Improved design and analysis of CRISPR knockout screens

Chen Hao Chen; Tengfei Xiao; Han Xu; Peng Jiang; Clifford A. Meyer; Wei Li; Myles Brown; X. Shirley Liu

doi:10.1093/bioinformatics/bty450

Improved design and analysis of CRISPR knockout screens

Chen Hao Chen, Tengfei Xiao, Han Xu, Peng Jiang, Clifford A. Meyer, Wei Li, Myles Brown, X. Shirley Liu

Research output: Contribution to journal › Article › peer-review

28 Scopus citations

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.

Original language	English (US)
Pages (from-to)	4095-4101
Number of pages	7
Journal	Bioinformatics
Volume	34
Issue number	23
DOIs	https://doi.org/10.1093/bioinformatics/bty450
State	Published - Dec 1 2018
Externally published	Yes

ASJC Scopus subject areas

Statistics and Probability
Biochemistry
Molecular Biology
Computer Science Applications
Computational Theory and Mathematics
Computational Mathematics

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10.1093/bioinformatics/bty450

Cite this

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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 {\textquoteleft}safe harbor{\textquoteright} 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.",

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AU - Chen, Chen Hao

AU - Xiao, Tengfei

AU - Xu, Han

AU - Jiang, Peng

AU - Meyer, Clifford A.

AU - Li, Wei

AU - Brown, Myles

AU - Liu, X. Shirley

PY - 2018/12/1

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N2 - 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.

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Improved design and analysis of CRISPR knockout screens

Abstract

ASJC Scopus subject areas

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