A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing

• Published in: eLife Vol. 9
• Main Authors: Zhang, Weina, Chen, Yu, Yang, Jiaqing, Zhang, Jing, Yu, Jiayu, Wang, Mengting, Zhao, Xiaodong, Wei, Ke, Wan, Xiaoping, Xu, Xiaojun, Jiang, Ying, Chen, Jiayu, Gao, Shaorong, Mao, Zhiyong
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 09.07.2020; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Animals; Blastocysts; Cell Biology; Cell cycle; Cells (Biology); Chromones - analysis; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Deoxyribonucleic acid; DNA; DNA damage; Efficiency; Embryonic development; Embryos; farrerol; Gene Editing; Gene expression; Gene targeting; Genes; Genetics and Genomics; Genome editing; Genomes; Genomics; HEK293 Cells; High-Throughput Screening Assays; Homologous recombination; homologous recombination (HR); Humans; Kinases; knock-in; Mice; Non-homologous end joining; non-homologous end joining (NHEJ); Stem cells; Tetracyclines; Tools and Resources; Mali; mouse; cell biology; genetics; CRISPR/Cas9; homologous recombination (HR); non-homologous end joining (NHEJ); farrerol; knock-in; genomics; human
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.56008

Directly modulating the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) - two independent pathways for repairing DNA double-strand breaks (DSBs) - has the potential to improve the efficiency of gene targeting by CRISPR/Cas9. Here, we have developed a rapid and easy-to-score screening approach for identifying small molecules that affect the choice between the two DSB repair pathways. Using this tool, we identified a small molecule, farrerol, that promotes HR but does not affect NHEJ. Further mechanistic studies indicate that farrerol functions through stimulating the recruitment of RAD51 to DSB sites. Importantly, we demonstrated that farrerol effectively promotes precise targeted integration in human cells, mouse cells and mouse embryos at multiple genomic loci. In addition, treating cells with farrerol did not have any obvious negative effect on genomic stability. Moreover, farrerol significantly improved the knock-in efficiency in blastocysts, and the subsequently generated knock-in mice retained the capacity for germline transmission.