TY - JOUR
T1 - An optimized genome-wide, virus-free CRISPR screen for mammalian cells
AU - Xiong, Kai
AU - Karottki, Karen Julie la Cour
AU - Hefzi, Hooman
AU - Li, Songyuan
AU - Grav, Lise Marie
AU - Li, Shangzhong
AU - Spahn, Philipp
AU - Lee, Jae Seong
AU - Ventina, Ildze
AU - Lee, Gyun Min
AU - Lewis, Nathan E.
AU - Kildegaard, Helene Faustrup
AU - Pedersen, Lasse Ebdrup
PY - 2021
Y1 - 2021
N2 - 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. CRISPR knockout screening has mostly been performed by using viruses to deliver the required components into cells. In this paper, Xiong et al. demonstrate a virus-free approach that reduces noise and broadens access to CRISPR-based screens.
AB - 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. CRISPR knockout screening has mostly been performed by using viruses to deliver the required components into cells. In this paper, Xiong et al. demonstrate a virus-free approach that reduces noise and broadens access to CRISPR-based screens.
U2 - 10.1016/j.crmeth.2021.100062
DO - 10.1016/j.crmeth.2021.100062
M3 - Journal article
C2 - 34935002
SN - 2667-2375
VL - 1
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 4
M1 - 100062
ER -