TY - JOUR T1 - Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens JF - bioRxiv DO - 10.1101/117341 SP - 117341 AU - Traver Hart AU - Amy Tong AU - Katie Chan AU - Jolanda Van Leeuwen AU - Ashwin Seetharaman AU - Michael Aregger AU - Megha Chandrashekhar AU - Nicole Hustedt AU - Sahil Seth AU - Avery Noonan AU - Andrea Habsid AU - Olga Sizova AU - Lyudmila Nedyalkova AU - Ryan Climie AU - Keith Lawson AU - Maria Augusta Sartori AU - Sabriyeh Alibeh AU - David Tieu AU - Sanna Masud AU - Patricia Mero AU - Alexander Weiss AU - Kevin R. Brown AU - Matej Usaj AU - Maximilian Billmann AU - Mahfuzur Rahman AU - Michael Constanzo AU - Chad L. Myers AU - Brenda J. Andrews AU - Charles Boone AU - Daniel Durocher AU - Jason Moffat Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/03/16/117341.abstract N2 - The 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. ER -