Abstract
It is highly desirable to identify gene’s function in a high-throughput fashion, and the CRISPR/Cas9 system has been harnessed to meet such a need. Here, we describe a general method to generate genome-scale lentiviral single-guide RNA (sgRNA) library and conduct a pooled function-based screening in human cells. This protocol would be of interest to researchers to rapidly identify genes in a variety of biological processes.
Similar content being viewed by others
References
Barrangou R et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712
Jinek M et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Mali P et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Zhou Y et al (2014) High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509:487–491
Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84
Shalem O et al (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87
Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32:267–273
Doench JG et al (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191
Doench JG et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32:1262–1267
Peng J, Zhou Y, Zhu S, Wei W (2015) High-throughput screens in mammalian cells using the CRISPR-Cas9 system. FEBS J 282:2089–2096
Li W et al (2014) MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 15:554
Acknowledgments
We thank J. Xi for providing the Cas9-encoding construct, and D. Trono for aid with the lentivirus packaging system. We thank Peking University High-throughput Sequencing Center operated by BIOPIC. This work was supported by funds from the National Basic Research Program of China (2010CB911800), the National Science Foundation of China (NSFC31170126, NSFC31070115), and the Peking-Tsinghua Centre for Life Sciences. Shiyou Zhu and Yuexin Zhou contributed equally to this work.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Zhu, S., Zhou, Y., Wei, W. (2017). Genome-Wide CRISPR/Cas9 Screening for High-Throughput Functional Genomics in Human Cells. In: Mossman, K. (eds) Innate Antiviral Immunity. Methods in Molecular Biology, vol 1656. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7237-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7237-1_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7236-4
Online ISBN: 978-1-4939-7237-1
eBook Packages: Springer Protocols