Dataset Information
Improved design and analysis of CRISPR knockout screens.
Ontology highlight
ABSTRACT: Motivation
Genome-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.Results
We 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 implementation
The MAGeCK workflow is available open source at https://bitbucket.org/liulab/mageck_nest under the MIT license.Supplementary information
Supplementary data are available at Bioinformatics online.
SUBMITTER: Chen CH
PROVIDER: S-EPMC6247926 | biostudies-literature | 2018 Dec
REPOSITORIES: biostudies-literature
Publications
Improved design and analysis of CRISPR knockout screens.
Bioinformatics (Oxford, England) 20181201 23
<h4>Motivation</h4>Genome-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.<h4>Results</h4>We 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 cont ...[more]