Functional studies of CRISPR RNA and its associated proteins

描述（由申请人提供）：最近发现，原核生物可以通过将外源DNA的短片段整合到有规则间隔的短回文重复序列（CRISPR’s）簇中来获得对病毒和质粒的抗性。然后，这些重复序列被转录并加工成用于指导破坏外来核酸的小向导RNA。这种机制与真核RNA干扰有许多相似之处，但与CRISPR应答相关的蛋白质在进化上与真核生物的对应物无关。我们的长期目标是了解crispr介导的原核生物耐药的生化和结构基础。这里的目标是确定从CRISPR转录本中产生引导RNA的机制。尽管最近取得了进展，但对这些机制的理解还很初级。我们的目标将通过对CRISPR转录本和CRISPR相关（cas）蛋白的生化、结构和细胞分析来实现。我们假设，在所有原核生物中，这一过程将需要多个cas蛋白的特异性和顺序作用，并且基本机制将是保守的。成功完成拟议的研究具有重要意义，因为它将增加我们对细菌对病毒和质粒的耐药性的了解。这两种遗传因素在致病菌的遗传中都起着重要的作用。

DESCRIPTION (provided by applicant): It has recently been discovered that prokaryotes can acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clusters of regularly interspaced short palindromic repeats (CRISPR's). These repeats are then transcribed and processed into small guide RNA's that are used to direct the destruction of foreign nucleic acid. This mechanism has many parallels with eukaryotic RNA interference but the proteins that are associated with the CRISPR response are evolutionarily unrelated to their eukaryotic counterparts. Our long-term goal is to understand the biochemical and structural basis of CRISPR-mediated resistance in prokaryotes. The objective here is to determine the mechanisms used to produce guide RNA's from CRISPR transcripts. Despite recent advances, understanding of these mechanisms is rudimentary. Our objective will be achieved through biochemical, structural and cell based analyses of CRISPR transcripts and the CRISPR-associated (cas) proteins. We hypothesize that in all prokaryotes this process will require the specific and sequential action of multiple cas proteins and that the fundamental mechanism will be conserved. Successful completion of the proposed studies is significant because it will increase our understanding of bacterial resistance to viruses and plasmids. Both of these genetic elements play important roles in the genetics of pathogenic bacteria.