CRISPR-Cas9 Based Gene Editing Method and System

Abstract

The present invention provides a CRISPR-Cas9-based gene editing method and system, which constructs a gRNA expression vector and a Cas9 expression vector; simultaneously introduces the gRNA expression vector, the Cas9 expression vector and a preset donor template into a target cell to form a Cas9-gRNA complex, and when the complex successfully binds to the target DNA sequence, a double-strand break is generated near the target DNA sequence; the double-strand break is repaired by using a repair mechanism; wherein: if the mutation site is located upstream of the PAM region, the donor template contains a synonymous mutation of all bases between the mutation site and the PAM region; if the mutation site is located downstream of the PAM region, the donor template contains a synonymous mutation of all bases between the gRNA complementary region and the mutation site, thereby achieving precise editing of the target gene locus, increasing the number of bases different from the original genome, and enhancing the editing window.

Description

Technical Field

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

[0002] The CRISPR-Cas (Clustered Regulatory Interspaced Short Palindromic Repeats-CRISPR associated) system is a technology widely used in gene editing, which can achieve precise editing of specific DNA sequences. The combination of the CRISPR-Cas system and the endogenous homology directed repair (HDR) technology has attracted much attention, and it can achieve the free conversion of N-N in base editing and has been widely used in the field of genome engineering. However, the efficient editing window for the free conversion of N-N bases in the genome by a single guide RNA (gRNA) of existing CRISPR-Cas base editing tools is narrow, which limits its application in complex genome editing. Especially in the case where multiple bases need to be edited simultaneously, the editing effect is not ideal and the editing accuracy decreases. Summary of the Invention

[0003] The present invention provides a gene editing method and system based on CRISPR-Cas9 to solve the defects of the traditional CRISPR-Cas system, such as narrow editing window, poor editing effect and decreased editing accuracy at a place far from the cleavage site of the gRNA region.

[0004] The present invention provides a gene editing method based on CRISPR-Cas9, including:

[0005] Constructing a gRNA expression vector and a Cas9 expression vector;

[0006] Simultaneously introducing the gRNA expression vector, the Cas9 expression vector and a preset donor template 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;

[0007] After the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence;

[0008] Using the homologous recombination-directed repair mechanism, repair the double-strand break through 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 synonymous mutations 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 synonymous mutations of all bases between the gRNA complementary region and the mutation site.

[0009] According to the CRISPR-Cas9-based gene editing method provided by the present invention, the steps of the repair include:

[0010] In the first round of editing, use the first donor template, which contains the target mutation of the mutation site and the synonymous mutation;

[0011] In the second round of editing, use the second donor template, which only contains the target mutation, and the region of the synonymous mutation is restored to the original genomic sequence.

[0012] According to the CRISPR-Cas9-based gene editing method provided by the present invention, the second donor template has a higher homology with the original genomic sequence than the first donor template, so as to preferentially restore the synonymous mutation to the original sequence through homologous recombination.

[0013] According to the CRISPR-Cas9-based gene editing method provided by the present invention, it further includes:

[0014] After the first round of editing, introduce an auxiliary plasmid, which is used for the gRNA targeting the selection marker of the first-round CRISPR plasmid;

[0015] Guide the Cas9 protein to cleave the first-round CRISPR plasmid through the gRNA of the marker to eliminate the first-round CRISPR plasmid.

[0016] 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, it further includes:

[0017] Perform pretreatment on the target cell, and the pretreatment includes at least one of cell electroporation, chemical transfection or viral vector transfection.

[0018] According to the CRISPR-Cas9 based gene editing method provided by the present invention, the preset donor template double-stranded oligonucleotide comprises a 40-base sequence homologous to both sides of the target DNA region; wherein: the preset donor template is a specific base substitution at the target DNA mutation site, and the bases in the synonymous mutation region are different from the original genomic sequence but encode the same amino acid.

[0019] The present invention also provides a CRISPR-Cas9 based gene editing system, comprising:

[0020] A gRNA expression vector for expressing a gRNA complementary to the target DNA sequence;

[0021] A Cas9 expression vector for expressing the Cas9 protein;

[0022] A donor template for repairing the double-strand break induced by Cas9 through homologous recombination repair;

[0023] 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 the target DNA sequence complementary to the gRNA in the target cell;

[0024] When the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence;

[0025] 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 upstream of the PAM region, the preset donor template comprises 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 comprises a synonymous mutation of all bases between the gRNA complementary region and the mutation site.

[0026] According to the CRISPR-Cas9 based gene editing system provided by the present invention, it further comprises: an auxiliary plasmid, and the repairing of the double-strand break through the preset donor template using the homologous recombination directed repair mechanism includes, in the first round of editing, using a first donor template, which comprises the target mutation of the mutation site and the synonymous mutation; in the second round of editing, using a second donor template, which only contains the target mutation, and the region of the synonymous mutation is restored to the original genomic sequence;

[0027] 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 gRNA of the marker to cut the first-round CRISPR plasmid, so as to eliminate the first-round CRISPR plasmid.

[0028] According to the CRISPR-Cas9-based gene editing system provided by the present invention, the preset donor template double-stranded oligonucleotide.

[0029] The CRISPR-Cas9-based gene editing method and system provided by the present invention construct a gRNA expression vector and a 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; 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 located 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 located 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. The present invention realizes precise editing of a target gene locus by ingeniously designing synonymous mutations on the donor template, and increases the number of bases different from the original genome, significantly improving the editing window of a single gRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0031] Figure 1 is a schematic flowchart of the CRISPR-Cas9-based gene editing method provided by the embodiment of the present invention;

[0032] Figure 2 is a schematic diagram of the donor DNA structure and the editing window provided by the embodiment of the present invention;

[0033] Figure 3 is a schematic diagram of the verification result of the single gRNA editing window provided by the embodiment of the present invention;

[0034] Figure 4 It is a schematic diagram for comparing the editing efficiency after expanding the synonymous mutation range provided by the embodiments of the present invention;

[0035] Figure 5 It is a schematic diagram of the process and efficiency of iterative editing to eliminate synonymous mutations provided by the embodiments of the present invention;

[0036] Figure 6 It is a schematic diagram for comparing the single traditional method and the rapid editing process of the auxiliary plasmid provided by the embodiments of the present invention;

[0037] Figure 7 It is a schematic diagram of the elimination effect of the auxiliary plasmid on different selection tags provided by the embodiments of the present invention;

[0038] Figure 8 It is a schematic diagram for evaluating the efficiency of the rapid editing method provided by the embodiments of the present invention. Detailed implementation manners

[0039] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0040] Figure 1 It is a flowchart of the CRISPR-Cas9-based gene editing method provided by the embodiments of the present invention. As Figure 1 shown, the CRISPR-Cas9-based gene editing method provided by the embodiments of the present invention includes:

[0041] Step 101, constructing a gRNA expression vector and a Cas9 expression vector;

[0042] Step 102, simultaneously introducing the gRNA expression vector, the Cas9 expression vector and a preset donor template 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;

[0043] 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;

[0044] Step 104: Repair the double-strand break by using the homologous recombination-directed repair mechanism through 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 synonymous mutations 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 synonymous mutations of all bases between the gRNA complementary region and the mutation site.

[0045] In the embodiment of the present invention, the donor DNA structure and the editing window are as Figure 2 shown. The verification result of the single gRNA editing window is as Figure 3 shown.

[0046] The editing window of a single guide RNA (gRNA) of traditional CRISPR-Cas base editing tools is narrow, which limits its application in complex genome editing. Especially in the case where multiple bases need to be edited simultaneously, the editing effect is not ideal and the editing accuracy decreases.

[0047] The gene editing method based on CRISPR-Cas9 provided in the embodiment of the present invention includes 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 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; repair the double-strand break by using the homologous recombination-directed repair mechanism through 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 synonymous mutations 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 synonymous mutations of all bases between the gRNA complementary region and the mutation site. The present invention realizes precise editing of the target gene locus by ingeniously designing synonymous mutations on the donor template, and increases the number of bases different from the original genome, significantly improving the editing window of a single gRNA.

[0048] Based on any of the above embodiments, the steps of the repair include:

[0049] In the first round of editing, use a first donor template, which contains the target mutation of the mutation site and the synonymous mutation;

[0050] In the second-round 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.

[0051] In the embodiments of the present invention, the homology of the second donor template to the original genomic sequence is higher than that of the first donor template, so as to preferentially restore the synonymous mutation to the original sequence by homologous recombination.

[0052] The embodiments of the present invention adopt a two-round iterative editing strategy and design two iterative editing schemes. Scheme 1: In the first-round editing, the Cas9 nuclease is used to induce double-strand breaks in the genome. When the donor repairs, all bases except the non-target bases between the target mutation base and the PAM region are subjected to synonymous mutations, so as to achieve the maximum base difference; in the second-round editing, the new donor DNA only performs specific base substitution on the target mutation base, and the rest is the same as the original genomic sequence, so that the non-target mutated amino acids after repair are restored to the original genomic sequence. Scheme 2: Only synonymous mutations are introduced in the target region in the first-round editing, and target mutations are introduced in the second-round editing while eliminating the synonymous mutations in the first round. Through the above two schemes, the "scar" of the synonymous mutation in the genome is successfully eliminated, effectively ensuring the minimum impact on protein expression. In addition to the ADE2 gene, the present invention also verified the point mutations of two essential genes ERG1 and ERG7 in the ergosterol synthesis pathway of Saccharomyces cerevisiae, and adopted Scheme 1 for editing. The experimental results show that the editing efficiencies of ADE2.a, ADE2.b, ERG1 K311R and ERG7 I705K are 100%, 98%, 90% and 83% respectively. Although the editing efficiency of essential genes is lower than that of non-essential genes, the overall editing efficiency is still satisfactory, fully demonstrating that the two-round CRISPR iterative editing method can effectively remove the "scar" of synonymous mutations.

[0053] In some embodiments of the present invention, the CRISPR-Cas9-based gene editing method further includes:

[0054] After the first-round editing, an auxiliary plasmid is introduced, and the auxiliary plasmid is used to target the gRNA of the selection marker of the first-round CRISPR plasmid;

[0055] The Cas9 protein is guided by the labeled gRNA to cleave the first-round CRISPR plasmid to eliminate the first-round CRISPR plasmid.

[0056] The embodiments of the present invention introduce a strategy of using an auxiliary plasmid, which is introduced between the first-round and second-round CRISPR plasmid editing. The auxiliary plasmid carries a specific gRNA expression cassette that can target the selection tags (such as URA3, HIS3, and LEU2, etc.) of the CRISPR plasmid, and cuts the first-round CRISPR plasmid through the gRNA expression cassette. During the cultivation in liquid medium, the effective elimination of the first-round CRISPR plasmid is achieved through the screening effect. The present invention selects three representative screening tags, URA3, HIS3, and LEU2, in Saccharomyces cerevisiae for verification, and designs a specific "gRNA expression cassette" targeting the first-round CRISPR plasmid to construct an auxiliary plasmid. After transforming the auxiliary plasmid into the first-round edited strain, it is cultured in the selective liquid medium of the auxiliary plasmid for 2 hours, and then spread on the YPD non-selective medium to obtain single colonies. These colonies are then streaked onto the corresponding auxotrophic solid medium to verify the elimination efficiency of the first-round edited plasmid. The results show that the elimination rate of plasmids with different tags is approximately 100%. After successfully eliminating the first-round plasmid and screening for 2 hours, the transformation of the second-round plasmid and donor DNA is immediately carried out, shortening the total editing process to approximately 9 days. Using this rapid plasmid elimination method to evaluate the editing efficiency of multiple genes (ADE2.a, ADE2.b, ERG1 K311R and ERG7 I705K ), the editing efficiencies are 100%, 100%, 87%, and 87% respectively, showing no significant difference compared with the traditional method.

[0057] As Figure 4 shown, by optimizing the synonymous substitution range, the base editing range of a single gRNA is effectively expanded. In the gRNA direction, at the ADE2 gene locus a, 69 bp away from the cleavage site, the single-base editing efficiency is increased from 18% to 95%; at locus b, 59 bp away from the cleavage site, the single-base editing efficiency is increased from 18% to 98%, and the overall editing efficiency in the gRNA direction is increased to more than 81%; in the PAM region direction, the overall editing efficiency is mostly increased to more than 70%, which is the high-efficiency base editing range of a single gRNA.

[0058] As Figure 5 shown, by using the iterative editing method, the genomic synonymous mutation "scars" are successfully eliminated, avoiding the potential interference of synonymous mutations on protein expression, providing a reliable guarantee for constructing stable engineering strains.

[0059] As Figure 6 、 7As shown, an auxiliary plasmid strategy is introduced, and an auxiliary plasmid carrying a specific gRNA expression cassette is introduced between the first-round and second-round CRISPR plasmid editing. The auxiliary plasmid targets a specific cleavage site of the selection tag of the first-round CRISPR plasmid and cleaves the selection tag through the action of an endonuclease. When cultured in a liquid medium, the cells containing the auxiliary plasmid cannot grow under the corresponding screening conditions due to the destruction of the selection tag, thereby effectively eliminating the first-round CRISPR plasmid and directly transforming the second-round editing plasmid and the donor DNA. This strategy significantly shortens the editing cycle, greatly reducing the time for losing the first-round CRISPR plasmid 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 the experimental cost.

[0060] 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 cells, it further includes:

[0061] Pre-treating the target cells, where the pre-treatment includes at least one of cell electroporation, chemical transfection, or viral vector transfection.

[0062] Based on any of the above embodiments, the preset donor template double-stranded oligonucleotide includes a sequence of 40 bases homologous to both sides of the target DNA region; wherein: the preset donor template has a specific base substitution at the target DNA mutation site, and the bases in the synonymous mutation region are different from the original genomic sequence but encode the same amino acid.

[0063] An embodiment of the present invention uses Saccharomyces cerevisiae as a model to provide a method for tracelessly expanding the efficient editing range of single bases based on CRISPR-Cas9 combined with HDR. This method uses a eukaryotic expression vector capable of expressing gRNA and Cas9 to achieve precise cleavage of the target gene locus; at the same time, a double-stranded oligonucleotide used as a donor template is used to introduce a desired mutation at the cleavage site. Among them, the base sequence of the double-stranded oligonucleotide is basically the same as 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 the base of the unmutated wild type; the other is that the target mutation and the synonymous mutation have occurred in the base region to be repaired. After repeating the experiment 3 times, after applying this method, as Figure 8As shown, the single-base mutation efficiency at positions 69 bp, 47 bp, and 29 bp from the cleavage site of the ADE2.a locus increased from 18%, 23%, and 42% to 95%, 85%, and 82% respectively; the single-base mutation efficiency at positions 59 bp, 42 bp, and 25 bp from the cleavage site of the ADE2.b locus increased from 18%, 55%, and 70% to 98%, 97%, and 93% respectively. Generally speaking, 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) was significantly improved. The maximum tested distance reached 69 bp with an editing efficiency of 95%, which was nearly seven times greater than the maximum distance of 10 bp in the mainstream literature.

[0064] It should be noted that the CRISPR base repair system provided in the embodiments of the present invention can not only repair single-base mutations, but also repair mutations of multiple bases in the relevant region, covering the mutual substitution between four bases. Those skilled in the art can make corresponding adjustments to this system according to actual needs, and it can be achieved without creative labor.

[0065] The gene editing method based on CRISPR-Cas9 provided by the embodiments of the present invention aims to provide a traceless iterative rapid genome base editing method based on CRISPR-Cas9. By expanding the synonymous mutation range (up to 69 bp), introducing two rounds of iterative editing to eliminate the residual synonymous mutations, and using the auxiliary plasmid strategy to shorten the editing cycle to about 9 days, the problems of narrow editing window, residual synonymous mutations, and long cycle in the prior art are solved. The principle of this method is based on the precise cleavage characteristics of the CRISPR-Cas9 system and the repair function of the HDR technology. By cleverly designing synonymous mutations on the donor template, precise editing of the target gene locus is achieved. In terms of expanding the synonymous mutation range, when the target mutated base is upstream of the PAM region, all bases between the target mutated base and the PAM region are subjected to synonymous mutations; if the target mutated base is downstream of the PAM region, all bases between the gRNA region and the target mutated site are subjected to synonymous mutations, thereby increasing the number of bases different from the original genome and significantly enhancing the editing window of a single gRNA. At the same time, with the help of a uniquely designed two-round iterative editing technology, in the first round, the maximum base difference is introduced or only synonymous mutations are introduced according to different needs, and in the second round, the target mutation is introduced and the synonymous mutations in the first round are eliminated, which can effectively eliminate the synonymous mutations in the genome and minimize the impact on protein expression. In addition, the embodiments of the present invention also introduce an auxiliary plasmid strategy. In this strategy, the auxiliary plasmid carries a specific gRNA expression cassette, and its design principle is based on the specific recognition of the sequences of the selection tags (such as URA3, HIS3, and LEU2, etc.) of the first-round CRISPR plasmid, and the selection tag is targeted and cleaved by the action of an endonuclease. During the culture process in liquid medium, the effective elimination of the first-round CRISPR plasmid is achieved through the screening effect, shortening the editing cycle to about 9 days and improving the efficiency of gene editing.

[0066] The gene editing system based on CRISPR-Cas9 provided by the present invention will be described below. The gene editing system based on CRISPR-Cas9 described below can be correspondingly referred to the gene editing method based on CRISPR-Cas9 described above.

[0067] The embodiments of the present invention also provide a gene editing system based on CRISPR-Cas9, including:

[0068] A gRNA expression vector for expressing a gRNA complementary to the target DNA sequence;

[0069] A Cas9 expression vector for expressing the Cas9 protein;

[0070] A donor template for repairing the double-strand break induced by Cas9 through homologous recombination repair;

[0071] 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, and the Cas9-gRNA complex searches for the target DNA sequence complementary to the gRNA in the target cells;

[0072] After the Cas9-gRNA complex successfully binds to the target DNA sequence, Cas9 generates a double-strand break near the target DNA sequence;

[0073] 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 located 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 located 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.

[0074] In the embodiment of the present invention, the gene editing system based on CRISPR-Cas9 further includes: an auxiliary plasmid. The repairing 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;

[0075] 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 cut the first-round CRISPR plasmid to eliminate the first-round CRISPR plasmid.

[0076] In the embodiment of the present invention, the preset donor template is a double-stranded oligonucleotide.

[0077] The embodiment of the present invention has the characteristics of scarless editing, can eliminate the traces that may be left by traditional editing methods, realizes precise and rapid base editing, provides an effective method supplement for the field of base editing of Saccharomyces cerevisiae, and also provides beneficial reference and inspiration for the gene editing research of other organisms.

[0078] The gene editing system based on CRISPR-Cas9 provided by the embodiments of the present invention constructs a gRNA expression vector and a Cas9 expression vector; simultaneously introduces the gRNA expression vector, the Cas9 expression vector and a preset donor template 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; utilizes 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. The embodiments of the present invention achieve precise editing of target gene sites by ingeniously designing synonymous mutations on the donor template, and increase the number of bases different from the original genome, significantly enhancing the editing window of a single gRNA.

[0079] Through the description of the above embodiments, those skilled in the art can clearly understand that each embodiment can be implemented by means of software plus a necessary general hardware platform, and of course, it can also be implemented by hardware. Based on such an understanding, the above technical solution, in essence, or the part that contributes to the related technology can be embodied in the form of a software product, and the computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, magnetic disk, optical disc, etc., including several instructions for causing 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.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate 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 break is 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; 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; Cas9 nuclease is used to induce double-strand breaks in the genome, and during donor repair, all bases between the target mutation base and the PAM region except the target base are subjected to synonymous mutations; 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 genome sequence; the new donor DNA only undergoes specific base replacement for the target mutation base, and the rest is the same as the original genome sequence, so that the non-target mutation amino acid is restored to the original genome sequence after repair.

2. The CRISPR-Cas9-based gene editing method according to claim 1, 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.

3. The CRISPR-Cas9-based gene editing method according to claim 1, 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.

4. 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.

5. 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.

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