Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

By Traver Hart, A. Tong, Katie Chan, Jolanda van Leeuwen, Ashwin Seetharaman, Michael Aregger, M. Chandrashekhar, Nicole Hustedt, S. Seth, Avery J. C. Noonan and 22 more. 2017

521 citations recorded by the source corpus.

The adaptation of CRISPR/SpCas9 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 reanalyze 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 algorithm to identify essential genes, we 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 then 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.

Subfields: Aging, Molecular Biology

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1. Pushmeet Kohli
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Citations in the corpus

• Evaluating drug targets through human loss-of-function genetic variation (2019, unknown): “Targets of negative drugs include 14 genes with lethal heterozygous knockout mouse phenotypes reported 23 and 6 reported as essential in human cell culture 24 .”
• Accurate proteome-wide missense variant effect prediction with AlphaMissense (2023, background): “Genesexper- imentallyidentifiedasessentialtocellsur-vivalacrossavarietyofhumancelllines( 46 ) showed a strong enrichment among the most-pathogenic decile of AlphaMissense.”
• Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens (2019, unknown): “Of the ADaM pan-cancer CF genes, 399 were previously defined as essential genes(Hart et al. 2015; Hart et al. 2017) and 125 are genes involved in essential cellular processes(Tzelepis et al. 2016; Wang et al. 2014) (Extended Data Fig.”