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

Kai Xiong,Karen Julie La Cour Karottki,Hooman Hefzi,Songyuan Li,Lise Marie Grav,Shangzhong Li,Philipp Spahn,Jae Seong Lee,Ildze Ventina,Gyun Min Lee,Nathan E Lewis,Helene Faustrup Kildegaard,Lasse Ebdrup Pedersen

doi:10.1016/j.crmeth.2021.100062

Abstract

SummaryPooled 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.

Highlights

CRISPR screens have been widely applied to decipher mammalian gene function at the genome scale, but most rely on lentiviral delivery methods (Adamson et al, 2016; Joung et al, 2017; Liu et al, 2018)
As we demonstrate in this paper, a recombinase-mediated cassette exchange (RMCE)-based, virus-free (VF) method can eliminate those concerns, and result in less noisy data
RMCE is a method in which one can move a piece of DNA, for example, from a plasmid, into a pre-established genome landing pad without causing large-scale disruptions that can occur with CRISPR-mediated targeted insertion

Summary

Introduction

CRISPR screens have been widely applied to decipher mammalian gene function at the genome scale, but most rely on lentiviral delivery methods (Adamson et al, 2016; Joung et al, 2017; Liu et al, 2018). Working with lentivirus requires specialized facilities as well as trained personnel, which are not available in all laboratories. In some industrial and medical facilities, it is likewise considered an increased risk to work with live viruses. RMCE is a method in which one can move a piece of DNA, for example, from a plasmid, into a pre-established genome landing pad without causing large-scale disruptions that can occur with CRISPR-mediated targeted insertion

Methods

Results

Discussion

Conclusion