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Improved design and analysis of CRISPR knockout screens

Improved design and analysis of CRISPR knockout screens MotivationGenome-wide clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 screen has been widely used to interrogate gene functions. However, the rules to design better libraries beg further refinement.ResultsWe found single guide RNA (sgRNA) outliers are characterized by higher G-nucleotide counts, especially in regions distal from the PAM motif and are associated with stronger off-target activities. Furthermore, using non-targeting sgRNAs as negative controls lead to strong bias, which can be mitigated by using sgRNAs targeting multiple ‘safe harbor’ regions. Custom-designed screens confirmed our findings and further revealed that 19 nt sgRNAs consistently gave the best signal-to-noise ratio. Collectively, our analysis motivated the design of a new genome-wide CRISPR/Cas9 screen library and uncovered some intriguing properties of the CRISPR-Cas9 system.Availability and implementationThe MAGeCK workflow is available open source at https://bitbucket.org/liulab/mageck_nest under the MIT license.Supplementary informationSupplementary data are available at Bioinformatics online. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioinformatics Oxford University Press

Improved design and analysis of CRISPR knockout screens

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Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
ISSN
1367-4803
eISSN
1460-2059
DOI
10.1093/bioinformatics/bty450
Publisher site
See Article on Publisher Site

Abstract

MotivationGenome-wide clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 screen has been widely used to interrogate gene functions. However, the rules to design better libraries beg further refinement.ResultsWe found single guide RNA (sgRNA) outliers are characterized by higher G-nucleotide counts, especially in regions distal from the PAM motif and are associated with stronger off-target activities. Furthermore, using non-targeting sgRNAs as negative controls lead to strong bias, which can be mitigated by using sgRNAs targeting multiple ‘safe harbor’ regions. Custom-designed screens confirmed our findings and further revealed that 19 nt sgRNAs consistently gave the best signal-to-noise ratio. Collectively, our analysis motivated the design of a new genome-wide CRISPR/Cas9 screen library and uncovered some intriguing properties of the CRISPR-Cas9 system.Availability and implementationThe MAGeCK workflow is available open source at https://bitbucket.org/liulab/mageck_nest under the MIT license.Supplementary informationSupplementary data are available at Bioinformatics online.

Journal

BioinformaticsOxford University Press

Published: Dec 1, 2018

References