Chou-Zheng Lucy, Hatoum-Aslan Asma
Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, USA.
Bio Protoc. 2017 Jun 5;7(11). doi: 10.21769/BioProtoc.2353.
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). and are bacterial residents on human skin that are also leading causes of antibiotic resistant infections (Lowy, 1998; National Nosocomial Infections Surveillance, 2004; Otto, 2009). Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Cao ., 2016), which have been shown to prevent plasmid transfer and protect against viral predators (Goldberg ., 2014; Hatoum-Aslan ., 2014; Samai ., 2015) in these organisms. Thus, gaining a mechanistic understanding of these systems in the native staphylococcal background can lead to important insights into the factors that impact the evolution and survival of these pathogens. Type III-A CRISPR-Cas systems encode a five-subunit effector complex called Cas10-Csm (Hatoum-Aslan ., 2013). Here, we describe a protocol for the expression and purification of Cas10-Csm from its native background or a heterologous background. The method consists of a two-step purification protocol involving Ni-affinity chromatography and a DNA affinity biotin pull-down, which together yield a pure preparation of the Cas10-Csm complex. This approach has been used previously to analyze the effects of mutations on Cas10-Csm complex integrity (Hatoum-Aslan ., 2014), crRNA formation (Hatoum-Aslan ., 2013), and to detect binding partners that directly interact with the core Cas10-Csm complex (Walker ., 2016). Importantly, this approach can be easily adapted for use in other species to probe and understand their native Type III-A CRISPR-Cas systems.
CRISPR-Cas（成簇规律间隔短回文重复序列- CRISPR相关蛋白）是一类原核生物免疫系统，能以序列特异性方式降解外源核酸。这些系统依赖于由Cas核酸酶和小CRISPR RNA（crRNA）组成的核糖核蛋白复合物。金黄色葡萄球菌和表皮葡萄球菌是人类皮肤上的常驻细菌，也是抗生素耐药性感染的主要原因（Lowy，1998；国家医院感染监测系统，2004；Otto，2009）。许多葡萄球菌拥有III-A型CRISPR-Cas系统（Marraffini和Sontheimer，2008；Cao等人，2016），已证明该系统可防止质粒转移并抵御这些生物体中的病毒捕食者（Goldberg等人，2014；Hatoum-Aslan等人，2014；Samai等人，2015）。因此，在天然葡萄球菌背景下对这些系统进行机制性了解，可深入洞察影响这些病原体进化和生存的因素。III-A型CRISPR-Cas系统编码一种名为Cas10-Csm的五亚基效应复合物（Hatoum-Aslan等人，2013）。在此，我们描述了一种从其天然金黄色葡萄球菌背景或异源表皮葡萄球菌背景中表达和纯化Cas10-Csm的方案。该方法包括两步纯化方案，涉及镍亲和色谱和DNA亲和生物素下拉法，共同产生Cas10-Csm复合物的纯制剂。此方法先前已用于分析突变对Cas10-Csm复合物完整性（Hatoum-Aslan等人，2014）、crRNA形成（Hatoum-Aslan等人，2013）的影响，并检测与核心Cas10-Csm复合物直接相互作用的结合伙伴（Walker等人，2016）。重要的是，该方法可轻松适用于其他葡萄球菌物种，以探究和了解其天然III-A型CRISPR-Cas系统。
