基于CRISPR/Cas9点突变技术构建HIV-1基因型耐药检测质控品
Construction of quality control materials for HIV-1 genotypic drug resistance testing based on CRISPR/Cas9 point mutation technique
丁梦君 1张鑫 2王宇 2姚均 2金聪1
作者信息
- 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;中国疾病预防控制中心性病艾滋病预防控制中心参比实验室,北京 102206
- 2. 中国疾病预防控制中心性病艾滋病预防控制中心参比实验室,北京 102206
- 折叠
摘要
目的 人外周血淋巴细胞株8E5可分泌无感染性HIV-1病毒颗粒,靶向于8E5细胞株基因组整合的HIV-1前病毒基因POL区,基于CRISPR/Cas9点突变技术构建含有POL区耐药突变位点的单克隆细胞株,可用于制备一种安全稳定的新型HIV-1基因型耐药检测质控品.方法 设计构建小向导RN A(single guide RNA,sgRNA)和Cas9共表达载体以及携带有目标突变位点的供体单链寡核苷酸(donor single-stranded oligonucleotides,Donor ssODN)共转染8E5细胞,通过流式微孔板分选获得转染成功的阳性单克隆细胞株,通过Sanger测序确认定点突变的编辑效果.对定点突变编辑成功的单克隆细胞株培养至第3代、5代和7代的细胞及细胞分泌到上清中的HIV病毒颗粒进行Sanger测序,验证定点突变的编辑效果.结果 成功构建双sgRNA和Cas9共表达载体,与携带有耐药突变位点Q151M的Donor ssODN共转染8E5细胞株的效果良好.对分选获得的共转染阳性单克隆细胞株进行靶位点测序,结果显示成功构建了携带有定点突变Q151M的纯合子单克隆细胞株8E5Q151M.细胞株8E5Q151M多次传代后的细胞及细胞分泌的无感染性HIV-1病毒颗粒可被持续检测出Q151M突变位点.结论 利用CRISPR/Cas9点突变技术成功构建稳定携带有Q151M耐药突变位点的细胞株8E5Q151M,为制备HIV-1基因型耐药检测质控品提供新的技术平台.
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 8E5Q151M cell line after transmission.Conclusions The cell line 8E5Q151M 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.
关键词
CRISPR/Cas9点突变技术/8E5细胞株/HIV-1基因型耐药检测/质控品Key words
CRISPR/Cas9 point mutation technology/8E5 cell line/HIV-1 genotypic drug resistance test/Quality control materials引用本文复制引用
基金项目
国家重点研发计划(2022YFC2305202)
出版年
2024
NETL
NSTL
万方数据