CRISPR/Cas9 Screening to Identify Conditionally Essential Genes in Human Cell Lines

2021 ◽  
pp. 29-42
Author(s):  
Kimberly S. Huggler ◽  
Nicholas J. Rossiter ◽  
Kyle M. Flickinger ◽  
Jason R. Cantor
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Essential Genes ◽  
Human Cell Lines

scholarly journals Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens

2017 ◽  
Author(s):  
Traver Hart ◽  
Amy Tong ◽  
Katie Chan ◽  
Jolanda Van Leeuwen ◽  
Ashwin Seetharaman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Viral Vectors ◽  
Human Cancer ◽  
Gene Knockout ◽  
Essential Genes ◽  
Human Cell Lines ◽  
Protein Coding ◽  
Mammalian Cell Lines ◽  
Genome Scale

AbstractThe adaptation of CRISPR/Cas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs), targeting human protein-coding genes and encoded in viral vectors, have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we re-analyze 17 genome-scale knockout screens in human cell lines from three research groups using three different genome-scale gRNA libraries, using the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm to identify essential genes, to refine and expand our previously defined set of human core essential genes, from 360 to 684 genes. We use this expanded set of reference Core Essential Genes (CEG2), plus empirical data from six CRISPR knockout screens, to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

scholarly journals One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines

2021 ◽  
pp. mbc.E21-02-0051
Author(s):  
Tsz Kwan Yeung ◽  
Ho Wai Lau ◽  
Hoi Tang Ma ◽  
Randy Y.C. Poon
Keyword(s):  
Gene Silencing ◽  
Cell Lines ◽  
Human Cell ◽  
Mammalian Cells ◽  
Essential Genes ◽  
Gene Inactivation ◽  
Human Cell Lines ◽  
Efficient Generation ◽  
Multiple Genes ◽  
One Step

Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.

Investigations of the influence of microgravity on the state of human cell lines

2004 ◽  
Vol 10 (5-6) ◽  
pp. 226-228
Author(s):  
L.M. Nosach ◽  
◽  
O.Yu. Povnitsa ◽  
V.L. Zhovnovata ◽  
◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
The State ◽  
Human Cell Lines

scholarly journals Dicalcin suppresses in vitro trophoblast attachment in human cell lines

2021 ◽  
Vol 570 ◽  
pp. 206-213
Author(s):  
Ryohei Saito ◽  
Hiromasa Satoh ◽  
Kayo Aoba ◽  
Hajime Hirasawa ◽  
Naofumi Miwa
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Human Cell Lines

scholarly journals Differential Effects of Cyanidin and Cyanidin-3-glucoside on Human Cell Lines

2011 ◽  
Vol 17 (6) ◽  
pp. 515-521 ◽  
Author(s):  
Masayuki TAKEUCHI ◽  
Katsuki OHTANI ◽  
Yanju MA ◽  
Sanae KATO ◽  
Shingo SEMBA ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Human Cell Lines ◽  
Differential Effects
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