WO2019237387A1 - Grna sequence for knocking out human act35 gene and use thereof - Google Patents

Abstract

Provided is a gRNA sequence for knocking out the human cell ACT35 gene. In cells, the gRNA is capable of specifically binding to the sense strand (438-457 bp) of the human ACT35 DNA, and forming a complex with Cas9, and specifically cleaving the nucleic acid sequence of the ACT35 gene. By using the cell non-recombinant end connection repair mechanism, frameshift mutation of the ACT35 gene occurs, so as to obtain ACT35 gene-knockout cells.

Description

GRNA sequence for knocking out human ACT35 gene and application thereof Technical field

 The invention belongs to the technical field of genetic engineering and gene editing, and particularly relates to a gRNA sequence for knocking out the human ACT35 gene and application thereof.

Background technique

ACT35 is a member of the TNF receptor superfamily, a type I transmembrane glycoprotein. The expression profile of ACT35 is limited to the surface of activated CD4 + and CD8 + T cells, and mainly CD4 + T cells. Human ACT35 ligand contains 183 amino acids (139 amino acids extracellularly, 21 amino acids transmembrane, 23 amino acids intracellular), and is a type II transmembrane glycoprotein. ACT35 / TXGP1 is an important pair of co-stimulatory molecules that play an important role in the body's immune response and various diseases. Their interactions can promote the activation, proliferation, and migration of CD + 4 T cells, extend their life span, and promote germination. The formation of centers and the differentiation of DCs mature.

technical problem

ACT35 plays an important role in the immunotherapy of tumors, and its potential clinical transformation value is great. It needs solid research before it can be put into practical application. However, the existing technology lacks the means of knocking out the ACT35 gene expression. Progress has created some obstacles.

Regularly spaced clustered short palindrome repeats Regularly Interspaced Short Palindromic Repeats (CRISPR) is a series of clustered DNA Sequences are a system that bacteria use to protect themselves against viruses and a genetic weapon against attackers. The Cas gene encodes a protein that contains nucleases, polymerases, helicases, and domains that bind to ribonucleic acid.

The RNA transcribed by CRISPR combines with the Cas protein to form a ribonucleoprotein complex that cooperates with the immune function of the CRISPR / Cas system to guide the Cas protein. Therefore, this RNA is also called guide RNA. When the invading virus DNA and gRNA sequences are identical, the Cas protein in the complex can cut the invading virus DNA to achieve the purpose of defense. Therefore, you only need to synthesize a guide RNA-oriented DNA sequence for the DNA sequence that needs to be edited. After being transferred into the host cell, the artificially constructed gRNA can guide the Cas9 protein to accurately cut the specific DNA sequence of the host cell to play a gene. The role of the editor.

Technical solutions

The purpose of the present invention is to overcome the defects existing in the prior art, provide a gRNA sequence for knocking out the human ACT35 gene, and construct a corresponding gene knockout vector px458-ACT35, so as to lay a foundation for the subsequent study of the function of the human ACT35 gene.

Its specific technical solution is:

A gRNA sequence for knocking out the ACT35 gene in human cells and application thereof include the following steps:

(1) Synthesize the target DNA sequence and its complementary strand artificially, recombine the nucleic acid fragment into the px458 plasmid after annealing to obtain the px458-ACT35 recombinant plasmid, transform E. coli NEBStable, screen and culture ampicillin, and select a monoclonal strain for sequencing identification;

(2) A large number of cultured and correctly sequenced Escherichia coli, endotoxin-free extraction of px458-ACT35 recombinant plasmid;

(3) The transfected cell culture medium was changed to a serum-free medium, and the px458-ACT35 recombinant plasmid was prepared into a transfection mixture with Opti-MEM medium and PEI, added to the transfected cell culture medium, and changed after 5 hours. After transfected cell culture medium was replaced with serum medium and cultured for 48 h, transfected cells with ACT35 gene knockout were obtained.

Beneficial effect

The gRNA sequence of the human ACT35 gene knockout provided by the present invention and its application can play an important role in the research and development of ACT35-related drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

 Figure 1 is a plasmid map of the px458 vector;

Figure 2 shows the results of Western Blot detection of ACT35 protein in control and experimental K562 cells.

Embodiments of the invention

The invention is further described below with reference to the drawings and specific embodiments.

E. coli NEBStable and T4 DNA ligase were purchased from NEB, px458 plasmid was purchased from Addgene, Bbs I endonuclease was purchased from Fermentas, PEI was purchased from Sichuan Best, Opti-MEM medium was purchased from Invitrogen, endotoxin-free plasmid extraction reagent The Endo-Free Plasmid Mini Kit was purchased from Omega bio-tek.

Example one  px458-ACT35 Plasmid construction

Synthetic nucleotide sequence 5’- CACCAACACGGTGTGCCGTCCGTG -3 ’, and its reverse complementary sequence 5’- AAACCACGGACGGCACACCGTGTT -3 '. Each of the two nucleotide sequences was prepared to 100 μmol / l with deionized bacteria water, placed in 600 ml of boiling water, and cooled and annealed at room temperature to form a double-stranded gRNA sequence.

Bbs I double-digested the px458 plasmid, mixed it with the gRNA sequence 1: 5 after recovery, and ligated T4 DNA ligase overnight at 16 ° C. Escherichia coli NEBStable was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing.

Example two K562 Cell transduction

A 100 μg / ml ampicillin-containing LB medium was used to culture a large number of correctly sequenced E. coli at 37 ° C. The px458-ACT35 recombinant plasmid was extracted without endotoxin.

K562 cells were seeded into a six-well plate one day before transfection at a seeding density of 50%. Transduction: Take 1 μg px458-ACT35 recombinant plasmid and 3 μl PEI (1 μg / μl) dissolved in 100 μl Opti-MEM medium, vortex and mix. The medium in the six-well plate was changed to serum-free medium, 2 ml per well, 600 μl per well was added to the transfection mixture, and 5 hours was replaced with DMEM medium preheated with 10% fetal bovine serum at 37 ° C. Incubate at 37 ° C for 5% CO2, 90% humidity for 48 h, and collect cells.

Example three Zh cell ACT35 Mutation identification

The genomic DNA of the cells collected in Example 2 was extracted, and then sequencing primers were designed based on the sequence of human ACT35 DNA 400-500 bp position to sequence the genomic DNA at the target position.

Example 4 Zh cell ACT35 Detection of protein expression

K562 cells without any treatment were used as the control group, and the K562 cells collected in Example 2 were used as the experimental group. 100-200 μl of 5 × SDS-PAGE loading buffer was added, and the mixture was boiled in boiling water for 5 minutes. 15 μl of the sample was loaded. SDS-PAGE protein electrophoresis. After the electrophoresis was completed, the protein was semi-dried and blocked with 10% skim milk powder for 2 h. The blocked PVDF membrane was placed in rabbit anti-human ACT35 antibody (1: 3000, Abcam / ab76000), and the buffer solution was 10% skim milk powder. Incubate on ice overnight, then rinse four times with rinsing buffer for 5 minutes, then transfer the membrane to 0.05M PBS, pH 7.2, 0.2% Tween-20, 1: 50000 diluted goat anti-rabbit secondary antibody buffer (Abcam / ab97051), incubate at room temperature for 60 min, and then rinse four times with rinsing buffer for 5 min each. After the rinsing was completed, the Western blot membrane was developed and detected by ECL, and the results are shown in FIG. 2. It can be seen that the ACT35 protein band cannot be detected by Western Blot in the ACT35 frameshift mutant K562 cells, while the ACT35 protein band appears in the control group, indicating that the gRNA sequence used for knocking out the ACT35 gene in human cells can achieve ACT35. Gene knockout.

Industrial applicability

The gRNA sequence of the human ACT35 gene knockout provided by the present invention and its application can play an important role in the research and development of ACT35-related drugs.

Claims (3)

A target DNA sequence of CRISPR / Cas9 guide RNA for knocking out the ACT35 gene of human cells, the sequence is 5'-AACACGGTGTGCCGTCCGTG-3 '. A method for knocking out the ACT35 gene in a human cell using the DNA sequence of claim 1, the specific steps are as follows: (1) Synthesize the target DNA sequence and its complementary strand artificially, recombine the nucleic acid fragment into the px458 plasmid after annealing to obtain the px458-ACT35 recombinant plasmid, transform E. coli NEBStable, screen and culture ampicillin, and select a monoclonal strain for sequencing and identification; (2) A large number of cultured and correctly sequenced Escherichia coli, endotoxin-free extraction of px458-ACT35 recombinant plasmid; (3) The transfected cell culture medium was changed to a serum-free medium, and the px458-ACT35 recombinant plasmid was prepared into a transfection mixture with Opti-MEM medium and PEI, added to the transfected cell culture medium, and changed after 5 hours. After transfected cell culture medium was replaced with serum medium and cultured for 48 h, transfected cells with ACT35 gene knockout were obtained. Use of the DNA sequence according to claim 1 in knocking out the ACT35 gene.