Related Experiment Video
Updated: Feb 9, 2026
Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells
Published on: January 31, 2025
Improved design and analysis of CRISPR knockout screens
Chen-Hao Chen1,2,3, Tengfei Xiao1,4, Han Xu1,2,5
1Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA.
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.
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