A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing
- Tongji University, China
- China Pharmaceutical University, China
Abstract
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.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 1, 2, 3, 4, 5, 6 and figure supplements contained within 'Source data files'. Primer sequences named 'Table 1-source data 1' are also included as 'Source data files'.
Article and author information
Author details
Funding
Chinese National Program on Key Basic Research Project (2018YFC2000100)
- Zhiyong Mao
the key project of the Science and Technology of Shanghai Municipality (19JC1415300)
- Shaorong Gao
the Shanghai Rising-Star Program (19QA1409600)
- Jiayu Chen
the Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015)
- Xiaoping Wan
the Young Elite Scientist Sponsorship Program by CAST (2018QNRC001)
- Jiayu Chen
Chinese National Program on Key Basic Research Project (2017YFA010330)
- Ying Jiang
Chinese National Program on Key Basic Research Project (2016YFA0100400)
- Shaorong Gao
the National Science Foundation of China (31871438)
- Zhiyong Mao
the National Science Foundation of China (81972457)
- Ying Jiang
the National Science Foundation of China (31721003)
- Shaorong Gao
the National Science Foundation of China (31871446)
- Jiayu Chen
the Fundamental Research Funds for the Central Universities, Program of Shanghai Academic Research Leader (19XD1403000)
- Zhiyong Mao
Shuguang Program" of Shanghai Education Development Foundation and Shanghai Municipal Education Commission" (19SG18)
- Zhiyong Mao
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: The specific-pathogen-free-grade mice, including C57BL/6n, ICR, and BDF1 mice, were housed in the animal facility at Tongji University. All the mice had free access to food and water. All the experiments were performed in accordance with the University of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Biological Research Ethics Committee of Tongji University, and the approved protocol number was TJLAC-019-095.
Reviewing Editor
- Wolf-Dietrich Heyer, University of California, Davis, United States
Version history
- Received: February 13, 2020
- Accepted: July 8, 2020
- Accepted Manuscript published: July 9, 2020 (version 1)
- Version of Record published: July 24, 2020 (version 2)
Copyright
© 2020, Zhang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing
eLife 9:e56008.
https://doi.org/10.7554/eLife.56008
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