CRISPR-Cas9-mediated exon site-directed mutagenesis method, donor DNA and its application

Abstract

The present application relates to the field of genetic engineering technology, and specifically discloses a CRISPR-Cas9-mediated exon site-directed mutagenesis method, donor DNA and its application. The CRISPR-Cas9-mediated exon site-directed mutagenesis method is as follows: inserting an artificial intron into the target exon, and using the cell homologous recombination repair mechanism to introduce a mutant base on the homologous arm to perform site-directed mutagenesis on the target exon; the artificial intron includes an intron recognition unit and a reporter gene expression unit inserted into the intron recognition unit; the intron recognition unit is used for the cell to recognize the artificial intron so as to cut the artificial intron during transcription; the reporter gene expression unit includes a reporter gene and a promoter that expresses the reporter gene. The present application can insert an artificial intron into any position of the gene sequence, thereby shortening the base distance between the restriction site and the editing site to obtain a smaller CT value, so that the efficiency of exon site-directed mutagenesis can be improved.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a CRISPR-Cas9-mediated exon site-directed mutagenesis method, donor DNA, and applications thereof. Background Art

[0002] Gene editing is a technique that precisely modifies, inserts or deletes the genome of an organism, thereby modifying the genetic shape. The earliest gene editing research randomly cuts DNA and targets exogenous DNA to specific sites on mammalian chromosomes through the cell's own homologous recombination repair mechanism. The CRISPR / Cas9 system uses the Cas9 nuclease and guide RNA (gRNA) complex to mediate DNA double-strand breaks at specific sites in the genome, and then uses donor DNA to repair the broken DNA through homologous recombination to complete site-specific gene editing, or exon-directed mutagenesis. Homologous recombination repair (HDR) and non-homologous end joining (NHEJ) are two mechanisms of DNA self-repair in cells. Since the HDR repair efficiency is much lower than that of NHEJ, the efficiency of exon-directed mutagenesis based on HDR is low. Therefore, improving the editing efficiency is an important prerequisite for the application of the HDR exon-directed mutagenesis method. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a CRISPR-Cas9-mediated exon site-directed mutagenesis method, donor DNA and its application, aiming to solve the problem of low efficiency of exon site-directed mutagenesis based on homologous recombination repair.

[0004] The technical solution of this application is as follows:

[0005] In a first aspect of the present application, a CRISPR-Cas9-mediated exon site-directed mutagenesis method is provided, wherein an artificial intron is inserted into a target exon, and a mutant base is introduced into a homologous arm using a cellular homologous recombination repair mechanism to perform site-directed mutagenesis on the target exon; wherein the artificial intron comprises an intron recognition unit and a reporter gene expression unit inserted into the intron recognition unit; the intron recognition unit is used for the cell to recognize the artificial intron so as to cut the artificial intron during transcription; and the reporter gene expression unit comprises a reporter gene and a promoter for expressing the reporter gene.

[0006] Preferably, the nucleic acid sequence of the intron recognition unit is a partial intron sequence of the human hemoglobin β subunit gene, as shown in SEQ ID No.1; or a partial sequence of the sequence shown in SEQ ID No.1, including the splicing junction sequence of the intron of the human hemoglobin β subunit gene.

[0007] Preferably, when the nucleic acid sequence of the intron recognition unit is a partial sequence of the sequence shown in SEQ ID No.1, the partial sequence of the sequence shown in SEQ ID No.1 is shown in SEQ ID No.2.

[0008] Preferably, the reporter gene is selected from one of a toxic gene, a fluorescent protein gene, and a drug resistance gene. The promoter is a pCMV promoter, and the reporter gene is a red fluorescent protein gene DsRed-T2A-BSD.

[0009] Preferably, the nucleic acid sequence of the artificial intron is shown as SEQ ID No.3.

[0010] Preferably, the CRISPR-Cas9-mediated exon site-directed mutagenesis method comprises the steps of: constructing a Cas9 plasmid and a donor plasmid, wherein the Cas9 plasmid comprises a guide RNA targeting a target exon, and the donor plasmid comprises a left homologous arm of the target exon, the artificial intron, and a right homologous arm of the target exon connected in sequence, and introducing a mutant base into the left homologous arm of the target exon and / or the right homologous arm of the target exon; using the donor plasmid as a template, obtaining double-stranded DNA by PCR amplification, and denaturing the double-stranded DNA into single-stranded DNA to obtain donor DNA; and transfecting the donor DNA and the Cas9 plasmid into cells to achieve site-directed mutagenesis of the exons in the cells.

[0011] Preferably, the method for constructing the Cas9 plasmid comprises the steps of: (1) designing and synthesizing a guide RNA targeting a target exon; (2) digesting the Lenit-CRISPR-V2 plasmid using a BsmBI endonuclease; and ligating the products of (1) and (2) using a T4 DNA ligase kit to obtain the Cas9 plasmid.

[0012] Preferably, the method for constructing the donor plasmid comprises the steps of: constructing a left homology arm and a right homology arm of the target exon; inserting the left homology arm and the right homology arm of the target exon into a plasmid containing an artificial intron to obtain the donor plasmid.

[0013] In a second aspect of the present application, a donor DNA is provided, wherein the donor DNA is a single-stranded DNA, and the donor DNA comprises the nucleic acid sequence of the homologous arm of the above-mentioned target exon and the nucleic acid sequence of the above-mentioned artificial intron.

[0014] The third aspect of the present application provides an application of the donor DNA exon site-directed mutagenesis according to the second aspect of the present application.

[0015] Beneficial effects of the present application: Based on the CRISPR-Cas9 technology, the present application constructs an artificial intron containing a reporter gene and a promoter driving the expression of the reporter gene, and inserts the artificial intron into the target exon. The artificial intron is removed by shearing during gene expression (in the process of the gene sequence being transcribed into precursor RNA), and the target exon can be site-directed mutagenesis by introducing the mutant base to be modified on the homologous arm without affecting the expression of the target exon. Compared with the existing exon site-directed mutagenesis method that requires the selection of an enzyme cutting site in the intron next to the target exon, the CRISPR-Cas9-mediated exon site-directed mutagenesis method (ARI-HDR) of the present application can insert an artificial intron into any position on the target exon, thereby shortening the base distance (conversiontract, CT) between the enzyme cutting site and the editing site to obtain a smaller CT value, thereby improving the efficiency of exon site-directed mutagenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0017] Figure 1 A schematic diagram of the process of the CRISPR-Cas9-mediated exon-directed mutagenesis method (ARI-HDR) provided in the embodiments of the present application;

[0018] Figure 2 A schematic diagram of the distance between the editing site and the restriction site of the GEIS exon site-directed mutagenesis method and the ARI-HDR exon site-directed mutagenesis method provided in the embodiments of the present application;

[0019] Figure 3 Screening analysis diagram of intron recognition unit provided in the embodiment of the present application:

[0020] A is a schematic diagram of inserting different intron recognition unit sequences into exons; B is an electrophoresis analysis diagram of the expression of fluorescent protein after inserting different intron recognition unit sequences; C is an analysis diagram of the expression of fluorescent protein after inserting different intron recognition unit sequences; D is a fluorescence microscopic observation diagram of cells after inserting different intron recognition unit sequences; E is a schematic diagram of truncation of HBB intron sequences; F is a gel electrophoresis analysis diagram of the expression of CopGFP protein after inserting HBB intron sequences of different lengths; G is a fluorescence microscopic observation diagram of cells after inserting HBB intron sequences of different lengths; H is a schematic diagram of the structure of artificial introns; I is a gel electrophoresis analysis diagram of the expression of CopGFP protein after inserting different reporter genes into artificial introns; J is a fluorescence microscopic observation diagram of cells after inserting different reporter genes into artificial introns; K is a schematic diagram of the reporter gene expression unit in the artificial intron;

[0021] Figure 4 The ARI-HDR exon site-directed mutagenesis method provided in the present application is FOXL2 Verification analysis diagram of gene editing:

[0022] A is the target FOXL2 Cas9 plasmid verification diagram of the gene; B is FOXL2 Gene expression analysis diagram; C is the expression analysis diagram of FOXL2 protein; D is the fluorescence microscopic observation diagram of cells with exon site-directed mutation at different sites;

[0023] Figure 5 The ARI-HDR exon site-directed mutagenesis method provided in the present application is FOXL2 Verification analysis diagram of gene editing:

[0024] A is FOXL2 Gene expression verification diagram; B is FOXL2 Sequencing results of site-directed mutation sites in gene exons;

[0025] Figure 6 This is a diagram analyzing the editing efficiency of the GEIS exon site-directed mutagenesis method and the ARI-HDR exon site-directed mutagenesis method provided in the examples of the present application. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0027] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0028] In the existing technology, in order to improve the efficiency of CRISPR / Cas9 and HDR-mediated exon site-directed mutagenesis, researchers have proposed methods such as inhibiting the NHEJ pathway, activating the HDR pathway, or controlling the cell cycle, all of which have improved the efficiency of HDR-mediated exon site-directed mutagenesis to a certain extent.

[0029] Previous studies have shown that donor DNA carrying screening markers can make edited cells carry fluorescence / drug resistance, and combined with drug screening / flow sorting technology, cells after exon site-directed mutation can be effectively enriched. In the above strategy, the screening marker must be located at the end of the open reading frame (ORF), which limits the application of this strategy. There is also a method to insert the screening marker into the intron region adjacent to the editing site, which expands the scope of marker application. However, this method cannot be implemented for genes without introns. In addition, this method also has the risk of affecting gene expression, so more optimized technology is still needed.

[0030] Based on this, the first aspect of the embodiment of the present application provides a CRISPR-Cas9-mediated exon site-directed mutagenesis method (ARI-HDR), specifically:

[0031] Insert an artificial intron into the target exon, and use the cell homologous recombination repair mechanism to introduce mutant bases on the homologous arm to perform site-directed mutation on the target exon. The artificial intron includes an intron recognition unit and a reporter gene expression unit inserted into the intron recognition unit. The intron recognition unit is used for the cell to recognize the artificial intron so as to cut the artificial intron during transcription. The reporter gene expression unit includes a reporter gene and a promoter that expresses the reporter gene.

[0032] The process is roughly as follows Figure 1As shown. By inserting a reporter gene and a promoter driving the expression of the reporter gene into the intron, an artificial intron is constructed. Based on the CRISPR-Cas9 technology, the Cas9 protein and the designed guide RNA (sgRNA) are used to recognize and cut the specific site of the target exon sequence, and by introducing the mutant bases that need to be modified on the homologous arm, the cell's own homologous recombination repair mechanism (Homology directed repair, HDR) is used to complete the site-directed mutagenesis of the exon.

[0033] In this process, artificial introns are inserted into exons that need to be site-directed mutated. The intron recognition unit in the artificial intron gives it the characteristics of an intron. During gene expression (when the gene sequence is transcribed into precursor RNA), the artificial intron is removed by shearing, so it does not affect the normal expression of the target exon. At the same time, since the artificial intron includes a reporter gene and a promoter that drives the expression of the reporter gene, when the cell repairs the gene sequence through the HDR mechanism using the homologous arm of the introduced mutant base, the artificial intron will be sheared, and the promoter in the artificial intron will drive the reporter gene to express, and then the expression of the reporter gene can be monitored to determine whether the exon site-directed mutation has been performed.

[0034] An existing exon-directed mutagenesis method (GEIS), such as Figure 2 As shown, the restriction site is selected in the intron of the gene sequence. If the sequence where the target exon is located does not contain the intron sequence, it cannot be implemented. In addition, in the GEIS technology, since the restriction site can only be set on the intron of the gene sequence, the distance between the restriction site and the target site that needs to be site-directed mutagenesis is large. According to the literature (S. Wang et al., Efficient gene editing through anintronic selection marker in cells. Cell Mol Life Sci 79, 111), the larger the base distance (conversion tract, CT) between the restriction site and the editing site, that is, the larger the CT value, the lower the efficiency of exon site-directed mutagenesis. Compared with the GEI exon site-directed mutagenesis method, the ARI-HDR exon site-directed mutagenesis method in this application can find restriction sites near the editing site, thereby obtaining a smaller CT value, so that the efficiency of exon site-directed mutagenesis can be improved.

[0035] In some embodiments, the nucleic acid sequence of the intron recognition unit is a partial intron sequence of the human hemoglobin β subunit gene, as shown in SEQ ID No.1; or a partial sequence of the sequence shown in SEQ ID No.1, including the splicing junction sequence of the intron of the human hemoglobin β subunit gene.

[0036] Through experiments, intron sequences that do not affect the expression of the inserted target exons were screened out, among which the partial intron sequence of the human hemoglobin β subunit gene (HBB) (as shown in SEQ ID No.1) ensures the expression of the target exons while not producing abnormal splicing products. In addition, by gradually truncating the sequence shown in SEQ ID No.1 while retaining its splicing junction sequence, experiments have shown that only a partial sequence of the sequence shown in SEQ ID No.1 can still make the inserted target exons express normally. The partial sequence of the sequence shown in SEQ ID No.1 includes the splicing junction sequence of the intron of the human hemoglobin β subunit gene.

[0037] In some embodiments, the nucleic acid sequence of the intron recognition unit is shown as SEQ ID No.2.

[0038] By performing progressive truncation on the sequence shown in SEQ ID No. 1 while retaining its splicing junction sequence, and verifying the expression of the exon gene, the sequence shown in SEQ ID No. 2 is finally obtained, which can ensure the normal expression of the inserted target exon while not producing abnormal indirect products.

[0039] By shortening the intron recognition unit in the artificial intron, the distance between the restriction site and the editing site can be further shortened, thereby improving the efficiency of exon site-directed mutagenesis. At the same time, shortening the sequence length of the intron recognition unit is also conducive to molecular-level operations during exon site-directed mutagenesis, reducing costs.

[0040] In some embodiments, the reporter gene is selected from one of a toxic gene, a fluorescent protein gene, and a drug resistance gene. When the reporter gene is a fluorescent protein gene, if the exon site-directed mutagenesis is successfully performed, the fluorescent protein will be expressed, and the fluorescent cells are observed by observing the fluorescence of the cells. The fluorescent cells are cells that have successfully achieved the exon site-directed mutagenesis. When the reporter gene is a drug resistance gene, the cells can be cultured in a culture medium containing specific antibiotics. The cells that survive after culture are cells expressing the resistance gene, proving that they have successfully achieved the exon site-directed mutagenesis.

[0041] In some embodiments, the promoter driving the expression of the reporter gene is a pCMV promoter, and the reporter gene is a red fluorescent protein gene DsRed-T2A-BSD.

[0042] In some embodiments, the nucleic acid sequence of the artificial intron is shown as SEQ ID No. 3, which includes a pCMV promoter, a red fluorescent protein gene DsRed-T2A-BSD, and an artificial intron recognition unit whose sequence is shown as SEQ ID No. 2.

[0043] In some embodiments, the CRISPR-Cas9-mediated exon-directed mutagenesis method comprises the steps of:

[0044] S1. Constructing Cas9 plasmid and donor plasmid: the Cas9 plasmid includes a guide RNA targeting the target exon, and the donor plasmid includes the left homology arm of the target exon, the artificial intron, and the right homology arm of the target exon connected in sequence, and the mutant base to be modified is introduced into the left homology arm of the target exon and / or the right homology arm of the target exon;

[0045] In some embodiments, the method for constructing a Cas9 plasmid comprises the steps of:

[0046] (1) Design and synthesize guide RNA targeting the target exon; (2) Digest the Lenit-CRISPR-V2 plasmid with BsmBI endonuclease; (3) Ligate the products of (1) and (2) using a T4 DNA ligase kit to obtain a Cas9 plasmid.

[0047] In some embodiments, the method for constructing a donor plasmid includes the steps of: constructing a left homologous arm of the target exon and a right homologous arm of the target exon; inserting the left homologous arm of the target exon and the right homologous arm of the target exon into a plasmid containing an artificial intron to obtain the donor plasmid.

[0048] Specifically, the method for constructing the donor plasmid comprises the steps of:

[0049] (1) The left homologous arm (Leftarm) and the right homologous arm (Rightarm) of the target exon are obtained by PCR amplification technology, wherein the bases to be modified are introduced into the Leftarm and the Rightarm; (2) The pUC-ARI plasmid is digested with AgeI endonuclease; (3) The Leftarm in (1) is connected with the product in (2) using T4 DNA ligase kit; (4) The product in (3) is assembled with the Rightarm in (1) using a seamless homologous recombination kit to obtain a donor plasmid.

[0050] S2. Constructing donor DNA: Using the donor plasmid as a template, amplifying the double-stranded DNA by PCR, and denaturing the double-stranded DNA into single-stranded DNA to obtain the donor DNA;

[0051] S3. Transfect the donor DNA and Cas9 plasmid into cells to obtain cells containing the target exon of gene modification, thereby achieving site-directed mutagenesis of the target exon.

[0052] According to a second aspect of an embodiment of the present application, a donor DNA is provided, which is a single-stranded DNA. The donor DNA includes the nucleic acid sequences of the left homologous arm of the target exon, the artificial intron, and the right homologous arm of the target exon, wherein the left homologous arm of the target exon and / or the right homologous arm of the target exon introduces a mutant base that needs to be modified.

[0053] The third aspect of the embodiment of the present application provides a use of the donor DNA of the second aspect of the embodiment of the present application in exon site-directed mutagenesis.

[0054] The following will provide further explanation through specific implementation methods.

[0055] Example 1

[0056] (1) Screening of artificial introns using vectors encoding fluorescent proteins. PIK3CA Gene, HBB Genes and NABP2 500-1000bp introns (HBB intron, NABP2 intron, PIK3CA intron) were selected from the genes and inserted into the copGreen expression frame of the plasmid pCDH-CMW-MCS-EF1-GreenPuro to construct three plasmids. The schematic diagrams of the intron-inserted parts of the three plasmids are shown in the figure. Figure 3 As shown in A, the plasmid pCDH-CMW-MCS-EF1-GreenPuro can express CopGFP protein. The constructed plasmid was transfected into HEK293T cells and cultured for 24 hours. The genome of the cells was then extracted using a genomic kit, and the cDNA of the CopGFP protein in the three constructed plasmids was identified by PCR and agarose gel electrophoresis. The results are shown in Figure 3 As shown in B. Figure 3 As can be seen in B, there is a single band in the lane of the agarose gel, indicating that the CopGFP mRNA in the plasmid with three introns inserted is transcribed normally and no abnormal transcripts are produced. The expression of fluorescent protein in cells was analyzed using qPCR and RT-PCR, and the results are as follows: Figure 3 As shown in C and D, from Figure 3 As shown in C and D, after the three introns were inserted, the CopGFP protein was expressed. And the expression level of CopGFP protein was the highest in the group with the intron of HBB gene inserted. At the same time, the cells were observed using a fluorescence microscope and the results were as follows: Figure 3As shown in D, NC represents the negative control group without introns inserted into the copGreen expression frame, and HBB, PIK3CA and NABP2 represent the experimental groups with three introns inserted into the copGreen expression frame. Figure 3 As shown in D, after the insertion of the three introns, the CopGFP protein can be expressed, and the fluorescence of the group with HBB intron and the group with NABP2 intron is basically the same as that of the NC group. Combined with the above tests, the expression level of CopGFP protein mediated by HBB intron is the highest, and it does not contain any abnormal indirect products. HBB intron can be used as a basic element for constructing artificial introns.

[0057] (2) On the basis of retaining the splicing adapter sequence at the 5' end of the HBB intron nucleic acid sequence, the HBB intron sequence was truncated, gradually shortening by 100 bp, to obtain the following: Figure 3 The full-length sequence of the HBB intron (FL), the HBB intron sequence shortened by 100bp (700bp), the HBB intron sequence shortened by 200bp (600bp), the HBB intron sequence shortened by 300bp (500bp), the HBB intron sequence shortened by 400bp (400bp), and the HBB intron sequence shortened by 500bp (300bp) shown in E. The six HBB intron sequences of different lengths were inserted into the CopGreen expression frame of pCDH-CMW-MCS-EF1-GreenPuro to construct six plasmids, which were transfected into HEK293T cells and cultured for 24h. The protein expression of CopGFP was detected by qPCR, and the results are shown in Figure 2. Figure 3 As shown in F. Figure 3 As can be seen in Figure F, after truncated introns of different lengths were inserted into the copGreen expression frame, the CopGFP protein was still able to express. The transfected cells were observed using a fluorescence microscope and the results were as follows: Figure 3 As shown in G, from Figure 3 As can be seen in Figure G, obvious green fluorescence can be observed in the 300bp, 500bp, and 600bp groups. Figure 3 The results in F and G show that the HBB intron (300bp group) truncated to 500bp can still efficiently mediate mRNA splicing without producing abnormal splicing products, and the CopGFP protein can still be well expressed.

[0058] (3) Based on (2), if Figure 3As shown in Figure 1, a marker gene (including promoter pCMV and reporter gene Select Marker) was inserted into the HBB intron truncated to 300 bp, wherein the reporter gene was selected from one of DsRed-T2A-BSD, Belo, Hygro, and EYFP. The constructed plasmid was transfected into HEK293T cells and cultured for 24 hours. The protein expression of CopGFP was detected by qPCR (the results are shown in Figure 1). Figure 3 The transfected cells were observed using a fluorescence microscope (as shown in Figure 1). Figure 3 J), combined with Figure 3 As shown in Figures I and J, after inserting the artificial intron carrying the promoter pCMV driving the DsRed-T2A-BSD marker gene, the CopGFP protein can be expressed and no abnormal transcripts are produced. Therefore, the promoter pCMV and the reporter gene DsRed-T2A-BSD are inserted into the intron selected above to obtain an artificial intron, the structure of which is shown in Figure 1. Figure 3 The sequence is shown in SEQ ID No. 3.

[0059] Example 2

[0060] The FOXL2 gene (without introns) was targeted and exon-directed mutagenesis was performed in HEK293T cells using the ARI-HDR exon-directed mutagenesis method. The primers used are shown in Table 1 .

[0061] Table 1 Primer nucleic acid sequence list

[0062]

[0063] The specific steps are as follows:

[0064] A. Constructing a target FOXL2 The Cas9 plasmid Cas9-FOXL2-sg gene sequence.

[0065] (A1) Primer sequence synthesis. Primers sense and antisense were dissolved in TE buffer to a final concentration of 20 μM. 10 μL of each was mixed and annealed using a thermal cycler (programmed at 95°C, 5 min), then cooled to room temperature at 2°C / min to obtain sgRNA.

[0066] (A2) According to the instructions, Lenti-CRISPR-V2 plasmid was digested with BsmBI-V2 endonuclease (purchased from NEB). The digestion system is shown in Table 2. After electrophoresis of the digestion product on 1% agarose gel, a band of about 10,000 bp was obtained (the result is shown in Figure 4 As shown in A in the middle, the gel was recovered using a gel recovery kit (purchased from TIANGEN).

[0067] Table 2 Lenti-CRISPR-V2 plasmid restriction enzyme system

[0068]

[0069] (A3) According to the instruction manual, use T4 DNA ligase (purchased from NEB) to connect the products of (A1) and (A2). The reaction system is shown in Table 3.

[0070] Table 3 Cas9-FOXL2-sg plasmid connection system

[0071]

[0072] The reaction system was placed at 16°C for 20 min.

[0073] (A4) Perform competent transformation on the product of (A3). Take 100 μL stbl3 competent E. coli cells (purchased from Kangti Biotechnology), add 10 μL of the product of (A3), place on ice for 30 minutes, heat shock at 42°C for 90 seconds using a metal bath, culture with 1 mL of non-resistant LB medium for 1 hour, centrifuge at 1000 rcf to obtain bacterial cells, break them up with 100 μL LB medium, and evenly spread them on LB agar plates containing 100 μg / L ampicillin. The plate opening is placed at 37°C for overnight culture.

[0074] (A5) A single clone was picked from the plate culture medium of (A4) and cultured in 15 mL LB culture medium containing 100 μg / L ampicillin. The plasmid was extracted from the cultured bacterial liquid and sequenced to confirm that the fragment was inserted into the vector, thus completing the construction of the plasmid Cas9-FOXL2-sg.

[0075] B. Construct the homology arms Leftarm and Rightarm of the target sequence.

[0076] (B1) According to the instruction manual, pUC-ARI plasmid was digested with AgeI restriction endonuclease. The digestion system is shown in Table 4.

[0077] Table 4 pUC-ARI restriction enzyme system

[0078]

[0079] The reaction system was placed at 37°C for 30 min. After the reaction, the solution was subjected to 1% agarose gel electrophoresis and recovered using a gel recovery kit.

[0080] (B2) The Leftarm gene was amplified by PCR using the primer pair FOXL2-left-F and FOXL2-Left-R and the genomic DNA of HEK293T as a template. The reaction system and reaction conditions are shown in Tables 5 and 6 below.

[0081] Table 5 PCR amplification reaction system of Leftarm gene

[0082]

[0083] Table 6 PCR amplification reaction procedure of Leftarm gene

[0084]

[0085] After the reaction was completed, the PCR product was subjected to 1% agarose gel electrophoresis and recovered using a gel recovery kit.

[0086] (B3) The products of (B1) and (B2) were assembled using a seamless homologous recombination kit (Yugong Biotechnology EG21202) to obtain a plasmid. The seamless homologous recombination reaction system is shown in Table 7.

[0087] Table 7 Seamless homologous recombination reaction system

[0088]

[0089] After the reaction system was reacted at 50°C for 30 minutes, the obtained product was transformed into stbl3 Escherichia coli competent cells. The specific steps were similar to (A4) and will not be repeated here. Finally, a plasmid with Leftarm successfully inserted was obtained. The constructed plasmid was digested with SalI endonuclease to obtain a linearized Leftarm plasmid. The digestion reaction system was similar to that in (B1) and will not be repeated here.

[0090] (B4) Rightarm was obtained by PCR amplification using primer pair FOXL2-Right-F and FOXL2-Right-R with HEK293T genomic DNA as template; Rightarm with A66V mutation was obtained by PCR amplification using primer pair FOXL2-Right-A66V- and FOXL2-Right-R with HEK293T genomic DNA as template; Rightarm with I80T mutation was obtained by PCR amplification using primer pair FOXL2-Right-I80T- and FOXL2-Right-R with HEK293T genomic DNA as template; Rightarm with I84N mutation was obtained by PCR amplification using primer pair FOXL2-Right-I84N-F and FOXL2-Right-R with HEK293T genomic DNA as template. The reaction system and reaction conditions were similar to those of (B2). After the reaction, the gel was recovered by 1% agarose gel electrophoresis using a gel recovery kit.

[0091] The four products in (B4) were assembled with the product of (B3) using a seamless homologous recombination kit (manufacturer) to obtain template plasmids Donor-FOXL2-WT, Donor-FOXL2-A66V, Donor-FOXL2-I80T, and Donor-FOXL2-I84N.

[0092] (B5) Preparation of single-stranded DNA template.

[0093] Primer synthesis: Primer donor-F and primer donor-R were dissolved in double distilled water to a final concentration of 10 mM. Using donor-F and donor-R as templates, and template plasmids Donor-FOXL2-WT, Donor-FOXL2-A66V, Donor-FOXL2-I80T, and Donor-FOXL2-I84N prepared in B as templates, PCR was performed. The reaction systems are shown in Tables 8 and 9.

[0094] Table 8 PCR reaction system

[0095]

[0096] Table 9 PCR reaction program

[0097]

[0098] The PCR product was subjected to 1% agarose gel electrophoresis to obtain a band of about 4000 bp, and the DNA was recovered in double distilled water using a gel recovery kit.

[0099] The obtained double-stranded DNA was subjected to high temperature denaturation treatment. NaCl was added to the recovered DNA solution so that the final concentration of NaCl in the solution was 100 mM. The mixed solution was incubated at 95°C for 10 minutes and quickly cooled to 4°C to obtain single-stranded DNA, which included FOXL2 The Lightarm and Rightarm of the gene, FOXL2 Lightarm and Rightarm, which have mutated the 66th amino acid of the gene, FOXL2 Lightarm and Rightarm, which have mutated the 80th amino acid of the gene, FOXL2 Lightarm and Rightarm, in which the 84th amino acid of the gene was mutated, were stored at -20°C for future use.

[0100] D. Transfect cells.

[0101] 10 μg of the Cas9-FOXL2-sg plasmid prepared in A and 500 ng of a single-stranded DNA (Lightarm and Rightarm) prepared in C were respectively transfected into HEK293T cells using Lipo3000 reagent. After 24 hours, the cells were killed with complete culture medium containing 0.6 μg / L puromycin. The cells obtained 72 hours after killing were cultured with complete culture medium (5% CO2, 37°C) for 72 hours.

[0102] E. Flow sorting.

[0103] (E1) Add an appropriate amount of PBS buffer to the culture dish containing the cells obtained in D, and wash the cells to remove dead cells. Aspirate the PBS buffer in the culture dish, add an appropriate amount of trypsin to the culture dish, and place it in a CO2 incubator for 1 minute. Then observe the cell morphology under a microscope. After confirming that the digestion is complete, add 2 times the volume of complete medium to terminate the digestion. Transfer the liquid in the culture dish to a 15mL centrifuge tube, centrifuge at 1000rpm for 5min, remove the supernatant, add an appropriate amount of PBS buffer to wash, and then centrifuge at 1000rpm for five minutes. After removing the supernatant, add an appropriate amount of PBS buffer to resuspend the cells.

[0104] (E2) Take the resuspended cell solution in (E1), filter the cells using a 40 μm pore cell filter that comes with a 5 mL flow cytometer, and analyze them using a BD FACSAriIII high-sensitivity flow cytometer for flow cytometric analysis and flow cytometric fluorescence sorting (FACS).

[0105] F. Cell monoclonal culture.

[0106] The flow-sorted cells in E were added to the plate at a concentration of 1.5 cells / well, and 10 μM small molecule inhibitors (Abmole Y-7632 was used to resist cell anoikis) were added to the medium in the plate. The cells were subcultured to establish a cell line.

[0107] right FOXL2 The gene expression was detected, and the results were as follows Figure 4 As shown in B. Figure 4 As can be seen in B, the cells of mutants A66V, I80T and I84N FOXL2 The mRNA expression of the gene is similar to that of wild-type cells. FOXL2 The mRNA expression of the gene was not much different; the expression of FOXL2 protein was detected, and the results were as follows Figure 4 As shown in C, from Figure 4 As shown in Figure C, the expression levels of FOXL2 protein in the cells of mutants A66V, I80T and I84N are almost the same as those in the wild-type cells. The cells were stained and observed using an immunofluorescence kit. The results are shown in Figure 5. Figure 4 As shown in D, from Figure 4 As can be seen in D, FOXL2 protein is normally expressed in cells of mutants A66V, I80T, and I84N. Figure 4 As can be seen from the results of B, C, and D, the ARI-HDR exon site-directed mutagenesis method of the present application is used to FOXL2 Site-directed mutation of genes will not affect FOXL2 Normal gene expression.

[0108] G, Monoclonal identification of exon site-directed mutations.

[0109] The cell line in F was used to extract genomic DNA using a genome extraction kit (yeasen 18700ES50), and primers FOXL2-gtest-F and pUC-R were used for PCR amplification, and the amplified primers were sequenced to determine the site-directed mutation of the cell exon. The PCR reaction system and reaction procedure are shown in Tables 10 and 11.

[0110] Table 10 PCR reaction system

[0111]

[0112] Table 11 PCR reaction program

[0113]

[0114] The PCR amplified products were analyzed by 1% agarose gel electrophoresis. Figure 5As shown in A. Figure 5 As can be seen in A, PCR amplified the bands of the specified size, indicating that the cells in the example have FOXL2 Gene. The sequencing results are as follows Figure 5 As shown in B, FoxL2-66-3 represents the mutation of the 66th amino acid of the FoxL2 protein (A66V) by the exon-directed mutagenesis method, from alanine to valine; FoxL2-80-2 represents the mutation of the 80th amino acid of the FoxL2 protein (I80T) by the exon-directed mutagenesis method; from isoleucine to threonine; FoxL2-84-5 represents the mutation of the 84th amino acid of the FoxL2 protein (I84N) by the exon-directed mutagenesis method, from isoleucine to asparagine. Figure 5 As can be seen from B, the exon site-directed mutagenesis method in this application was successfully used to FOXL2 Site-directed mutagenesis of gene exons.

[0115] Combination Figure 4 Neutralization Figure 5 The results show that the exon-directed mutagenesis method of the present invention successfully achieved the target gene expression under the condition that the expression level of FOXL2 protein remained unchanged. FOXL2 Site-directed mutations in the exons of the gene caused changes in the subcellular localization of FOXL2 protein.

[0116] Example 3

[0117] The ARI-HDR exon-directed mutagenesis method was used to edit the RELA gene and EGFR gene. The specific steps include:

[0118] synthesis EGFR gRNA of the gene (5'-GCTGGCCAAACTGCTGGGTG-3'), RELA The Lenti-CRISPR-V2 plasmid was digested with BsmBI-V2 endonuclease (purchased from NEB) and ligated with T4 DNA ligase (purchased from NEB). EGFR gRNA and RELA The gRNA of the gene was connected to the Lenti-CRISPR-V2 plasmid cut by enzyme to obtain plasmid Cas9-EGFR-sg and plasmid Cas9-RELA-sg. The construction process is similar to that of Example 2 and will not be repeated here.

[0119] The target sequence was constructed using a method similar to that in Example 2 ( EGFR Genes and RELAThe homology arms Leftarm and Rightarm of the gene), and the primers used are shown in Table 12.

[0120] Table 12 Primer Nucleotide Sequence List

[0121]

[0122] Plasmid Cas9-EGFR-sg and EGFR The homologous arms Leftarm and Rightarm of the gene were transfected into HRK293T cells using Lipo3000 reagent, and the plasmids Cas9-RELA-sg and RELA The homologous arms Leftarm and Rightarm of the gene were transfected into HRK293T cells using Lipo3000 reagent. After 24 hours, the cells were screened and killed with complete medium containing 0.6μg / L puromycin. The cells obtained after 72 hours of screening were cultured with complete medium at 5% CO2 and 37°C for 72 hours. After culturing with complete medium for another 72 hours, the cell population was sorted with a BD-AriaIII flow cytometer to screen the cell population with red fluorescent protein.

[0123] The genomic DNA was extracted from the screened cell population using a genomic extraction kit (yeasen 18700ES50), and amplified by PCR using primers of the nucleic acid sequence shown in Table 13.

[0124] Table 13 Primer nucleic acid sequences

[0125]

[0126] According to the GEIS exon site-directed mutagenesis method in the literature (S. Wang et al., Efficient gene editing through an intronicselection marker in cells. Cell Mol Life Sci 79, 111), RELA Genes and EGFR The efficiency of gene editing is comparable to that of the ARI-HDR exon site-directed mutagenesis method determined in Example 3. RELA Genes and EGFR The efficiency of gene editing was compared. Figure 6 As shown, from Figure 6 It can be seen that the ARI-HDR exon site-directed mutagenesis method RELA Genes and EGFR The efficiency of gene editing is significantly higher than that of using GEIS exon-directed mutagenesis. RELA Genes and EGFR The efficiency of gene editing.

[0127] The GEIS exon site-directed mutagenesis method must select introns near the editing site as reporter elements, while the exon site-directed mutagenesis method in this application can artificially design reporter elements near the editing site, the base distance (conversion tract, CT value) between the restriction site and the editing site will be shorter, and the editing efficiency will be higher.

[0128] In summary, based on the CRISPR-Cas9 technology, the present application constructs an artificial intron containing a reporter gene and a promoter that expresses the reporter gene, and inserts the artificial intron into the target exon. The artificial intron is removed by shearing during gene expression (in the process of the gene sequence being transcribed into precursor RNA), and the exon site-directed mutation of the target site can be completed by introducing the mutant base to be modified on the homologous arm without affecting the expression of the target exon. Compared with the existing exon site-directed mutation method that requires the selection of an enzyme cleavage site in the intron of the DNA sequence where the target exon is located, the CRISPR-Cas9-mediated exon site-directed mutation method (ARI-HDR) of the present application can insert an artificial intron into any position of the target exon, thereby shortening the base distance (conversiontract, CT) between the enzyme cleavage site and the editing site to obtain a smaller CT value, thereby improving the efficiency of exon site-directed mutation.

[0129] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A CRISPR-Cas9-mediated exon site-directed mutagenesis method, characterized in that: The exon site-directed mutagenesis method is to insert an artificial intron into the target exon in human cells, and use the cell homologous recombination repair mechanism to introduce a mutant base on the homology arm to perform site-directed mutagenesis on the target exon; The artificial intron includes an intron recognition unit and a reporter gene expression unit inserted into the intron recognition unit, and the nucleic acid sequence of the intron recognition unit is a partial intron sequence of a human hemoglobin β subunit gene, as shown in SEQ ID No. 1; The intron recognition unit is used for the cell to recognize the artificial intron so as to cut the artificial intron during transcription; The reporter gene expression unit comprises a reporter gene and a promoter for expressing the reporter gene; The CRISPR-Cas9-mediated exon-directed mutagenesis method was performed in vitro.

2. The CRISPR-Cas9-mediated exon site-directed mutagenesis method according to claim 1, characterized in that: The nucleic acid sequence of the intron recognition unit is a partial sequence of the sequence shown in SEQ ID No.1, and the partial sequence is obtained by truncating 500bp under the condition of retaining the splicing junction sequence of the intron including the human hemoglobin β subunit gene, and the sequence is shown in SEQ ID No.

2.

3. The CRISPR-Cas9-mediated exon site-directed mutagenesis method according to claim 2, characterized in that: The reporter gene is selected from one of a toxic gene, a fluorescent protein gene, and a drug resistance gene; The promoter for expressing the reporter gene is the pCMV promoter, and the reporter gene is the red fluorescent protein gene DsRed-T2A-BSD.

4. The CRISPR-Cas9-mediated exon site-directed mutagenesis method according to claim 3, characterized in that: When the nucleic acid sequence of the intron recognition unit is the sequence shown in SEQ ID No.2, the nucleic acid sequence of the artificial intron is the sequence shown in SEQ ID No.

3.

5. The CRISPR-Cas9-mediated exon site-directed mutagenesis method according to any one of claims 1 to 4, characterized in that: Includes steps: Constructing a Cas9 plasmid and a donor plasmid, wherein the Cas9 plasmid includes a guide RNA targeting a target exon, and the donor plasmid includes a left homologous arm of the target exon, the artificial intron, and a right homologous arm of the target exon connected in sequence, and a mutant base is introduced into the left homologous arm of the target exon and / or the right homologous arm of the target exon; Using the donor plasmid as a template, amplifying by PCR to obtain double-stranded DNA, and denaturing the double-stranded DNA into single-stranded DNA to obtain donor DNA; The donor DNA and Cas9 plasmid are transfected into cells to achieve site-directed mutation of the exons in the cells.

6. The CRISPR-Cas9-mediated exon site-directed mutagenesis method according to claim 5, characterized in that: The method for constructing the Cas9 plasmid comprises the steps of: (1) Design and synthesize guide RNA targeting the target exon; (2) Digest the Lenit-CRISPR-V2 plasmid with BsmBI endonuclease; The product of (1) and the product of (2) were ligated using a T4 DNA ligase kit to obtain the Cas9 plasmid.

7. The CRISPR-Cas9-mediated exon site-directed mutagenesis method according to claim 5, characterized in that: The construction method of the donor plasmid comprises the steps of: Constructing the left homology arm of the target exon and the right homology arm of the target exon; The left homology arm of the target exon and the right homology arm of the target exon are inserted into a plasmid containing an artificial intron to obtain the donor plasmid.

8. A donor DNA, characterized in that The donor DNA is a single-stranded DNA, and the donor DNA comprises the nucleic acid sequence of the homology arm of the target exon according to claim 1 and the nucleic acid sequence of the artificial intron according to claim 1.

9. Use of the donor DNA according to claim 8 in exon site-directed mutagenesis, wherein the use is performed in vitro.

Images