RT Journal Article
SR Electronic
T1 Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 117341
DO 10.1101/117341
A1 Traver Hart
A1 Amy Tong
A1 Katie Chan
A1 Jolanda Van Leeuwen
A1 Ashwin Seetharaman
A1 Michael Aregger
A1 Megha Chandrashekhar
A1 Nicole Hustedt
A1 Sahil Seth
A1 Avery Noonan
A1 Andrea Habsid
A1 Olga Sizova
A1 Lyudmila Nedyalkova
A1 Ryan Climie
A1 Keith Lawson
A1 Maria Augusta Sartori
A1 Sabriyeh Alibeh
A1 David Tieu
A1 Sanna Masud
A1 Patricia Mero
A1 Alexander Weiss
A1 Kevin R. Brown
A1 Matej Usaj
A1 Maximilian Billmann
A1 Mahfuzur Rahman
A1 Michael Constanzo
A1 Chad L. Myers
A1 Brenda J. Andrews
A1 Charles Boone
A1 Daniel Durocher
A1 Jason Moffat
YR 2017
UL http://biorxiv.org/content/early/2017/03/16/117341.abstract
AB 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.