Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada.
Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto, 661 University Avenue, 14th Floor, Toronto, ON M5G 1M1, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada.
STAR Protoc. 2023 Mar 17;4(1):101933. doi: 10.1016/j.xpro.2022.101933. Epub 2022 Dec 26.
Here, we describe a protocol for purifying functional clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Staphylococcus aureus within 24 h and over 90% purity. SaCas9 purification begins with immobilized metal affinity chromatography, followed by cation exchange chromatography, and ended with centrifugal concentrators. The simplicity, cost-effectiveness, and reproducibility of such protocols will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating CRISPR research.
在这里，我们描述了一种在 24 小时内从金黄色葡萄球菌中纯化具有功能的簇状规则间隔短回文重复序列（CRISPR）相关蛋白 9（Cas9）的方案，纯度超过 90%。SaCas9 的纯化从固定化金属亲和层析开始，然后进行阳离子交换层析，最后使用离心浓缩器。这些方案的简单性、成本效益和可重复性将使普通实验室能够生产大量的 Cas9 蛋白，从而进一步加速 CRISPR 研究。
