阅读说明：本技术 一种减少CRISPR-Cas9基因编辑中双链DNA片段串联的方法及其应用 (Method for reducing tandem connection of double-stranded DNA (deoxyribonucleic acid) fragments in CRISPR-Cas9 gene editing and application thereof ) 是由 官敏 朱石磊 林梓凡 于 2020-08-04 设计创作，主要内容包括：本发明提供一种减少CRISPR-Cas9基因编辑中双链DNA片段串联的方法及其应用。所述方法包括如下步骤：在双链DNA的5′端连接自杀基因,3′端连接自杀基因的启动子,再将所述双链DNA与CRISPR-Cas9反应体系混合,转入待编辑的细胞中完成CRISPR-Cas9基因编辑。将自杀基因元件及其启动子元件分别连接在dsDNA的两端,当发生DNA串联时,启动子和自杀基因串联在一起,诱导自杀基因表达,从而杀死宿主细胞,使宿主细胞死亡从而消除DNA串联现象。(The invention provides a method for reducing tandem connection of double-stranded DNA (deoxyribonucleic acid) fragments in CRISPR-Cas9 gene editing and application thereof. The method comprises the following steps: connecting a suicide gene at the 5 'end of the double-stranded DNA, connecting a suicide gene promoter at the 3' end, mixing the double-stranded DNA with a CRISPR-Cas9 reaction system, and transferring the double-stranded DNA into a cell to be edited to finish CRISPR-Cas9 gene editing. The suicide gene element and the promoter element thereof are respectively connected to two ends of dsDNA, when the DNA tandem connection occurs, the promoter and the suicide gene are connected in series to induce the suicide gene to express, thereby killing host cells, leading the host cells to die and eliminating the DNA tandem connection phenomenon.)

1. A method of reducing tandem of double-stranded DNA fragments in CRISPR-Cas9 gene editing, comprising the steps of:

connecting a suicide gene at the 5 'end of the double-stranded DNA, connecting a suicide gene promoter at the 3' end, mixing the double-stranded DNA with a CRISPR-Cas9 reaction system, and transferring the double-stranded DNA into a cell to be edited to finish CRISPR-Cas9 gene editing.

2. The method of claim 1, wherein the suicide gene comprises DTA or TK, and the promoter of the suicide gene comprises any one of CAG, CMV, EF1a or PGK.

3. The method of claim 2, wherein the suicide gene is DTA and the promoter of the suicide gene is CMV.

4. The method according to claim 3, wherein the nucleotide sequence of the suicide gene is shown as SEQ ID No.1, and the nucleotide sequence of the promoter of the suicide gene is shown as SEQ ID No. 2.

5. The method according to claim 1, wherein the concentration of the double-stranded DNA mixed with the CRISPR-Cas9 reaction system is 2-10 ng/μ L;

the CRISPR-Cas9 reaction system includes sgRNA and Cas9 mRNA;

the working concentration of the sgRNA is 2-8 ng/mu L;

the working concentration of the Cas9 mRNA is 5-15 ng/. mu.L.

6. The method according to claim 1, wherein the double-stranded DNA is obtained by digesting the vector with restriction enzyme I and restriction enzyme II, wherein the restriction enzyme I and the restriction enzyme II are homologous overhang non-homocerclase or non-homologous overhang enzyme.

7. The method according to claim 6, wherein the restriction enzyme I and the restriction enzyme II are non-homomeric overhang enzymes, the cohesive end formed after the restriction enzyme I is cut by the restriction enzyme I is 5 '-3', and the cohesive end formed after the restriction enzyme II is cut by the restriction enzyme II is 3 '-5';

or the restriction enzyme I and the restriction enzyme II are non-homologous overhang enzymes, the cohesive end formed after the restriction enzyme I is cut by the enzyme I is 3 '-5', and the cohesive end formed after the restriction enzyme II is cut by the enzyme II is 5 '-3'.

8. The method of claim 1, wherein the cell to be edited comprises a mouse zygote.

9. Method according to claim 1, characterized in that it comprises the following steps:

connecting a suicide gene at the 5 'end of the double-stranded DNA, connecting a suicide gene promoter at the 3' end, and mixing the double-stranded DNA with a CRISPR-Cas9 reaction system;

the working concentration of sgRNA in the CRISPR-Cas9 reaction system is 2-8 ng/mu L, the working concentration of Cas9 mRNA is 5-15 ng/mu L, and the concentration of the double-stranded DNA mixed with the CRISPR-Cas9 reaction system is 2-10 ng/mu L;

then, the cells are transferred into mouse fertilized eggs to obtain gene-edited fertilized egg cells.

10. Use of the method of any one of claims 1 to 9 in constructing a CRISPR-Cas9 gene editing method.

Technical Field

The invention relates to the field of genetic engineering, and relates to a method for reducing tandem connection of double-stranded DNA (deoxyribonucleic acid) fragments in CRISPR-Cas9 gene editing.

Background

Suicide gene (suicide gene) refers to a gene of some viruses or bacteria, which is introduced into a target cell, and the expressed product is a toxic substance or can catalyze a nontoxic precursor to be converted into a toxic substance, thereby causing a recipient cell carrying the gene to be killed. DTA and TK are two suicide genes commonly used in eukaryotic cells, wherein DTA can generate toxic substances without adding a substrate, and TK can play a suicide role without adding a substrate. At present, suicide genes are often used for treating tumors and infectious diseases, and are also often used as negative screens in genetic engineering.

With the intensive research and interpretation of Non-Homologous end joining (NHEJ) and Homologous Recombination (HR) repair methods, DNA repair mechanisms are gradually applied to gene editing techniques for the targeted modification of genes. Researchers use nuclease technology to purposefully break the double strand of DNA, and use this mechanism to regulate the expression of a target gene or introduce a selectable marker.

CRISPR-Cas9-mediated homology directed DNA repair is the first method of precise gene editing in a variety of model organisms including mice and humans. The Cas9 protein cuts a DNA Double strand to form DNA Double Strand Breaks (DSBs), and a NHEJ repair mechanism is utilized to randomly delete or insert a base to cause frameshift mutation, so that a target gene is knocked out; introducing a section of exogenous gene while shearing, and accessing the exogenous gene into DSBs sites by NHEJ to realize gene insertion; or the two ends are respectively provided with a shearing site, the middle segment is free, and the two ends are connected through an NHEJ mechanism to form large-segment gene knockout. Meanwhile, a foreign gene and a homologous sequence are added during shearing, and the gene can be accurately knocked in and replaced at a fixed point under the action of an HR mechanism. The method is widely used in the biomedical field at present, so that the editing is more efficient and the sequence is more specific.

Nevertheless, this technique still has drawbacks. Researches show that in the process of establishing six knockout mouse models under different conditions, the donor DNA template has multiple unnecessary head-tail tandem phenomena. In most cases, these multiple integration events are not recognized by traditional PCR analysis, which seriously affects the accuracy of the technique (see Skryabin BV, et al. Pervasive head-to-tails of DNA templates determined CRISPR-Cas9-mediated genome editing events. Sci adv. 2020;6(7): eaax 2941.).

In the CRISPR-Cas9 gene targeting process, the recombinant DNA fragments have a head-to-tail tandem phenomenon, and the tandem phenomenon causes the gene targeting failure. In the field, Southern, qPCR, PCR and other methods are commonly used to detect whether there is a tandem, but no effective method for reducing the tandem has been found.

Therefore, the suicide gene is used for reducing the tandem rate of the recombinant DNA fragment in the CRISPR-Cas9 gene targeting, and the method has important significance for the development and the improvement of the CRISPR-Cas9 gene editing technology.

Disclosure of Invention

Suicide gene (suicide gene) refers to a gene of some viruses or bacteria, which is introduced into a target cell, and the expressed product is a toxic substance or can catalyze a nontoxic precursor to be converted into a toxic substance, thereby causing a recipient cell carrying the gene to be killed. DTA and TK are two suicide genes commonly used in eukaryotic cells, wherein DTA can generate toxic substances without adding a substrate, and TK can play a suicide role without adding a substrate. At present, suicide genes are often used for treating tumors and infectious diseases, and are also often used as negative screens in genetic engineering.

With the intensive research and interpretation of Non-Homologous end joining (NHEJ) and Homologous Recombination (HR) repair methods, DNA repair mechanisms are gradually applied to gene editing techniques for the targeted modification of genes. Researchers use nuclease technology to purposefully break the double strand of DNA, and use this mechanism to regulate the expression of a target gene or introduce a selectable marker.

CRISPR-Cas9-mediated homology directed DNA repair is the first method of precise gene editing in a variety of model organisms including mice and humans. The Cas9 protein cuts a DNA Double strand to form DNA Double Strand Breaks (DSBs), and a NHEJ repair mechanism is utilized to randomly delete or insert a base to cause frameshift mutation, so that a target gene is knocked out; introducing a section of exogenous gene while shearing, and accessing the exogenous gene into DSBs sites by NHEJ to realize gene insertion; or the two ends are respectively provided with a shearing site, the middle segment is free, and the two ends are connected through an NHEJ mechanism to form large-segment gene knockout. Meanwhile, a foreign gene and a homologous sequence are added during shearing, and the gene can be accurately knocked in and replaced at a fixed point under the action of an HR mechanism. The method is widely used in the biomedical field at present, so that the editing is more efficient and the sequence is more specific.

Nevertheless, this technique still has drawbacks. Researches show that in the process of establishing six knockout mouse models under different conditions, the donor DNA template has multiple unnecessary head-tail tandem phenomena. In most cases, these multiple integration events are not recognized by conventional PCR analysis, which seriously affects the accuracy of the technique (see Skryabin BV, et al. Pervasive head-to-tail insertions of DNA templates determined CRISPR-Cas9-mediated genome editing events. Sci adv. 2020;6(7): eaax 2941.).

In the CRISPR-Cas9 gene targeting process, the recombinant DNA fragments have a head-to-tail tandem phenomenon, and the tandem phenomenon causes the gene targeting failure. In the field, Southern, qPCR, PCR and other methods are commonly used to detect whether there is a tandem, but no effective method for reducing the tandem has been found.

Therefore, the suicide gene is used for reducing the tandem rate of the recombinant DNA fragment in the CRISPR-Cas9 gene targeting, and the method has important significance for the development and the improvement of the CRISPR-Cas9 gene editing technology.

Drawings

FIG. 1 is a schematic diagram of the structure of dsDNA fragments constructed in example 1.

FIG. 2 is a micrograph of dsDNA injected into pronuclei as in example 1.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

In the following examples, female (C57 BL/6JGpt, 4 weeks old), male (C57 BL/6JGpt, >8 weeks old) mice: all produced by Jiangsu Jiejiaokang Biotech limited;

in the following examples, reagents used included: hyaluronidase working solution, DPBS working solution, injection buffer solution, PMSG/HCG working solution, M16, M2, 75% alcohol and mineral oil; the apparatus used comprises: inverted microscope, micro-operation system, micro sample loader, needle drawing instrument, needle forging instrument, and CO₂Incubator, refrigerator, stereomicroscope; the consumables used include: GD-1 glass needle, microsyringe, 3.5cm dish, fixed tube, fallopian tube, 1mL syringe, surgical instrument, etc.; are all commercially available from conventional sources.

In the following examples, the remaining steps can be performed by means of techniques commonly used in the art, such as digestion, purification, etc., except for specific descriptions.