阅读说明：本技术 高通量crispr基因编辑工具载体构建方法 (Construction method of high-throughput CRISPR gene editing tool vector ) 是由 杨延辉 林�源 田静 何龙 杨林 王大军 杨丽 唐静 李晓雨 马凯 杨玉玛 刘 于 2021-09-19 设计创作，主要内容包括：本发明公开了一种高通量CRISPR基因编辑工具载体构建方法,包括如下步骤：S1、引物设计及合成；S2、引物退火；S3、sgRNA重组质粒构建；S4、转化得到24份2μl的混合物；S5、筛选单克隆；S6、质粒提取及测序。该方法优化了重组质粒的构建步骤和转化实验的孵育步骤,同时缩短了质粒构建和转化实验的时间提高了转化效率和阳性率,减少了实验成本,在筛选单克隆过程中替换了传统的涂布棒法,为最终实现自动化、高通量sgRNA载体构建奠定了基础；解决了酶切和连接的矛盾,将不可能统一的体系实现了统一,该方法不仅能够应用于基因编辑sgRNA的构建,还可以应用于载体构建的无缝克隆技术。(The invention discloses a construction method of a high-throughput CRISPR gene editing tool vector, which comprises the following steps: s1, designing and synthesizing a primer; s2, primer annealing; s3, constructing sgRNA recombinant plasmids; s4, obtaining 24 parts of 2 mu l mixture through conversion; s5, screening single clones; s6, plasmid extraction and sequencing. The method optimizes the construction steps of the recombinant plasmid and the incubation steps of the transformation experiment, shortens the time of the plasmid construction and the transformation experiment, improves the transformation efficiency and the positive rate, reduces the experiment cost, replaces the traditional coating rod method in the process of screening monoclonal, and lays a foundation for finally realizing the construction of the automated and high-flux sgRNA vector; the method solves the contradiction between enzyme digestion and connection, realizes unification of systems which cannot be unified, and can be applied to the construction of sgRNA for gene editing and the seamless cloning technology for vector construction.)

1. A construction method of a high-throughput CRISPR gene editing tool vector is characterized in that: the method comprises the following steps:

s1, designing and synthesizing a primer;

s2, primer annealing;

s3, constructing sgRNA recombinant plasmids;

s4, conversion to give 24 parts of a 2 μ l mixture:

s41, transferring 24 parts of 2. mu.l of each ligation-derived mixture into 24 parts of 50. mu.l of competent cell Escherichia coli DH 5. alpha. and thawing DH 5. alpha. in advance on ice for 3-5min to obtain qualified cells, followed by gently mixing;

s42, incubating the mixture on ice for 30 min;

s43, thermally shocking for 90S at 42 ℃, then immediately putting the tube back on ice, and keeping on the ice for 5 min;

s5, coating and screening a monoclonal antibody;

s51, adding 100 mu l of LB liquid culture medium into each of 24 transformation liquid in the step S43, diluting by 3 times, taking out solid culture medium poured in advance and containing a benzyl penicillin 24-hole plate, wherein each hole contains 800 mu l-1000 mu l of culture medium, placing 3-4 sterile glass beads with the diameter of 1mm in each hole, adding 10 mu l of corresponding culture medium into each hole, covering a 24-hole plate cover, slowly shaking in a vertical and horizontal mode for 3-5 times respectively, taking out the glass beads, and finally placing the 24-hole plate in an incubator at 37 ℃ for overnight culture (<18 h);

s52, observing whether the plate has bacterial colony on the next day;

s6, plasmid extraction and sequencing.

2. The method for constructing the high-throughput CRISPR gene editing tool vector as claimed in claim 1, wherein the method comprises the following steps: the primer annealing in the step S2 specifically comprises the following steps: placing the system in a PCR instrument at 100 ℃ for 10min, and then placing the system at room temperature for natural cooling to room temperature; primer T1. mu.L (100. mu. mol/L), Primer B1. mu.L (100. mu. mol/L), ddH₂O98. mu.L, total volume 100. mu.L.

3. The method for constructing the high-throughput CRISPR gene editing tool vector as claimed in claim 1, wherein the method comprises the following steps: the sgRNA recombinant plasmid in the step S3 comprises lentiCRISPRV20.3 μ L (151ng/μ L), target gene 0.5 μ L (1 μmol/L), T4DNA ligase buffer solution 0.2 μ L, endonuclease Bsmbi 0.1 μ L, Hi-T4^TMDNA thermostable ligase 0.1. mu.l, distilled water 0.8. mu.l;

placing the sgRNA recombinant plasmid in a PCR instrument, and performing PCR at 50 ℃ for 20 min; after three cycles of 10min at 37 ℃ the empty plasmid in the system was completely excised 30min at 50 ℃.

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to a construction method of a high-throughput CRISPR gene editing tool vector.

Background

The CRISPR-Cas9 gene editing technology is the most widely applied gene operation tool at present. Vector construction to ligate sgrnas is the most basic and important experimental procedure for this technology. However, classical vector construction has the disadvantages of complicated steps, time consumption and incapability of realizing high throughput. Therefore, a construction method of a high-throughput CRISPR gene editing tool vector is provided.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a construction method of a high-throughput CRISPR gene editing tool vector.

In order to achieve the purpose, the invention provides the following technical scheme: a construction method of a high-throughput CRISPR gene editing tool vector comprises the following steps:

s1, designing and synthesizing a primer;

s2, primer annealing: placing the system in a PCR instrument at 100 ℃ for 10min, and then placing the system at room temperature for natural cooling to room temperature; (Primer T1. mu.L (100. mu. mol/L), Primer B1. mu.L (100. mu. mol/L), ddH2O 98. mu.L, total 100. mu.L);

s3, construction of sgRNA recombinant plasmids:

each reaction system

S4, transformation:

s41, transferring 24 parts of each ligation-derived 2. mu.l mixture into 24 parts of 50. mu.l competent cell E.coli DH 5. alpha. cells, thawing DH 5. alpha. cells on ice in advance (3-5min) as qualified cells, and gently mixing;

s42, incubating the mixture on ice for 30 min;

s43, thermally shocking for 90S at 42 ℃, and immediately putting the tube back on ice; keeping it on ice for 5 min;

s5, coating and screening a monoclonal antibody;

s51, adding 100 ul LB liquid culture medium (dilution multiple is 3 times) into each of the 24 transformation liquid in the step S43, taking out the solid culture medium (each well has about 800 ul-1000 ul culture medium) of 24-well plate containing ampicillin poured in advance, placing 3-4 aseptic glass beads with diameter of 1mm in each well, adding 10 ul of diluted culture medium into each well, covering the 24-well plate, slowly shaking 3-5 times in a vertical and horizontal manner respectively, taking out the glass beads, and finally placing the 24-well plate in an incubator at 37 ℃ for overnight culture (<18 h);

s52, observing whether the plate has bacterial colony on the next day;

s6, plasmid extraction and sequencing.

The invention develops a technology for realizing enzyme digestion connection in a micro reaction system by adopting heat-resistant T4 ligase, and optimizes the construction method of the sgRNA vector edited by the CRISPR-Cas9 gene by overcoming the incompatible contradiction of enzyme digestion and connection. The cloning method is proved to have high positive rate, time saving and cost saving, and can be completed by a PCR instrument. The method lays a foundation for realizing automatic and high-throughput construction of the sgRNA vector and development of a seamless connection method kit.

Compared with the prior art, the invention has the beneficial effects that: compared with the prior art, the method optimizes the construction steps of recombinant plasmids and the incubation steps of transformation experiments, shortens the time of vector construction and transformation, improves the transformation efficiency and the positive rate, reduces the volume of experimental reagents, reduces the experimental cost, and lays a foundation for finally realizing the construction of an automated and high-throughput sgRNA vector;

the method solves the contradiction between enzyme digestion and connection, realizes unification of systems which cannot be unified, and can be applied to the construction of sgRNA for gene editing and the seamless cloning technology for vector construction.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A construction method of a high-throughput CRISPR gene editing tool vector comprises the following steps:

s1, designing and synthesizing a primer;

s2, primer annealing: placing the system in a PCR instrument at 100 ℃ for 10min, and then placing the system at room temperature for natural cooling to room temperature; (Primer T1. mu.L (100. mu. mol/L), Primer B1. mu.L (100. mu. mol/L), ddH2O 98. mu.L, total 100. mu.L);

s3, construction of sgRNA recombinant plasmids:

each reaction system

S4, transformation:

s41, transferring 24 parts of each ligation-derived 2. mu.l mixture into 24 parts of 50. mu.l competent cell E.coli DH 5. alpha. cells, thawing DH 5. alpha. cells on ice in advance (3-5min) as qualified cells, and gently mixing;

s42, incubating the mixture on ice for 30 min;

s43, thermally shocking for 90S at 42 ℃, and immediately putting the tube back on ice; keeping it on ice for 5 min;

s5, coating and screening a monoclonal antibody;

s51, adding 100 ul LB liquid culture medium (dilution multiple is 3 times) into each of the 24 transformation liquid in the step S43, taking out the solid culture medium (each well has about 800 ul-1000 ul culture medium) of 24-well plate containing ampicillin poured in advance, placing 3-4 aseptic glass beads with diameter of 1mm in each well, adding 10 ul of diluted culture medium into each well, covering the 24-well plate, slowly shaking 3-5 times in a vertical and horizontal manner respectively, taking out the glass beads, and finally placing the 24-well plate in an incubator at 37 ℃ for overnight culture (<18 h);

s52, observing whether the plate has bacterial colony on the next day;

s6, plasmid extraction and sequencing.

Adding a primer, a vector, a restriction endonuclease, T4 ligase, T4 ligase buffer solution and deionized water into a reaction system according to a certain proportion, and connecting the vector and the primer through the dual functions of the endonuclease and the T4 ligase at a certain temperature to finally obtain the recombinant vector.

Specifically verifying: verifying recombinant colonies, selecting 5 colonies randomly from the two groups of plates A and B, and separately scribing on a plate containing ampicillin to verify whether the recombinant colonies are transformed into plasmids or not, wherein five lines of the two plates have bacterial growth to prove that the whole plasmids are transformed into competent bacteria;

sequencing and identification of target genes 3 single colony strains selected from group A and group B were sent to Beijing Rui Boxing Ke Biotech limited to use primers for target regions: gagggcctatttcccatgatt, respectively; the fragment of interest (25/26bp) was sequenced in 6 single colonies and the sequencing was aligned with the empty plasmid sequence.

In summary, the following steps: compared with the prior art, the construction method of the high-throughput CRISPR gene editing tool vector optimizes the construction steps of recombinant plasmids and the incubation steps of transformation experiments, shortens the time of plasmid construction and transformation experiments, improves the transformation efficiency and the positive rate, reduces the experiment cost, replaces the traditional coating rod method in the process of screening single clones, and lays a foundation for finally realizing the construction of an automated and high-throughput sgRNA vector;

the method solves the contradiction between enzyme digestion and connection, realizes unification of systems which cannot be unified, and can be applied to the construction of sgRNA for gene editing and the seamless cloning technology for vector construction.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.