基于CRISPR/cas9文库筛选的慢病毒库包装方法研究  

Methods of packaging lentivirus library for CRISPR/cas9 -based screening

作　　者：刘铁柱[1] 李阿茜 李乃哲 曲园园 李川[1] 张全福[1] 刘洋[1] 李德新[1] 梁米芳[1] 王世文[1] Liu Tiezhu;Li Aqian;Li Naizhe;Qu Yuanyuan;Li Chuan;Zhang Quanfu;Liu Yang;Li Dexin;Liang Mifang;Wang Shiwen(National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒性出血热室,北京102206

出　　处：《中华实验和临床病毒学杂志》2019年第2期207-211,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的探讨基于CRISPR/cas9(Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9)文库筛选的慢病毒库包装优化条件。方法采用RT-PCR、酶联免疫吸附试验(ELISA法)和免疫荧光法,对不同质粒配比、不同收毒时间以及不同量的Lipofectamine 3000 reagent包装出的慢病毒滴度进行检测,最后通过高通量测序技术评价慢病毒库的sgRNA(single guide RNA,单链引导RNA)覆盖度和reads数分布。结果当质粒配比psPAX2∶pMD2.0G∶Lentivirus library=2∶1∶1时,慢病毒滴度较高;当加入Lipofectamine 3000 reagent的量为10 μl时,慢病毒滴度较高。两种情况,RT-PCR和ELISA法结果一致。免疫荧光显示,收毒时间为60 h时慢病毒滴度较高。经Ion Torrent高通量测序,按照本研究的最优条件包装出的慢病毒库覆盖度达99.3%,sgRNA的reads数符合正态分布。结论本研究探索了慢病毒库包装的优化条件,为下一步CRISPR/cas9文库筛选提供保证。Objective To obtain the optimum of lentiviral library packaging based on CRISPR/cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9). Methods Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence antibody (IFA) and enzyme linked immunosorbent assay (ELISA) were used to detect the lentivirus titers in condition of different ratio of packaging plasmids, different addition of lipofectamine 3000 reagent and different time points post-transfection. Then, high-throughput sequencing was performed to evaluate the representation and distribution of single guide (sg)RNAs in the library. Results The lentivirus titer was the highest when the molar ratio of psPAX2∶pMD2.0G∶Lentivirus library was 2∶1∶1, and the optimum addition of Lipofectamine 3000 reagent was 10 μl, while the result of ELISA were correspondent to that of RT-PCR. The IFA result showed that the lentivirus titer was the highest at 60 h post-transfecion. The coverage of sgRNAs in the lentivirus library packaged with the optimum we obtained was 99.3%, and the read counts of sgRNAs was observed in a normal distribution. Conclusions The optimal lentivirus library packaging was obtained, and this can provide basis for CRISPR/cas9-based screening.

关 键 词：CRISPR/cas9文库筛选 慢病毒库包装 高通量测序技术 

分 类 号：Q78[生物学—分子生物学]