Construction of quality control materials for HIV-1 genotypic drug resistance testing based on CRISPR/Cas9 point mutation technique
10.3760/cma.j.cn112866-20240425-00069
- VernacularTitle:基于CRISPR/Cas9点突变技术构建HIV-1基因型耐药检测质控品
- Author:
Mengjun DING
1
;
Xin ZHANG
;
Yu WANG
;
Jun YAO
;
Cong JIN
Author Information
1. 中国疾病预防控制中心性病艾滋病预防控制中心 传染病溯源与智能决策全国重点实验室，北京 102206
- Keywords:
CRISPR/Cas9 point mutation technology;
8E5 cell line;
HIV-1 genotypic drug resistance test;
Quality control materials
- From:
Chinese Journal of Experimental and Clinical Virology
2024;38(3):231-238
- CountryChina
- Language:Chinese
-
Abstract:
Objective:The human peripheral blood lymphocyte cell line 8E5 is capable of secreting non-infectious HIV-1 viral particles. By targeting the POL region of the HIV-1 proviral gene integrated into the genome of 8E5 cell line and constructing a monoclonal cell line containing a drug resistance mutation site in the POL region using CRISPR/Cas9 point mutation technology, safe and stable HIV-1 genotypic drug resistance test quality control materials can be prepared.Methods:8E5 cells were co-transfected with sgRNA (single guide RNA) and Cas9 coexpression vector and Donor ssODN (donor single-stranded oligonucleotides) carrying the target mutation sites. The positive monoclonal cell lines were obtained through flow microtiter plate sorting, and the editing efficacy of the targeted mutations was validated by Sanger sequencing. Sanger sequencing was performed to verify the editing effect of the targeted mutations on the HIV virus particles secreted into the supernatant of the monoclonal cell lines cultured to the 3rd, 5th and 7th generations.Results:A double sgRNA and Cas9 coexpression vector was successfully constructed and co-transfected with a Donor ssODN carrying the drug-resistant mutation site Q151M to the 8E5 cell line, resulting in the desired outcome. The sequencing result of the target site confirmed the successful mutation at the resistance site and the establishment of a monoclonal homozygous cell line. The Q151M mutation site was detected in non-infectious HIV-1 virus particles secreted by the 8E5 Q151M cell line after transmission. Conclusions:The cell line 8E5 Q151M was successfully constructed using CRISPR/Cas9 point mutation technology to stably carry the Q151M drug resistance mutation site, which provides a new technological platform for the preparation of quality-control materials for testing HIV-1 genotypic drug resistance.
