acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat 540

agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc 600

ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg 660

tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct 720

ttgaaaaaca cgatgataat atggccacaa ccatggtgag caagggcgag gagctgttca 780

ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 840

tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 900

ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 960

agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 1020

ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 1080

gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 1140

acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 1200

acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 1260

acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 1320

gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 1380

aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 1440

tcactctcgg catggacgag ctgtacaagt aaagcgaatt cctagagctc gctgatcagc 1500

ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 1560

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 1620

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 1680

ggattgggaa gagaatagca ggcatgctgg gga 1713

<210> 4

<211> 6246

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta 60

gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac 120

gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact 180

atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga 240

aaggacgaaa caccgggtat gtcgggaacc tctccgtttt agagctagaa atagcaagtt 300

aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt 360

ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt 420

ctgcagacaa atggctctag aggtaccggt atgtcgggaa cctctccagg tgcgattaag 480

ggatctgtag ggcgcagtag tccagggttt ccttgatgat gtcatactta tcctgtccct 540

tttttttcca cagctcgcgg ttgaggacaa actcttcgcg gtctttccag tggggatcga 600

cggtatcgta gagtcgaggc cgctctagcg gatctgcccc tctccctccc ccccccctaa 660

cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc 720

caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 780

gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt 840

gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 900

caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 960

agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 1020

aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 1080

accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 1140

gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 1200

acgatgataa tatggccaca accatggtga gcaagggcga ggagctgttc accggggtgg 1260

tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 1320

agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 1380

agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 1440

gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 1500

acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 1560

tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 1620

aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 1680

tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 1740

aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 1800

ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 1860

acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 1920

gcatggacga gctgtacaag taaagcgaat tcctagagct cgctgatcag cctcgactgt 1980

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 2040

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 2100

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 2160

agagaatagc aggcatgctg gggagcagga agagtcctga ggcggaaaga accagctgtg 2220

gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 2280

aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg 2340

cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc 2400

gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 2460

tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg 2520

aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat 2580

cgtttcgctc tagagtggcc acaaccatga ccgagtacaa gcccacggtg cgcctcgcca 2640

cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg 2700

ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac 2760

tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg 2820

cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg 2880

gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc 2940

tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc 3000

ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg 3060

agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg 3120

agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt 3180

gcatgacccg caagcccggt gcctgagata aacgcgggac tctggggttc gaaatgaccg 3240

accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa 3300

ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 3360

tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa 3420

taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg 3480

ggtcgtttgt tcggcgcgcc gatctcttaa ggcaggaacc cctagtgatg gagttggcca 3540

ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 3600

cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc 3660

tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca 3720

accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3780

cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3840

tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 3900

ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 3960

tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4020

taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt 4080

tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 4140

aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag 4200

tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 4260

cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 4320

cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 4380

cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 4440

aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 4500

ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 4560

aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 4620

ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 4680

gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 4740

ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 4800

ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 4860

gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 4920

aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 4980

gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 5040

aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 5100

caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 5160

tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 5220

acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 5280

gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 5340

agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 5400

gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 5460

ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 5520

taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 5580

agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 5640

aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 5700

ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 5760

gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 5820

aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 5880

aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 5940

gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6000

aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 6060

aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 6120

cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6180

cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 6240

tgctca 6246

<210> 5

<211> 7079

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60

ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120

aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360

ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480

gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540

caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600

caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660

cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720

tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat 780

tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt 840

aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900

cacctattgg tcttactgac atccactttg cctttctctc cacaggggta ccgaagccgc 960

tagtcgacac cggtgccacc atggactata aggaccacga cggagactac aaggatcatg 1020

atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta 1080

tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact 1140

ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc 1200

tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca 1260

gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac 1320

ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg 1380

acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc 1440

ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca 1500

tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct 1560

atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga 1620

accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc 1680

tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca 1740

gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga 1800

atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca 1860

acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc 1920

tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga 1980

acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg 2040

cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc 2100

tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga 2160

gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt 2220

tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca 2280

gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc 2340

acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg 2400

acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc 2460

tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc 2520

cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga 2580

tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt 2640

acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga 2700

gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga 2760

ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct 2820

tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc 2880

acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca 2940

ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac 3000

ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga 3060

gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt 3120

ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc 3180

agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg 3240

gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga 3300

agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca 3360

agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga 3420

agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga 3480

tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact 3540

acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg 3600

actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca 3660

aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg 3720

tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga 3780

gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg 3840

gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc 3900

tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag 3960

tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag 4020

tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa 4080

ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg 4140

tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca 4200

agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg 4260

gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg 4320

ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg 4380

tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga 4440

acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg 4500

acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca 4560

agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg 4620

agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga 4680

tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg 4740

cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact 4800

tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga 4860

aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg 4920

agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca 4980

acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc 5040

tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga 5100

ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc 5160

tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga 5220

aaaaggccgg ccaggcaaaa aagaaaaagg aattcctaga gctcgctgat cagcctcgac 5280

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 5340

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 5400

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 5460

ggaagagaat agcaggcatg ctggggagcg gccgccgtga catgtgagca aaaggccagc 5520

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5580

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5640

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5700

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5760

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5820

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5880

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5940

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 6000

ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 6060

gctcttgatc cggcaaacaa accacgctgg tagcggtggt ttttttgttt gcaagcagca 6120

gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 6180

cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgccgtctc agaagaactc 6240

gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac 6300

gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc 6360

tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg 6420

gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc 6480

gccgtcgggc atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg 6540

ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc 6600

gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg 6660

ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag 6720

atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc 6780

gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc 6840

ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg 6900

cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata 6960

gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat 7020

cataatatta ttgaagcatt tatcagggtt cgtctcgtcc cggtctcctc ccatgcatg 7079

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtaatgtag accatcaagc 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtggacagac ttaactaggt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggactttaat tattaggtga 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tatcaagagt cgcctcgaga 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccttggagtg tgtattaagt 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcttaaccgt aatcagcctc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgtgggaata tagagaaatt 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgatacttgt tcaagggctc 20

Claims

1. A targeted gene capture system independent of homologous recombination, the system comprising the following modules:

an sgRNA expression module recognizing a Cas9 protein recognition site, comprising a promoter 1 and an sgRNA1 operably linked downstream of the promoter 1, the sgRNA1 being capable of recognizing a Cas9 protein recognition site for guiding a Cas9 protein to cleave a vector into linearity;

a Cas9 protein expression module comprising promoter 2 and a Cas9 gene operably linked downstream of said promoter 2;

an sgRNA expression module that recognizes a gene capture target site, comprising a promoter 1 and a sgRNA2 operably linked downstream of the promoter 1, the sgRNA2 capable of recognizing the gene capture target site.

2. The targeted gene capture system independent of homologous recombination according to claim 1, wherein the gene capture system further comprises a gene capture screening module comprising a promoter 3 and a drug screening gene for screening cells in which the gene capture module is successfully inserted into the target site;

preferably, the promoter 3 is an SV40 promoter, and the drug screening gene is a Puro resistance gene.

3. The targeted gene capture system independent of homologous recombination of claim 2, wherein the gene capture screening module is operably linked downstream of the gene capture module, and the sgRNA expression module that recognizes the Cas9 protein recognition site and the Cas9 protein recognition site module are sequentially and operably linked upstream of the gene capture module in an upstream-to-downstream direction.

4. The targeted gene capture system independent of homologous recombination according to any one of claims 1 to 3, wherein the promoter 1 is a U6 promoter and the promoter 2 is a CMV promoter.

5. The homologous recombination-independent targeted gene capture system of claim 1, wherein the nucleotide sequence for Cas9 protein recognition is shown in SEQ ID No.1, the sequence of sgRNA1 is shown in SEQ ID No.2, and/or the sequence of the gene capture module is shown in SEQ ID No. 3.

6. The targeted gene capture system independent of homologous recombination according to any one of claims 1-5, wherein the Cas9 protein recognition site module, the sgRNA expression module that recognizes the Cas9 protein recognition site, and the gene capture module are located on a first vector;

preferably, the Cas9 protein expression module and the sgRNA expression module that recognizes the gene-trapping target site are located on a second vector and a third vector, respectively.

7. A targeted gene trap system capable of reverting and conditional knockout, which comprises the targeted gene trap system of any one of claims 1-5, and further comprises a Cre enzyme-mediated FlEx conversion module, wherein the FlEx conversion module comprises a loxP/lox2272 sequence and a Cre enzyme expression element, and the loxP/lox2272 sequence is respectively positioned between the gene trap module and the Cas9 protein recognition site module and downstream of the gene trap screening module.

8. The recoverable and conditional knockout targeted gene capture system of claim 7, wherein the Cre enzyme expression element is separately transferred to the cell when targeted gene capture requires recovery or conditional knockout.

9. The recoverable and conditional knockout targeted gene capture system of claim 7 or 8, wherein the Cas9 protein recognition site module, the gene capture screening module are located on a first vector; the sgRNA expression module that recognizes the Cas9 protein recognition site is located on a second vector;

preferably, the Cas9 protein expression module and the sgRNA expression module that recognizes the gene-trapping target site are located on a third vector and a fourth vector, respectively.

10. Use of a targeted gene capture system independent of homologous recombination according to any one of claims 1 to 6 or of a recoverable and conditional knockout targeted gene capture system according to any one of claims 7 to 9 for targeted gene capture, animal model preparation or drug screening.