Expression and Purification of the Cas10-Csm Complex from Staphylococci
Expression and Purification of the Cas10-Csm Complex from Staphylococci
复制标题
DOI：
10.21769/bioprotoc.2353
复制
发表时间：
2017-06-05
期刊：
影响因子：
0.8
通讯作者：
Hatoum-Aslan, Asma
中科院分区：
文献类型：
--
作者：
Chou-Zheng, Lucy;Hatoum-Aslan, Asma
CRISPR-Cas(簇状规则间隔短回文重复序列-CRISPR相关蛋白)是一类以序列特异性方式降解外源核酸的原核免疫系统。这些系统依赖于由Cas核酸酶和小CRISPR RNAs(CrRNAs)组成的核糖核蛋白复合体。表皮葡萄球菌和金黄色葡萄球菌是人类皮肤上的细菌，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，2004；Otto，2009)。许多葡萄球菌拥有III-A型CRISPR-CAS系统(Marraffini和Sonthemer，2008；曹等人，2016)，该系统已被证明在这些生物体中可以防止质粒转移和抵御病毒捕食者(Goldberg等人，2014；Hatoum-Aslan等人，2014；Samai等人，2015)。因此，在自然葡萄球菌背景下对这些系统进行机制理解可以导致对影响这些病原体的进化和生存的因素的重要洞察。III-A型CRISPR-CAS系统编码一个称为Cas10-CSM的五亚单位效应复合体(Hatoum-Aslan等人，2013年)。在这里，我们描述了一种从其天然的表皮葡萄球菌背景或异源金黄色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，包括Ni2+亲和层析和DNA亲和生物素下拉，两者共同产生了Cas10-CSM络合物的纯制备。这一方法先前已被用于分析突变对Cas10-CSM复合体完整性(Hatoum-Aslan等人，2014年)、crRNA形成(Hatoum-Aslan等人，2013年)的影响，并用于检测直接与核心Cas10-CSM复合体相互作用的结合伙伴(Walker等人，2016年)。重要的是，这种方法可以很容易地适用于其他葡萄球菌物种，以探测和了解他们的天然III-A型CRISPR-CAS系统。
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). Staphylococcus epidermidis and Staphylococcus aureus 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 et al., 2016), which have been shown to prevent plasmid transfer and protect against viral predators (Goldberg et al., 2014; Hatoum-Aslan et al., 2014; Samai et al., 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 et al., 2013). Here, we describe a protocol for the expression and purification of Cas10-Csm from its native S. epidermidis background or a heterologous S. aureus background. The method consists of a two-step purification protocol involving Ni2+-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 et al., 2014), crRNA formation (Hatoum-Aslan et al., 2013), and to detect binding partners that directly interact with the core Cas10-Csm complex (Walker et al., 2016). Importantly, this approach can be easily adapted for use in other Staphylococcus species to probe and understand their native Type III-A CRISPR-Cas systems.