Methods of packaging lentivirus library for CRISPR/cas9 -based screening
10.3760/cma.j.issn.1003-9279.2019.02.020
- VernacularTitle: 基于CRISPR/cas9文库筛选的慢病毒库包装方法研究
- Author:
Tiezhu LIU
1
;
Aqian LI
;
Naizhe LI
;
Yuanyuan QU
;
Chuan LI
;
Quanfu ZHANG
;
Yang LIU
;
Dexin LI
;
Mifang LIANG
;
Shiwen WANG
Author Information
1. 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
- Publication Type:Journal Article
- Keywords:
CRISPR/cas9 screening;
Lentivirus library packaging;
High-throughput sequencing
- From:
Chinese Journal of Experimental and Clinical Virology
2019;33(2):207-211
- CountryChina
- Language:Chinese
-
Abstract:
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.
