High-throughput screens in mammalian cells using the CRISPR-Cas9 system.
As a powerful genome-editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR)-clustered regularly interspaced short palindromic repeats-associated protein 9 (Cas9) system has been quickly developed into a large-scale function-based screening strategy in mammalian cells. This new type of genetic library is constructed through the lentiviral delivery of single-guide RNA collections that direct Cas9 or inactive dead Cas9 fused with effectors to interrogate gene function or regulate gene transcription in targeted cells. Compared with RNA interference screening, the CRISPR-Cas9 system demonstrates much higher levels of effectiveness and reliability with respect to both loss-of-function and gain-of-function screening. Unlike the RNA interference strategy, a CRISPR-Cas9 library can target both protein-coding sequences and regulatory elements, including promoters, enhancers and elements transcribing microRNAs and long noncoding RNAs. This powerful genetic tool will undoubtedly accelerate the mechanistic discovery of various biological processes. In this mini review, we summarize the general procedure of CRISPR-Cas9 library mediated functional screening, system optimization strategies and applications of this new genetic toolkit.
Biodynamic Optical Imaging Centre (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Jingyu
J
Peng
Biodynamic Optical Imaging Centre (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Yuexin
Y
Zhou
Biodynamic Optical Imaging Centre (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Shiyou
S
Zhu
Biodynamic Optical Imaging Centre (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Wensheng
W
Wei
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001681:Biological Assay; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics*; D055785:Gene Knockdown Techniques; D015723:Gene Library; D005818:Genetic Engineering; D006801:Humans; D034622:RNA Interference
D001426:Bacterial Proteins; D004720:Endonucleases
CRISPR-Cas9 system; high-throughput; knockout; screening; sgRNA