Improved design and analysis of CRISPR knockout screens

Bioinformatics. 2018 Dec 1;34(23):4095-4101. doi: 10.1093/bioinformatics/bty450.


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 under the MIT license.

Supplementary information: Supplementary data are available at Bioinformatics online.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Computational Biology
  • Gene Library*
  • Genome
  • RNA, Guide / genetics*


  • RNA, Guide