An optimized genome-wide, virus-free CRISPR screen for mammalian cells

Cell Reports: Methods

Jae Seong Lee¹  Gyun Min Lee²  Songyuan Li²  Nathan E. Lewis³  Karen Julie la Cour Karottki⁴  Shangzhong Li⁴  Helene Faustrup Kildegaard⁴  Lise Marie Grav⁴  Kai Xiong⁴  Lasse Ebdrup Pedersen⁴  Philipp Spahn⁵  Ildze Ventina⁵  Hooman Hefzi⁵ 

[1] Department of Bioengineering, University of California at San Diego, La Jolla, CA, USA;Department of Pediatrics, University of California at San Diego, La Jolla, CA, USA;Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea;The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark;The Novo Nordisk Foundation Center for Biosustainability, University of California at San Diego, La Jolla, CA, USA;

关键词: CRISPR;    Cas9;    pooled screen;    virus free;    genome wide;    CHO;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability. Motivation: Although lentivirus-based delivery of genome-wide CRISPR screen components has proven successful, there are situations in, e.g., industry and hospitals where working with live viruses is difficult or simply not an option. For those situations we have developed an alternative to virus-based, genome-wide CRISPR screens that retains compatibility with the software tools developed for analyzing the results, takes a similar amount of time, and offers improved signal-to-noise ratio.

【 授权许可】

Unknown