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

热纤梭菌 CRISPR-Cas I-B 型 DNA 干扰复合物的表征

• 批准号: 206943638
• 负责人: Professor Dr. Lennart Randau
• 金额: --
• 依托单位: Arbeitsgruppe Genetik
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/206943638?language=en
• 关键词: Characterization; CRISPR; Cas; type; DNA

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.

大肠杆菌和细菌可以包含适应性CRISPR-Cas免疫系统。这些不同的系统利用与Cas蛋白复合物结合的小CRISPR RNA来检测病毒攻击并降解外源DNA。我们建议研究热纤梭菌的I-B型DNA干扰复合物。我们已经建立了这种核糖核蛋白复合物（称为Cascade）的所有五个亚基（Cas 3、Cas 5、Cas 6、Cas 7、Cas 8b）的重组生产和CRISPR RNA转录物的体外产生。我们注意到，亚型特异性蛋白Cas 8b以两个片段产生，并且这种片段化模式在其他Cas 8b蛋白中也是保守的。绘制了Cas 8b切割位点。我们将对现有重组复合物的蛋白质和核酸组分进行突变研究，以研究单个Cas亚基在Cascade功能中的作用。我们的目标是阐明（i）DNA靶点选择和（ii）CRISPR RNA加载到Cascade中的机制和特异性。其中一个重点将放在亚型特异性大亚基Cas 8b在这些过程中的未知作用上。我们认为，观察到的该蛋白质的保守片段化产生了在不同CRISPR-Cas亚型的干扰复合物中发现的难以捉摸的小亚基。我们将研究Cas 8b片段化的机制，并将遵循指向自切割反应的初步结果。阻止内部切割的Cas 8b突变体以及代表潜在的小亚基和大亚基的蛋白质片段将被组装到DNA干扰复合物中。将使用体外DNA结合和切割测定来推断Cascade小亚基和大亚基在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型之间的DNA干扰复合物的底物选择性，I-A型是以前在我们的实验室从古菌Thermoproteus tenax重建。我们计划与DFG Forschergruppe FOR 1680的成员合作，以（i）绘制CRISPR RNA和C.热纤Cas蛋白质的质谱分析和（ii）在体内研究DNA干扰复合物的小和大亚基的功能。不同DNA干扰机制的比较应该阐明（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。