Characterization of the CRISPR-Cas type I-B DNA interference complex from Clostridiumthermocellum

古生菌和细菌可以含有适应性CRISPR-Cas免疫系统。这些不同的系统利用与Cas蛋白复合体结合的小CRISPR RNA来检测病毒攻击和降解外来DNA。我们建议研究来自热细胞梭菌的I-B型DNA干扰复合体。我们已经建立了这种核糖核蛋白复合体(称为Cascade)的所有五个亚基(Cas3、Cas5、Cas6、Cas7、Cas8b)的重组生产和CRISPR RNA转录本的体外生成。我们注意到，亚型特异性蛋白Cas8b在两个片段中产生，这种片段模式在其他Cas8b蛋白中也是保守的。绘制了Cas8b裂解位点的图谱。我们将对可用的重组复合体的蛋白质和核酸成分进行突变研究，以调查单个CaS亚单位在级联功能中的作用(S)。我们的目标是解开(S)的机制和(I)DNA靶标选择和(Ii)CRISPR RNA加载到级联中的特异性。其中一个重点将放在亚型特有的大亚基Cas8b在这些过程中的未知作用上。我们认为，观察到的该蛋白的保守片段创造了在不同CRISPR-Cas亚型的干扰复合体中发现的难以捉摸的小亚基。我们将研究Cas8b的碎裂机制，并将遵循指向自切割反应的初步结果。防止内部切割的Cas8b突变体以及代表潜在大小亚基的蛋白质片段将组装成DNA干扰复合体。在体外，DNA结合和切割分析将被用来推断小的和大的级联亚基在DNA靶标识别中的作用。这些研究应该使我们能够比较I-B型和I-A型DNA干扰复合体的底物选择性，I-A型干扰复合体是我们实验室以前从考古子Thermoprous tenax重组而来的。我们计划与DFG Forschergruppe FOR1680的成员合作，(I)通过质谱学绘制CRISPR RNAs和Cas蛋白的相互作用位点，并(Ii)研究DNA干扰复合体的小亚基和大亚基在体内的功能。不同DNA干扰机制的比较应该揭示(I)CRISPR-Cas系统在进化过程中多样化的分子基础，以及(Ii)DNA靶标选择的灵活性程度，例如CRISPR-Cas介导的基因组工程所需的灵活性。

Archaea and bacteria can contain adaptive CRISPR-Cas immune systems. These diverse systems utilize small CRISPR RNAs bound to a Cas protein complex to detect viral attacks and to degrade the foreign DNA. We propose to investigate the type I-B DNA interference complex from Clostridium thermocellum. We have established the recombinant production of all five subunits (Cas3, Cas5, Cas6, Cas7, Cas8b) of this ribonucleoprotein complex (termed Cascade) and the in vitro generation of CRISPR RNA transcripts. We noted that the subtype-specific protein Cas8b is produced in two fragments and that this fragmentation pattern is also conserved in other Cas8b proteins. The Cas8b cleavage site was mapped. We will apply mutational studies of the protein and nucleic acid components of the available recombinant complex to investigate the role(s) of the individual Cas subunits in Cascade functionality. We aim to unravel the mechanism(s) and the specificity of (i) DNA target selection and (ii) CRISPR RNAs loading into Cascade. One focus will be placed on the unknown role of the subtype-specific large subunit Cas8b in these processes. We propose that the observed conserved fragmentation of this protein creates the elusive small subunit found in interference complexes of different CRISPR-Cas subtypes. We will investigate the mechanism of Cas8b fragmentation and will follow preliminary results that point at a self-cleavage reaction. Cas8b mutants that prevent internal cleavage as well as protein fragments that represent potential small and large subunits will be assembled into the DNA interference complex. In vitro DNA binding and cleavage assays will be used to deduce the roles of the small and the large Cascade subunits in DNA target recognition. These studies should enable us to compare substrate selectivity of DNA interference complexes between type I-B and type I-A which was previously reconstituted in our laboratory from the archaeon Thermoproteus tenax. We plan to collaborate with members of the DFG Forschergruppe FOR1680 to (i) map the interaction sites of CRISPR RNAs and C. thermocellum Cas proteins via mass-spectrometry and to (ii) study the functionality of small and large subunits of DNA interference complexes in vivo. The comparison of different DNA interference mechanisms should shed light on (i) the molecular bases for the diversification of CRISPR-Cas systems during evolution and (ii) the degree of flexibility in DNA target selection which is e.g. needed for CRISPR-Cas-mediated genome engineering.