WO2019237395A1

WO2019237395A1 - Method for targeted knockout of human cd357 gene by applying crispr/cas9 system

Info

Publication number: WO2019237395A1
Application number: PCT/CN2018/091725
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2018-06-16
Filing date: 2018-06-16
Publication date: 2019-12-19

Abstract

Provided is a method for the targeted knockout of a human CD357 gene by applying a CRISPR/Cas9 system. The method comprises: designing a gRNA according to the PAM design principle of gRNA by means of analyzing the specifics of a human CD357 gene sequence, linking the gRNA with a px459 vector to obtain a recombinant vector for the targeted knockout of the human CD357 gene, and using the gRNA in Jurkat cells to guide a CRISPR/Cas9 system to knock out the human CD357 gene.

Description

Method for targeted knockout of human CD357 gene using CRISPR / Cas9 system

Technical field

The invention relates to the field of molecular biology, in particular to a method for targeted knockout of human CD357 gene by using CRISPR / Cas9 system.

Background technique

CD357 is the eighteenth member of the tumor necrosis factor receptor (TNFR) superfamily. It is a surface molecule on CD4 + CD25 + Treg cells derived from thymus and its ligand is TNFSF18. TNFSF18 / CD357 has many important biological activities, including cell proliferation, differentiation, and survival.

technical problem

The TNFSF18 / CD357 system participates in the role of Treg cells in immunoregulation, plays an important role in tumor immunotherapy, and has good clinical transformation prospects, but the prior art lacks a means to target CD357 gene knockout. Related research Progress has caused certain obstacles.

Technical solutions

In view of the above problems, the present invention provides a method for targeted knockout of human CD357 gene by using CRISPR / Cas9 system. The present invention is implemented as follows:

A method for using the CRISPR / Cas9 system to target the knockout of human CD357 gene. The specific steps are as follows:

(1) Construct the pxCD357 vector as follows:

a According to the sequence of human CD357 gene and the PAM design rules of gRNA, design gRNA targeting CD357;

b. The px459 plasmid was digested by Bbs I, and the digested product was recovered by electrophoresis;

c. ligating the gRNA targeting CD357 with the px459 vector after digestion with T4 DNA ligase to obtain the pxCD357 vector;

d. Transforming the pxCD357 vector into E. coli Stbl3, cultivating and picking out the monoclonal bacteria for sequencing identification.

(2) Take the sequenced recombinant Stbl3 E. coli, and incubate at 37 ° C, 250 rpm for 14 h, then extract the pxCD357 vector with an endotoxin-free plasmid extraction kit.

(3) Liposomes transduce pxCD357 into the recipient cells. The recipient cells were screened for 7 days at a working concentration of puromycin to expand the recipient cells and extract genomic DNA. The PCR products were recovered by electrophoresis after PCR amplification, and then T7 Endonuclease was used. I Digest the product and identify it by electrophoresis.

Further, the reaction temperature of the BBs I digestion system was 37 ° C., the time was 2 hours, and the digestion system was: 2 μg of px459 plasmid, 3 μl of 10 × FastDigest Buffer, 3 μl of Bbs I, and made up to 30 μl with water.

Further, the liposome is Lipofectamine 2000.

Further, the recipient cells are selected from Jurkat cells.

Further, the T7 Endonuclease I reaction system is: 1 μl 10 × T7E1 Buffer, 1 μl T7 Endonuclease I, 1 μg of recovered PCR product, made up to 10 μl with water. The reaction conditions were: 37 ° C water bath for 1 h.

The above-mentioned method using the CRISPR / Cas9 system to target human CD357 gene knockout can be used for human CD357 gene knockout.

Beneficial effect

The method for applying the CRISPR / Cas9 system to target the knockout of human CD357 gene provided by the present invention provides an experimental technology platform for further exploring the role of CD357 gene, and can be used in the research and development of drugs related to abnormal expression of CD357 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the T7 Endonuclease of Jurkat cells in the control and experimental groups. I test results chart.

Embodiments of the invention

实施例一靶向敲除基因克隆载体的构建Example 1 Construction of a Targeted Knockout Gene Cloning Vector

a. According to the sequence of human CD357 gene and the PAM design rules of gRNA, a gRNA targeting CD357 was designed, and its upstream sequence was 5’-ACCGATGGCACAGCACGGGGCGAT -3 ', the downstream sequence is 5'-AAACATCGCCCCGTGCTGTGCCAT-3'. After dissolving, 5 μl of each was mixed, heated at 95 ° C. for 5 minutes, and then naturally cooled to room temperature to form a double-stranded DNA with Bbs I sticky ends.

b. The px459 plasmid was digested with Bbs I, and the digested product was subjected to agarose gel electrophoresis. The gel was recovered and the concentration was determined.

c. Use T4 DNA ligase to ligate a and b products. The ligated product was then transformed into E. coli Stbl3 and identified by sequencing.

实施例二无内毒素质粒Example 2 Endotoxin-free plasmid DNADNA 的制备Preparation

The correct strain was sequenced and identified in Example 1, and placed in an LB liquid medium having an ampicillin concentration of 100 μg / ml, and cultured at 250 rpm and 37 ° C with shaking for 12-16 hours. Collect the bacterial solution by centrifugation at 4 ° C and 10,000 rpm, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the endotoxin-free pxCD357 plasmid.

实施例三Example three JurkatJurkat 细胞的转导Cell transduction

One day before transduction, Jurkat cells were seeded in a 35 mm petri dish with a seeding density of 50% and DMEM containing 10% fetal bovine serum (FBS) under 37 ° C and 5% CO2. On the day of transduction, 2 μg of pxCD357 plasmid was introduced according to the instructions of the Lipofectamine 2000 kit. 48 hours after transfection, 1 μg / ml puromycin was added to screen for 7 d. After the screening was completed, the concentration of puromycin was reduced to 0.5 μg / ml and the cells were expanded.

实施例四Example 4 T7 Endonuclease I T7 Endonuclease I 试验检测转导效果Test to detect transduction effect

Untreated Jurkat cells and Jurkat cells introduced with pxCD357 plasmid were inoculated into six-well plates. After the cells were full, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to amplify the expected sites of gene editing. The PCR product was recovered.

After re-annealing the PCR products, T7 Endonuclease I was digested for 1 h at 37 ° C and then subjected to agarose gel electrophoresis. The results are shown in Figure 1. Compared with the control group, the experimental group showed two distinct cutting strips. The band indicates that the gRNA sequence targeting the CD357 gene guided the CRISPR / Cas9 system to successfully edit the human CD357 gene.

Industrial applicability

Claims

A method for applying the CRISPR / Cas9 system to target the human CD357 gene is characterized in that it includes the following steps:

(1) Construct the pxCD357 vector as follows:

A. According to the sequence of human CD357 gene and the PAM design rules of gRNA, design the gRNA targeting CD357;

B. Bbs I digests the px459 plasmid and recovers the digested product by electrophoresis;

C. The T4 DNA ligase is used to ligate the gRNA targeting CD357 with the digested px459 vector to obtain the pxCD357 vector;

D. Transforming the pxCD357 vector into E. coli Stbl3, culturing and picking out the monoclonal bacteria for sequencing identification.

(2) Take the sequenced recombinant Stbl3 E. coli, and incubate at 37 ° C, 250 rpm for 14 h, then extract the pxCD357 vector with an endotoxin-free plasmid extraction kit.

(3) Liposomes transduce pxCD357 into the recipient cells. The recipient cells were screened for 7 days at a working concentration of puromycin to expand the recipient cells and extract genomic DNA. The PCR products were recovered by electrophoresis after PCR amplification, and then T7 Endonuclease I was used. The product was digested with enzyme and identified by electrophoresis.
The method according to claim 1, wherein the upstream sequence of the gRNA targeting CD357 is 5'- ACCGATGGCACAGCACGGGGCGAT -3 ', the downstream sequence is 5'-AAACATCGCCCCGTGCTGTGCCAT-3'.
The method for applying the CRISPR / Cas9 system to target human CD357 gene knockout according to claim 1, wherein the liposome is Lipofectamine 2000.
The method for applying the CRISPR / Cas9 system to target human CD357 gene knockout according to claim 1, wherein the recipient cell is a Jurkat cell.
The method for applying the CRISPR / Cas9 system to target the human CD357 gene according to any one of claims 1-4, wherein the method for applying the CRISPR / Cas9 system to target the human CD357 gene can be applied to humans. CD357 gene knockout.