Chen-Hao Chen1,2,3, Tengfei Xiao1,4, Han Xu1,2,5, Peng Jiang1,2, Clifford A Meyer1,2, Wei Li1,2,6,7, Myles Brown1,4, X Shirley Liu1,2. 1. Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA. 2. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, MA, USA. 3. Biological and Biomedical Science Program, Harvard Medical School, Boston, MA, USA. 4. Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA. 5. Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA. 6. Center for Genetic Medicine Research, Children's National Health System, Washington, DC, USA. 7. Department of Genomics and Precision Medicine, The George Washington School of Medicine and Health Sciences, Washington, DC, USA.
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.
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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