TY - JOUR
T1 - Evaluation and design of genome-wide CRISPR/SpCas9 knockout screens
AU - Hart, Traver
AU - Tong, Amy Hin Yan
AU - Chan, Katie
AU - Van Leeuwen, Jolanda
AU - Seetharaman, Ashwin
AU - Aregger, Michael
AU - Chandrashekhar, Megha
AU - Hustedt, Nicole
AU - Seth, Sahil
AU - Noonan, Avery
AU - Habsid, Andrea
AU - Sizova, Olga
AU - Nedyalkova, Lyudmila
AU - Climie, Ryan
AU - Tworzyanski, Leanne
AU - Lawson, Keith
AU - Sartori, Maria Augusta
AU - Alibeh, Sabriyeh
AU - Tieu, David
AU - Masud, Sanna
AU - Mero, Patricia
AU - Weiss, Alexander
AU - Brown, Kevin R.
AU - Usaj, Matej
AU - Billmann, Maximilian
AU - Rahman, Mahfuzur
AU - Constanzo, Michael
AU - Myers, Chad L.
AU - Andrews, Brenda J.
AU - Boone, Charles
AU - Durocher, Daniel
AU - Moffat, Jason
N1 - Publisher Copyright:
© 2017 Hart et al.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - CRISPR/Cas9
KW - Cancer cell lines
KW - Core essential genes
KW - Genetic screens
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U2 - 10.1534/g3.117.041277
DO - 10.1534/g3.117.041277
M3 - Article
C2 - 28655737
AN - SCOPUS:85027283916
SN - 2160-1836
VL - 7
SP - 2719
EP - 2727
JO - G3: Genes, Genomes, Genetics
JF - G3: Genes, Genomes, Genetics
IS - 8
ER -