期刊
ACS SYNTHETIC BIOLOGY
卷 11, 期 1, 页码 53-60出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00340
关键词
I-SceI; CRISPR; endonuclease; gene-editing; positive selection; directed evolution
资金
- Ralph W. and Grace M. Showalter Trust [41000622]
- Purdue Research Foundation [60000025]
- USDA National Institute of Food and Agriculture [S1041]
- Purdue's Colleges of Engineering and Agriculture
- University of Delaware's College of Engineering
Prokaryote genomes encode diverse programmable DNA endonucleases with significant potential for biotechnology and gene editing. A positive selection screen based on the homing endonuclease I-SceI has been developed and validated to measure the relative activity of endonucleases and enrich for more active variants. This system may be applied in high throughput to characterize novel programmable endonucleases and evolve endonuclease function.
Prokaryote genomes encode diverse programmable DNA endonucleases with significant potential for biotechnology and gene editing. However, these endonucleases differ significantly in their properties, which must be screened and measured. While positive selection screens based on ccdB and barnase have been developed to evaluate such proteins, their high levels of toxicity make them challenging to use. Here, we develop and validate a more robust positive selection screen based on the homing endonuclease I-SceI. Candidate endonucleases target and cure the I-SceI expression plasmid preventing induction of I-SceI-mediated double strand DNA breaks that lead to cell death in E. coli. We validated this screen to measure the relative activity of SpCas9, xCas9, and eSpCas9 and demonstrated an ability to enrich for more active endonuclease variants from a mixed population. This system may be applied in high throughput to rapidly characterize novel programmable endonucleases and be adapted for directed evolution of endonuclease function.
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