阅读说明：本技术 精确大片段基因删除结合终止密码子***的基因敲除法 (Gene knock-out method combining precise large-fragment gene deletion with stop codon insertion ) 是由 赵培 康雅虹 罗莹 于 2019-09-20 设计创作，主要内容包括：本发明属于基因工程的基因编辑技术领域,更具体地涉及一种高效的基因敲除法,即精确大片段基因删除结合终止密码子插入的基因敲除法。本发明旨在解决目前基因精确敲除中长片段敲除的难度大、周期长的问题。首先确定5’sgRNA靶位点位于起始密码子附近(100bp以内),3’端sgRNA靶位点位于5’端sgRNA靶位点与终止密码子之间任何位置,sgRNA靶位点的编辑效率分数高并且脱靶效应接近于0；其次确定被删除序列的5’和3’端点位于5’sgRNA靶序列和3’sgRNA靶序列中,修复模板带有终止密码子。最后将5’端上游sgRNA质粒和3’端下游sgRNA质粒与所述Cas9质粒、共编辑标记质粒、报告基因质粒和修复模板制得显微注射DNA混合液体系一起注入雌雄同体线虫体内,统计其基因编辑效率并验证筛选功能与全基因敲除法的一致性,发现本发明提供的技术方案基因编辑效率高、性价比好和成功率高的优点。(The invention belongs to the technical field of gene editing of genetic engineering, and particularly relates to a high-efficiency gene knock-out method, namely a gene knock-out method combining precise large-fragment gene deletion with stop codon insertion. The invention aims to solve the problems of high difficulty and long period of knockout of long fragments in the current gene precise knockout. Firstly, determining that a 5 ' sgRNA target site is positioned near a start codon (within 100 bp), a 3 ' sgRNA target site is positioned at any position between a 5 ' sgRNA target site and a stop codon, the editing efficiency score of the sgRNA target site is high, and the off-target effect is close to 0; secondly, the 5 'and 3' end points of the deleted sequence are determined to be positioned in the 5 'sgRNA target sequence and the 3' sgRNA target sequence, and the repair template is provided with a stop codon. And finally, preparing a microinjection DNA mixed solution system by the upstream sgRNA plasmid at the 5 'end, the downstream sgRNA plasmid at the 3' end, the Cas9 plasmid, the co-editing marker plasmid, the reporter gene plasmid and the repair template, injecting the microinjection DNA mixed solution system into the hermaphrodite nematode, counting the gene editing efficiency of the hermaphrodite nematode, and verifying the consistency of the screening function and the whole gene knock-out method.)

1. A method for gene knock-out by combining precise large-fragment gene deletion with stop codon insertion, which is characterized by comprising the following steps:

Preparing a Cas9 plasmid, a co-editing marker plasmid, a reporter gene plasmid and hermaphrodite nematodes;

Determining the target point sequences of the sgRNA at the 5 'end and the 3' end;

Determining and constructing a repair template;

constructing an upstream sgRNA plasmid at a 5 'end and a downstream sgRNA plasmid at a 3' end;

preparing a microinjection DNA mixed solution: mixing the 5 'end upstream sgRNA plasmid and the 3' end downstream sgRNA plasmid, the Cas9 plasmid, the co-editing marker plasmid, the reporter gene plasmid and the repair template to obtain the microinjection DNA mixed solution;

microinjection: injecting the microinjection DNA mixed solution into gonads of hermaphrodite nematodes to obtain P0 generation nematodes, and culturing to obtain F1 generation nematodes; and

And screening and verifying the target mutation.

2. The gene knock-out method of claim 1, wherein the determined 5 'and 3' sgRNA target site sequences comprise the steps of:

Searching sgRNA target sites within 100bp near the initiation codon from a sgRNA efficiency prediction website; selecting a sgRNA target site with high editing efficiency specificity fraction and low off-target effect to obtain a 5' end sgRNA target site sequence;

searching sgRNA target sites with high specific scores of editing efficiency and low off-target effect between a 5' sgRNA target site sequence and a stop codon from a sgRNA efficiency prediction website; and obtaining the 3' sgRNA target site sequence.

3. The gene knock-out method of claim 1, wherein the 5 'and 3' ends of the precisely large-fragment gene deletion sequences are located in the 5 'sgRNA target sequence and the 3' sgRNA target sequence, respectively.

4. The gene knock-out method of claim 1, wherein the repair template is an oligonucleotide chain.

5. The method of claim 4, wherein the repair template comprises a stop codon.

6. The gene knock-out method of claim 4, wherein the stop codon on the repair template is flanked by sequences of 35-50bp 5 'upstream and 3' downstream, respectively, outside the deletion sequence of the precise large fragment gene.

7. The gene knock-out method of claim 1, wherein the microinjection DNA mixture system comprises the following:

Cas9 plasmid, 50 ng/. mu.L;

Co-editing marker plasmid, 50 ng/. mu.L;

5' end upstream sgRNA plasmid, 50 ng/. mu.L;

3' end downstream sgRNA plasmid, 50 ng/. mu.L;

reporter plasmid, 20 ng/. mu.L; and

Repair template, 20 ng/. mu.L.

Technical Field

The invention belongs to the technical field of gene editing of genetic engineering, and particularly relates to a high-efficiency gene knock-out method, namely a gene knock-out method combining precise large-fragment gene deletion with stop codon insertion.

Background

CRISPR/Cas9 gene editing is one of the current highly efficient gene targeting operation technologies, has the advantages of short period, safety, reliability, low cost and the like, and is very suitable for constructing various animal models.

the gene knockout efficiency obtained by the CRISPR/Cas9 system is very high, and the following two modes exist at present: the first editing mode is as follows: deleting the complete gene sequence from the initiation codon ("ATG") to the termination codon ("TAG") to obtain a whole gene deletion mutant; the second editing mode is as follows: and (3) editing genes by using one sgRNA in a region which is relatively close to the initiation codon, and randomly generating knock-in deletion mutation so as to obtain a frame shift mutant.

the advantage of the first editing approach is that it can be ensured that the resulting mutants are full knockout mutants, since the entire gene sequence is completely deleted (see the entire gene deletion in FIG. 1). The disadvantages are that for the gene with longer coding sequence (from the start codon to the stop codon), for example, the gene with the sequence length of more than 6kb, the efficiency of deleting large fragments is lower, and the screening difficulty is higher. In many cases, suitable sgRNA target sites are not necessarily available for editing at the 5 'end and 3' end of the deleted gene, and thus sgRNA target sites located farther away from the start codon and the stop codon are required, which is inefficient.

The advantage of the second editing approach is that random gene indel mutants can be easily generated with one sgRNA (see random gene deletion in fig. 2). The disadvantages are: (1) random gene indel mutations generated by single sgRNA editing are very short in length (a few nucleotide base pairs) and cannot be easily screened by the change in PCR fragment length when there is no phenotypic change; (2) because the generated insertion deletion mutation is random, the influence on a cDNA sequence cannot be predicted in advance, whether frame shift expression is caused or not can be generated in advance, or a stop codon is generated in advance, so that the gene expression is stopped in advance; (3) whether frameshift expression disrupts the function of the protein is still unknown, and it is not certain whether it is a knockout mutant or not.

in view of the limitation of the second editing method, it is difficult to widely popularize the method of generating gene knockout by frame shift mutation. Therefore, the editing mode of accurate knockout through whole gene deletion is still the most common gene deletion method at present, and the analysis shows that the difficulty of whole gene long fragment knockout is high, and the long period limits the use range of complete gene knockout.

Disclosure of Invention

In view of the above problems in the background art, the present invention aims to provide a gene knock-out method combining precise large-fragment gene deletion with insertion of a stop codon, so as to solve the problems of difficult and long cycle of long-fragment knockout in precise gene knock-out.

To achieve the above object, the inventors provide a gene knock-out method for precise large fragment gene deletion in combination with stop codon insertion (see large fragment deletion stop method in fig. 3), comprising the steps of:

Preparing a Cas9 plasmid, a co-editing marker plasmid, a reporter gene plasmid and hermaphrodite nematodes;

Determining the sequences of the 5 'and 3' sgRNA target sites: the 5 ' sgRNA target site is positioned near the initiation codon (within 100 bp), the 3 ' sgRNA target site is positioned at any position between the 5 ' sgRNA target site and the termination codon, the editing efficiency specificity score of the sgRNA target site is high, and the off-target effect is close to 0;

Determining and constructing a repair template;

constructing an upstream sgRNA plasmid at a 5 'end and a downstream sgRNA plasmid at a 3' end;

Preparing a microinjection DNA mixed solution: mixing the 5 'end upstream sgRNA plasmid and the 3' end downstream sgRNA plasmid, the Cas9 plasmid, the co-editing marker plasmid, the reporter gene plasmid and the repair template to obtain the microinjection DNA mixed solution;

Microinjection: injecting the microinjection DNA mixed solution into gonads of hermaphrodite nematodes to obtain P0 generation nematodes, and culturing to obtain F1 generation nematodes; and screening and verifying the target mutation.

Different from the prior art, the technical scheme at least has the following beneficial effects:

1) When the 5 'end sgRNA target site and the 3' end sgRNA target site are selected, the site with high editing efficiency of the sgRNA target site is preferentially considered, the sgRNA target site which is very close to a start codon and a stop codon is not necessarily required to be selected, the problem that the sgRNA target site which is very close to the start codon or the stop codon (within 30 bp) is low in efficiency is avoided, and in addition, the risk that a non-frame-shift mutation is generated, and a knockout mutant cannot be obtained is also avoided;

2) For a gene with a long coding sequence, all the coding sequences do not need to be deleted, and only the coding sequences with the length of less than 6kb need to be deleted, so that the editing efficiency of gene deletion is higher;

3) Because the sgRNA target site with high editing efficiency specificity fraction and low off-target effect is selected, and the 5 'end point and the 3' end point of the deleted sequence are positioned in the 5 'sgRNA target sequence and the 3' sgRNA target sequence, oligonucleotide chain can be used as a template, no plasmid is required to be constructed as the template, and the cost is greatly reduced;

4) When the gene coding sequence of a larger segment is deleted, a stop codon is inserted at the position after deletion, so that the gene is terminated in advance or mRNA is degraded, and the purpose of knocking out the gene is achieved.

Drawings

FIG. 1 is a schematic diagram of a gene knock-out method using a whole gene deletion method according to the prior art;

FIG. 2 is a schematic diagram of a gene knock-out method using random gene deletion in the background art;

FIG. 3 is a schematic diagram of a gene knock-out method for precise large fragment gene deletion in combination with stop codon insertion according to an embodiment;

FIG. 4 is a diagram showing the editing process of the wah-1 gene complete gene knock-out method in the background art;

FIG. 5 is a schematic diagram of a gene knock-out method for deletion of the wah-1 gene in combination with insertion of a stop codon according to an embodiment of the present invention;

FIG. 6 is a schematic diagram showing the editing of the gsk-3 gene by the whole gene knockout method in the background art;

FIG. 7 is a schematic diagram of a gene knock-out method combining gene deletion of the gsk-3 gene with insertion of a stop codon according to an embodiment of the present invention;

FIG. 8 is a schematic diagram of editing the tat-5 gene complete gene knock-out method in the background art;

FIG. 9 is a schematic diagram of a gene knock-out method combining gene deletion of tat-5 gene with insertion of stop codon according to the embodiment of the present invention.

Detailed Description

The gene knock-out method for the precise deletion of large fragment genes combined with the insertion of stop codons provided by the present invention is described in detail below.

A method for gene knock-out of a precise large fragment gene deletion in combination with a stop codon insertion comprising the steps of:

Preparing a Cas9 plasmid, a co-editing marker plasmid, a reporter gene plasmid and hermaphrodite nematodes;

Determining the target point sequences of the sgRNA at the 5 'end and the 3' end;

Determining and constructing a repair template;

Constructing an upstream sgRNA plasmid at a 5 'end and a downstream sgRNA plasmid at a 3' end;

preparing a microinjection DNA mixed solution: mixing the 5 'end upstream sgRNA plasmid and the 3' end downstream sgRNA plasmid, the Cas9 plasmid, the co-editing marker plasmid, the reporter gene plasmid and the repair template to obtain the microinjection DNA mixed solution;

Microinjection: injecting the microinjection DNA mixed solution into gonads of hermaphrodite nematodes to obtain P0 generation nematodes, and culturing to obtain F1 generation nematodes; and screening and verifying the target mutation.

The gene knock-out method combining precise large-fragment gene deletion with stop codon insertion is essentially that two gene editing behaviors of large-fragment gene deletion and stop codon insertion are involved in one gene editing at the same time.

The Cas9 plasmid is a plasmid for expressing Streptococcus pyogenes Cas9 (Cas 9 for short); the co-editing marker plasmid is used for identifying sgRNA plasmids of co-editing sgRNA sites; the reporter gene plasmid refers to Psur-5, sur-5, NLS, GFP plasmid; the recombinant protein is used for identifying a 5 'sgRNA target site sequence, a 3' sgRNA target site sequence, an sgRNA plasmid skeleton for co-editing a target site sequence and a common hermaphrodite nematode for gene editing, nematode lysate is a common buffer solution disclosed in a nematode research laboratory, and the above materials can be directly purchased.

selecting a sgRNA target site with highest specific score of editing efficiency and off-target effect of 0 or close to 0 at the 5 'end near the initiation codon, namely a 5' end sgRNA target site sequence; the 3 'sgRNA target site is positioned at any position between the 5' sgRNA target site and the stop codon, the specific score of the editing efficiency is high, and the off-target effect is close to 0. The levels of the specific scores of the editing efficiency are relatively high and low, the target editing genes are different, the predicted specific scores of the editing efficiency are possibly different, and in the same group of data, the target site corresponding to the highest specific score of the editing efficiency is selected as the corresponding sgRNA target site.

At present, a plurality of programs based on big data algorithm are used for predicting the efficiency and the applicability of sgRNA in gene editing, and in the specific embodiment of the invention, a website (http:// crispr. mit. edu/or http:// crispor. force. net) is adopted to obtain the target site sequences of the sgRNA at the 5 'end and the 3' end. But based on the possible error between the prediction efficiency and the actual efficiency of the prediction software, the invention also combines the sequencing results of PCR and sequencing company for verification. The gene-editing nematode was subjected to PCR using upstream and downstream primers whose binding sites were 500bp from the deleted sequence 300 and then sent to the sequencing company for sequencing.

In the invention, because the sgRNA target sites with high editing efficiency fraction and low off-target effect are preferably selected, and the 5 'end point and the 3' end point of the deleted sequence are positioned in the 5 'sgRNA target sequence and the 3' sgRNA target sequence, the gene editing cost can be greatly reduced by directly adopting an oligonucleotide chain as a template without constructing a plasmid. In a further preferred embodiment of the present invention, the repair template is an oligonucleotide chain.

The present invention is different from available technology in that it has relatively great gene coding sequence eliminated and has inserted stop codon in the position after deletion to result in early gene termination or mRNA degradation and thus gene knock-out. To achieve the above object, a repair template is used which is specifically different from a conventional repair template, and as a further preferred embodiment of the present invention, the repair template comprises a stop codon.

As a further preferred embodiment of the present invention, the stop codon on the repair template is flanked by 5 'upstream and 3' downstream sequences of 35-50bp, respectively, outside the deleted sequence. The template sequences of 35-50bp outside the deleted sequence are also called recombination arms, namely, the stop codon of the repair template and the recombination arms of 35-50bp on both sides in the scheme provided by the invention, and the sequences of the recombination arms must be the same as the sequences of 5 'upstream and 3' downstream outside the deleted sequence.

In a further preferred embodiment of the present invention, the microinjection DNA mixture system includes the following:

Cas9 plasmid, 50 ng/. mu.L;

Co-editing marker plasmid, 50 ng/. mu.L;

5' end upstream sgRNA plasmid, 50 ng/. mu.L;

3' end downstream sgRNA plasmid, 50 ng/. mu.L;

reporter plasmid, 20 ng/. mu.L; and

Repair template, 20 ng/. mu.L.

In addition, the microinjection DNA mixed solution system also contains 20 ng/mu L of template matched with the co-editing marker plasmid.

The co-editing system for screening the target mutation is a ben-164sgRNA co-editing system, and the heterozygosis or homozygosis target editing gene mutation nematode is screened by performing a conventional gene editing experiment.

And (5) sequencing to verify whether the homozygous target gene mutation nematode is correct.

to explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application.