CRISPR-Cas9-based gene editing method and system

Abstract

The invention provides a gene editing method and system based on CRISPR-Cas9. The gene editing method comprises the following steps: constructing a gRNA expression vector and a Cas9 expression vector; simultaneously introducing the gRNA expression vector, the Cas9 expression vector and a preset donor template into a target cell to form a Cas9-gRNA compound, and after the compound is successfully combined to a target DNA sequence, generating double-strand breakage near the target DNA sequence; repairing the double-chain fracture by utilizing a repairing mechanism; wherein if the mutation site is located at the upstream of the PAM region, the donor template contains synonymous mutation of all basic groups between the mutation site and the PAM region; if the mutation site is located at the downstream of the PAM region, the donor template contains synonymous mutation of all basic groups between the gRNA complementary region and the mutation site, so that accurate editing of the target gene site is realized, the number of basic groups different from that of the original genome is increased, and an editing window is improved.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a gene editing method and system based on CRISPR-Cas9. Background Technology

[0002] CRISPR-Cas (Clustered Regulatory Interspaced Short PalindromicRepeats-CRISPR associated) system is a widely used technology for gene editing, which can achieve precise editing of specific DNA sequences. The combination of CRISPR-Cas system and homology-directed repair (HDR) technology has attracted much attention. It can achieve NN free conversion in base editing and has been widely used in the field of genome engineering. However, the single guide RNA (gRNA) of the existing CRISPR-Cas base editing tool has a narrow efficient editing window for free conversion of NN bases in the genome, which limits its application in complex genome editing. Especially when multiple bases need to be edited at the same time, the editing effect is not ideal and the editing accuracy is reduced. SUMMARY OF THE INVENTION

[0003] The present invention provides a gene editing method and system based on CRISPR-Cas9, which is used to solve the defects of the traditional CRISPR-Cas system, such as narrow editing window, unsatisfactory editing effect and reduced editing accuracy at places far away from the cutting site of the gRNA region.

[0004] The present invention provides a gene editing method based on CRISPR-Cas9, comprising: Construct gRNA expression vector and Cas9 expression vector; The gRNA expression vector, the Cas9 expression vector and the preset donor template are simultaneously introduced into the target cell to form a Cas9-gRNA complex in the target cell, and the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; When the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; The double-strand breaks are repaired by the preset donor template using the homologous recombination directed repair mechanism; wherein: if the mutation site of the target DNA sequence is located upstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the gRNA complementary region and the mutation site.

[0005] According to the CRISPR-Cas9-based gene editing method provided by the present invention, the repair step comprises: In the first round of editing, a first donor template is used, which contains the target mutation of the mutation site and the synonymous mutation; In the second round of editing, a second donor template is used, which contains only the target mutation, and the region of the synonymous mutation is restored to the original genomic sequence.

[0006] According to the CRISPR-Cas9-based gene editing method provided by the present invention, the second donor template has a higher homology to the original genome sequence than the first donor template, so as to preferentially restore the synonymous mutation to the original sequence through homologous recombination.

[0007] The CRISPR-Cas9-based gene editing method provided by the present invention also includes: After the first round of editing, a helper plasmid is introduced, which is used to target the gRNA of the first round CRISPR plasmid selection marker; The Cas9 protein is guided by the labeled gRNA to cut the first round of CRISPR plasmid to eliminate the first round of CRISPR plasmid.

[0008] According to the CRISPR-Cas9-based gene editing method provided by the present invention, before simultaneously introducing the gRNA expression vector, the Cas9 expression vector and the preset donor template into the target cell, the method further comprises: Pre-treating the target cells, wherein the pre-treatment includes at least one of cell electroporation, chemical transfection or viral vector transfection.

[0009] According to the CRISPR-Cas9-based gene editing method provided by the present invention, the preset donor template double-stranded oligonucleotide comprises a sequence of 40 bases homologous to both sides of the target DNA region; wherein: the preset donor template is a specific base replacement at the target DNA mutation site, and the bases in the synonymous mutation region are different from the original genome sequence but encode the same amino acid.

[0010] The present invention also provides a gene editing system based on CRISPR-Cas9, comprising: gRNA expression vector, used to express gRNA complementary to the target DNA sequence; Cas9 expression vector, used to express Cas9 protein; Donor template, used to repair Cas9-induced double-strand breaks by homologous recombination; Wherein, the gRNA expression vector, the Cas9 expression vector and the preset donor template are simultaneously introduced into the target cell to form a Cas9-gRNA complex in the target cell, and the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; When the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; Using the homologous recombination directed repair mechanism, the double-strand break is repaired through the preset donor template; wherein: if the mutation site of the target DNA sequence is located upstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the gRNA complementary region and the mutation site.

[0011] The CRISPR-Cas9-based gene editing system provided by the present invention further comprises: an auxiliary plasmid, wherein the repair of double-strand breaks by the preset donor template using the homologous recombination directed repair mechanism comprises using a first donor template in the first round of editing, which contains the target mutation of the mutation site and the synonymous mutation; and using a second donor template in the second round of editing, which contains only the target mutation, and the region of the synonymous mutation is restored to the original genome sequence; The auxiliary plasmid is used to target the gRNA of the first round CRISPR plasmid selection marker; the Cas9 protein is guided by the marker gRNA to cut the first round CRISPR plasmid to eliminate the first round CRISPR plasmid.

[0012] According to the CRISPR-Cas9-based gene editing system provided by the present invention, the preset donor template is a double-stranded oligonucleotide.

[0013] The CRISPR-Cas9-based gene editing method and system provided by the present invention are as follows: a gRNA expression vector and a Cas9 expression vector are constructed; the gRNA expression vector, the Cas9 expression vector and a preset donor template are simultaneously introduced into a target cell to form a Cas9-gRNA complex in the target cell, and the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; when the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; the double-strand break is repaired by the preset donor template using a homologous recombination directed repair mechanism; wherein: if the mutation site of the target DNA sequence is located upstream of a PAM region, the preset donor template contains synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template contains synonymous mutations of all bases between a gRNA complementary region and the mutation site. The present invention achieves precise editing of the target gene site by cleverly designing synonymous mutations on the donor template, and increases the number of bases that are different from the original genome, significantly improving the editing window of a single gRNA. Brief Description of the Figures

[0014] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 is a schematic diagram of a CRISPR-Cas9-based gene editing method provided in an embodiment of the present invention; Figure 2 is a schematic diagram of the donor DNA structure and editing window provided by an embodiment of the present invention; Figure 3 is a schematic diagram of the verification results of the single gRNA editing window provided in an embodiment of the present invention; Figure 4 is a schematic diagram for comparing editing efficiencies after expanding the scope of synonymous mutations provided by an embodiment of the present invention; Figure 5 is a schematic diagram of the process and efficiency of iterative editing to eliminate synonymous mutations provided by an embodiment of the present invention; Figure 6 is a schematic diagram comparing the single traditional method and the auxiliary plasmid rapid editing process provided by the embodiment of the present invention; Figure 7This is a schematic diagram of the elimination effect of the helper plasmid provided in the embodiment of the present invention on different selection tags; Figure 8 This is a schematic diagram of the efficiency evaluation of the quick editing method provided by an embodiment of the present invention. Specific implementation method

[0016] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] Figure 1 A flow chart of a gene editing method based on CRISPR-Cas9 provided in an embodiment of the present invention, such as Figure 1 As shown, the CRISPR-Cas9-based gene editing method provided in the embodiment of the present invention includes: Step 101, constructing gRNA expression vector and Cas9 expression vector; Step 102, introducing the gRNA expression vector, the Cas9 expression vector and the preset donor template into the target cell at the same time to form a Cas9-gRNA complex in the target cell, and the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; Step 103, when the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; Step 104, using the homologous recombination directed repair mechanism to repair the double-strand break through the preset donor template; wherein: if the mutation site of the target DNA sequence is located upstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the gRNA complementary region and the mutation site.

[0018] In the embodiment of the present invention, the donor DNA structure and editing window are as follows Figure 2 . The single gRNA editing window verification results are shown in Figure 3 As shown.

[0019] ​The single guide RNA (gRNA) editing window of traditional CRISPR-Cas base editing tools is narrow, which limits its application in complex genome editing. Especially when multiple bases need to be edited simultaneously, the editing effect is not ideal and the editing accuracy is reduced.

[0020] The CRISPR-Cas9-based gene editing method provided in an embodiment of the present invention comprises constructing a gRNA expression vector and a Cas9 expression vector; introducing the gRNA expression vector, the Cas9 expression vector and a preset donor template into a target cell at the same time to form a Cas9-gRNA complex in the target cell, wherein the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; when the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; and repairs the double-strand break by the preset donor template using a homologous recombination directed repair mechanism; wherein: if the mutation site of the target DNA sequence is located upstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the gRNA complementary region and the mutation site. The present invention achieves precise editing of the target gene site by cleverly designing synonymous mutations on the donor template, and increases the number of bases that are different from the original genome, significantly improving the editing window of a single gRNA.

[0021] Based on any of the above embodiments, the repairing steps include: In the first round of editing, a first donor template is used, which contains the target mutation of the mutation site and the synonymous mutation; In the second round of editing, a second donor template is used, which contains only the target mutation, and the region of the synonymous mutation is restored to the original genomic sequence.

[0022] In an embodiment of the present invention, the second donor template has a higher homology to the original genome sequence than the first donor template, so as to preferentially restore the synonymous mutation to the original sequence through homologous recombination.

[0023] The embodiment of the present invention adopts a two-round iterative editing strategy and designs two iterative editing schemes. Scheme 1: In the first round of editing, Cas9 nuclease is used to induce double-strand breaks in the genome. During donor repair, all bases except the non-target bases between the target mutant base and the PAM region are subjected to synonymous mutations, thereby achieving the maximum base difference; in the second round of editing, the new donor DNA only performs specific base replacement for the target mutant base, and the rest is the same as the original genome sequence, so that the non-target mutant amino acids are restored to the original genome sequence after repair. Scheme 2: The first round of editing only introduces synonymous mutations in the target region, and the second round of editing introduces target mutations and eliminates the synonymous mutations in the first round at the same time. Through the above two schemes, the "scars" of synonymous mutations in the genome are successfully eliminated, effectively ensuring the minimum effect on protein expression. In addition to the ADE2 gene, the present invention also verifies the point mutations of two essential genes ERG1 and ERG7 in the ergosterol synthesis pathway of Saccharomyces cerevisiae, and uses Scheme 1 for editing. The experimental results show that ADE2.a, ADE2.b, ERG1 K311R and ERG7 I705K The editing efficiencies of the two rounds of CRISPR were 100%, 98%, 90% and 83% respectively. Although the editing efficiency of essential genes was lower than that of non-essential genes, the overall editing efficiency was still satisfactory, which fully demonstrated that the two-round CRISPR iterative editing method can effectively remove synonymous mutation "scars".

[0024] In some embodiments of the present invention, the CRISPR-Cas9-based gene editing method further comprises: After the first round of editing, a helper plasmid is introduced, which is used to target the gRNA of the first round CRISPR plasmid selection marker; The Cas9 protein is guided by the labeled gRNA to cut the first round of CRISPR plasmid to eliminate the first round of CRISPR plasmid.

[0025] The embodiment of the present invention introduces the strategy of auxiliary plasmids, and introduces auxiliary plasmids between the first and second rounds of CRISPR plasmid editing. The auxiliary plasmid carries a specific gRNA expression cassette, which can target the selection tags of CRISPR plasmids (such as URA3, HIS3 and LEU2, etc.), and cuts the first round of CRISPR plasmids through the gRNA expression cassette. During the liquid culture medium culture process, the first round of CRISPR plasmids are effectively eliminated by screening. The present invention selects three representative screening tags of URA3, HIS3 and LEU2 in Saccharomyces cerevisiae for verification, and designs a specific "gRNA expression cassette" for the first round of CRISPR plasmids to construct auxiliary plasmids. After the auxiliary plasmid is transformed into the first round editing strain, it is cultured in the auxiliary plasmid selective liquid culture medium for 2 hours, and then spread on the YPD non-selective culture medium to obtain a single colony, and then these colonies are streaked to the corresponding nutrient-deficient solid culture medium to verify the elimination efficiency of the first round of editing plasmids. The results show that the elimination rate of different tag plasmids is about 100%. After successful elimination of the first round of plasmids and 2 hours of screening, the second round of plasmid and donor DNA transformation was immediately performed, shortening the total editing process to approximately 9 days. This rapid plasmid elimination method was used to evaluate multiple genes (ADE2.a, ADE2.b, ERG1 K311R and ERG7 I705K ) were 100%, 100%, 87% and 87% respectively, with no significant difference compared with the traditional method.

[0026] If Figure 4 As shown in the figure, by optimizing the synonymous substitution range, the base editing range of a single gRNA is effectively expanded. In the gRNA direction, in the ADE2 gene site a, 69bp away from the cutting site, the single base editing efficiency increased from 18% to 95%; the single base editing efficiency of site b, 59bp away from the cutting site, increased from 18% to 98%, and the overall editing efficiency in the gRNA direction increased to more than 81%; the overall editing efficiency in the PAM region direction was mostly increased to more than 70%, and the single gRNA had a high-efficiency base editing range.

[0027] If Figure 5 As shown in the figure, the iterative editing method was used to successfully eliminate the "scar" of synonymous mutations in the genome, avoiding the potential interference of synonymous mutations on protein expression, and providing a reliable guarantee for the construction of stable engineering strains.

[0028] If Figure 6 、 7As shown, the auxiliary plasmid strategy is introduced to introduce an auxiliary plasmid carrying a specific gRNA expression cassette between the first and second rounds of CRISPR plasmid editing. The auxiliary plasmid targets the specific cleavage site of the selection tag of the first round of CRISPR plasmid and cuts the selection tag through the action of nuclease. When cultured in liquid culture medium, cells containing the auxiliary plasmid cannot grow under the corresponding screening conditions because the selection tag is destroyed, thereby effectively eliminating the first round of CRISPR plasmids and directly transforming the second round of editing plasmids and donor DNA. This strategy significantly shortens the editing cycle, greatly shortening the time of losing the first round of CRISPR plasmids from the original 6 days to 1 day, and reducing the entire round of iterative editing time from the original 14 days to 9 days, improving the efficiency of gene editing and reducing experimental costs.

[0029] Based on any of the above embodiments, before simultaneously introducing the gRNA expression vector, the Cas9 expression vector and the preset donor template into the target cell, the method further includes: Pre-treating the target cells, wherein the pre-treatment includes at least one of cell electroporation, chemical transfection or viral vector transfection.

[0030] Based on any of the above embodiments, the preset donor template double-stranded oligonucleotide comprises a sequence of 40 bases homologous to both sides of the target DNA region; wherein: the preset donor template is a specific base replacement at the target DNA mutation site, and the bases in the synonymous mutation region are different from the original genome sequence but encode the same amino acid.

[0031] The embodiment of the present invention uses Saccharomyces cerevisiae as a model and provides a method for seamlessly expanding the range of efficient single-base editing based on CRISPR-Cas9 combined with HDR. The method uses a eukaryotic expression vector that can express gRNA and Cas9 to achieve precise cutting of the target gene site; at the same time, a double-stranded oligonucleotide is used as a donor template to introduce the desired mutation at the cutting site. Among them, the base sequence of the double-stranded oligonucleotide is basically consistent with the base sequence between 40 bases on both sides of the base region to be repaired, but there are two key differences: one is that on the donor template, the base corresponding to the base to be repaired is an unmutated wild-type base; the other is that the target mutation and synonymous mutation occurred in the base region to be repaired. After repeating the experiment three times, after applying this method, such as Figure 8As shown, the single-base mutation efficiency of ADE2.a sites at 69bp, 47bp and 29bp from the cleavage site increased from 18%, 23% and 42% to 95%, 85% and 82%, respectively; the single-base mutation efficiency of ADE2.b sites at 59bp, 42bp and 25bp from the cleavage site increased from 18%, 55% and 70% to 98%, 97% and 93%, respectively. In general, by expanding the scope of synonymous substitution, the single-base editing efficiency of a single gRNA at a relatively long distance (especially upstream of the PAM region) is significantly improved, with the maximum tested distance reaching 69bp and the editing efficiency being 95%, which is nearly seven times larger than the maximum distance of 10bp in mainstream literature.

[0032] It should be noted that the CRISPR base repair system provided in the embodiment of the present invention can not only repair single base mutations, but also repair mutations of multiple bases in related regions, covering the mutual substitution between four bases. Those skilled in the art can make corresponding adjustments to the system according to actual needs, and can be realized without creative labor.

[0033] The CRISPR-Cas9-based gene editing method provided in the embodiment of the present invention aims to provide a CRISPR-Cas9-based traceless iterative rapid genome base editing method, which expands the synonymous mutation range (maximum 69bp), introduces two rounds of iterative editing to eliminate the residual synonymous mutation, and shortens the editing cycle to about 9 days by using the auxiliary plasmid strategy to solve the problems of narrow editing window, residual synonymous mutation and long cycle in the prior art. The principle of this method is based on the precise cutting characteristics of the CRISPR-Cas9 system and the repair function of the HDR technology, and the precise editing of the target gene site is achieved by cleverly designing synonymous mutations on the donor template. In terms of expanding the range of synonymous mutations, when the target mutation base is upstream of the PAM region, all bases between the target mutation base and the PAM region are subjected to synonymous mutations; if the target mutation base is downstream of the PAM region, all bases between the gRNA region and the target mutation site are subjected to synonymous mutations, thereby increasing the number of bases different from the original genome, and significantly improving the editing window of a single gRNA. At the same time, with the help of a uniquely designed two-round iterative editing technology, the first round introduces the maximum base difference or only introduces synonymous mutations according to different needs, and the second round introduces target mutations and eliminates the synonymous mutations in the first round, which can effectively eliminate synonymous mutations in the genome and minimize the impact on protein expression. In addition, the embodiment of the present invention also introduces an auxiliary plasmid strategy, in which the auxiliary plasmid carries a specific gRNA expression cassette. The design principle is based on the specific recognition of the sequence of the first-round CRISPR plasmid selection tag (such as URA3, HIS3 and LEU2, etc.), and the selection tag is targeted for cutting by nuclease action. During the liquid culture medium culture process, the first-round CRISPR plasmid is effectively eliminated by screening, shortening the editing cycle to about 9 days, and improving the efficiency of gene editing.

[0034] The following is a description of the CRISPR-Cas9-based gene editing system provided by the present invention. The CRISPR-Cas9-based gene editing system described below and the CRISPR-Cas9-based gene editing method described above can be referenced to each other.

[0035] The embodiment of the present invention also provides a CRISPR-Cas9-based gene editing system including: gRNA expression vector, used to express gRNA complementary to the target DNA sequence; Cas9 expression vector, used to express Cas9 protein; Donor template, used to repair Cas9-induced double-strand breaks by homologous recombination; Among them, the gRNA expression vector, the Cas9 expression vector, and the preset donor template are simultaneously introduced into the target cells to form a Cas9-gRNA complex in the target cells. The Cas9-gRNA complex searches for the target DNA sequence complementary to the gRNA in the target cells; After the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; Using the homologous recombination-directed repair mechanism, the double-strand break is repaired by the preset donor template; wherein: if the mutation site of the target DNA sequence is upstream of the PAM region, the preset donor template contains a synonymous mutation of all bases between the mutation site and the PAM region; if the mutation site is downstream of the PAM region, the preset donor template contains a synonymous mutation of all bases between the gRNA complementary region and the mutation site.

[0036] In the embodiments of the present invention, the CRISPR-Cas9-based gene editing system further includes: an auxiliary plasmid. The repair of the double-strand break by the preset donor template using the homologous recombination-directed repair mechanism includes using a first donor template containing the target mutation and the synonymous mutation of the mutation site in the first round of editing; in the second round of editing, using a second donor template containing only the target mutation, and the region of the synonymous mutation is restored to the original genomic sequence; The auxiliary plasmid is used for the gRNA targeting the selection marker of the first-round CRISPR plasmid; the Cas9 protein is guided by the labeled gRNA to cleave the first-round CRISPR plasmid to eliminate the first-round CRISPR plasmid.

[0037] In the embodiments of the present invention, the preset donor template is a double-stranded oligonucleotide.

[0038] The embodiments of the present invention have the characteristics of scarless editing, can eliminate the traces that may be left by traditional editing methods, achieve precise and rapid base editing, provide an effective method supplement for the field of base editing of Saccharomyces cerevisiae, and also provide useful reference and inspiration for gene editing research of other organisms.

[0039] The CRISPR-Cas9-based gene editing system provided in an embodiment of the present invention comprises constructing a gRNA expression vector and a Cas9 expression vector; introducing the gRNA expression vector, the Cas9 expression vector and a preset donor template into a target cell at the same time to form a Cas9-gRNA complex in the target cell, wherein the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; when the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; and repairs the double-strand break by the preset donor template using a homologous recombination directed repair mechanism; wherein: if the mutation site of the target DNA sequence is located upstream of the PAM region, the preset donor template comprises synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template comprises synonymous mutations of all bases between the gRNA complementary region and the mutation site. The embodiment of the present invention achieves precise editing of the target gene site by cleverly designing synonymous mutations on the donor template, and increases the number of bases that are different from the original genome, significantly improving the editing window of a single gRNA.

[0040] Through the description of the above implementation methods, those skilled in the art can clearly understand that each implementation method can be implemented by means of software plus a necessary general hardware platform, or of course by hardware. Based on this understanding, the above technical solution, in essence, or the part that contributes to the relevant technology, can be embodied in the form of a software product, which can be stored in a computer-readable storage medium, such as ROM / RAM, a disk, an optical disk, etc., and includes a number of instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in each embodiment or some parts of the embodiments.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene editing method based on CRISPR-Cas9, characterized in that: include: Construct gRNA expression vector and Cas9 expression vector; The gRNA expression vector, the Cas9 expression vector, and a preset donor template are simultaneously introduced into a target cell to form a Cas9-gRNA complex in the target cell, and the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; When the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; The double-strand breaks are repaired by the preset donor template using the homologous recombination directed repair mechanism; wherein: if the mutation site of the target DNA sequence is located upstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the gRNA complementary region and the mutation site.

2. The CRISPR-Cas9-based gene editing method according to claim 1, characterized in that: The repair steps include: In the first round of editing, a first donor template is used, which contains the target mutation of the mutation site and the synonymous mutation; In the second round of editing, a second donor template is used, which contains only the target mutation, and the region of the synonymous mutation is restored to the original genomic sequence.

3. The CRISPR-Cas9-based gene editing method according to claim 2, characterized in that: The second donor template has a higher homology to the original genomic sequence than the first donor template, so as to preferentially restore the synonymous mutation to the original sequence through homologous recombination.

4. The CRISPR-Cas9-based gene editing method according to claim 2, characterized in that: Also includes: After the first round of editing, a helper plasmid is introduced, which is used to target the gRNA of the first round CRISPR plasmid selection marker; The labeled gRNA guides the Cas9 protein to cut the first round of CRISPR plasmid to eliminate the first round of CRISPR plasmid.

5. The CRISPR-Cas9-based gene editing method according to claim 1, characterized in that: Before simultaneously introducing the gRNA expression vector, the Cas9 expression vector and the preset donor template into the target cell, the method further comprises: The target cells are pretreated, and the pretreatment includes at least one of cell electroporation, chemical transfection or viral vector transfection.

6. The CRISPR-Cas9-based gene editing method according to claim 1, characterized in that: The preset donor template is a double-stranded oligonucleotide, which contains a 40-base sequence homologous to both sides of the target DNA region; wherein: the preset donor template is a specific base replacement at the target DNA mutation site, and the bases in the synonymous mutation region are different from the original genome sequence but encode the same amino acid.

7. A gene editing system based on CRISPR-Cas9, characterized in that: include: gRNA expression vector, used to express gRNA complementary to the target DNA sequence; Cas9 expression vector, used to express Cas9 protein; Donor template for repairing Cas9-induced double-strand breaks by homologous recombination; The gRNA expression vector, the Cas9 expression vector, and a preset donor template are simultaneously introduced into the target cell to form a Cas9-gRNA complex in the target cell, and the Cas9-gRNA complex searches for a target DNA sequence complementary to the gRNA in the target cell; When the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence; The double-strand breaks are repaired by the preset donor template using the homologous recombination directed repair mechanism; wherein: if the mutation site of the target DNA sequence is located upstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the preset donor template contains synonymous mutations of all bases between the gRNA complementary region and the mutation site.

8. The CRISPR-Cas9-based gene editing system according to claim 7, characterized in that: Also includes: A helper plasmid, wherein the repair of the double-strand break by the preset donor template using the homologous recombination directed repair mechanism includes using a first donor template in a first round of editing, which contains the target mutation of the mutation site and the synonymous mutation; and using a second donor template in a second round of editing, which contains only the target mutation, and the region of the synonymous mutation is restored to the original genome sequence; The auxiliary plasmid is used to target the gRNA marked by the first round CRISPR plasmid selection; the Cas9 protein is guided by the marked gRNA to cut the first round CRISPR plasmid to eliminate the first round CRISPR plasmid.

9. The CRISPR-Cas9-based gene editing system according to claim 7, characterized in that: The preset donor template is a double-stranded oligonucleotide.

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