Lee Kok Zhi, Mechikoff Michael A, Parasa Mrugesh Krishna, Rankin Tyler J, Pandolfi Paula, Fitzgerald Kevin S, Hillman Ethan T, Solomon Kevin V
Department of Agricultural and Biological Engineering, Purdue University, 225 South University Street, West Lafayette, Indiana 47907-2093, United States.
Weldon School of Biomedical Engineering, Purdue University, 206 S Martin Jischke Drive, West Lafayette, Indiana 47907-2093, United States.
ACS Synth Biol. 2022 Jan 21;11(1):53-60. doi: 10.1021/acssynbio.1c00340. Epub 2022 Jan 10.
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 . 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.
原核生物基因组编码多种可编程的DNA内切核酸酶，在生物技术和基因编辑方面具有巨大潜力。然而，这些内切核酸酶的特性差异很大，必须进行筛选和测定。虽然已经开发出基于ccdB和芽孢杆菌RNA酶的阳性选择筛选方法来评估此类蛋白质，但它们的高毒性使其使用具有挑战性。在此，我们开发并验证了一种基于归巢内切核酸酶I-SceI的更强大的阳性选择筛选方法。候选内切核酸酶靶向并消除I-SceI表达质粒，防止诱导I-SceI介导的双链DNA断裂，而这种断裂会导致细胞死亡。我们验证了该筛选方法可用于测量SpCas9、xCas9和eSpCas9的相对活性，并证明了能够从混合群体中富集活性更高的内切核酸酶变体。该系统可用于高通量快速表征新型可编程内切核酸酶，并适用于内切核酸酶功能的定向进化。
