Cross-talk and extensive rewiring of CRISPR-Cas systems with the cellular regulatory machinery in cyanobacteria

通过这一建议，我们旨在分析蓝藻中的CRISPR-Cas系统，重点是它们与宿主调控机制的相互作用。作为模式生物，我们选择了聚球藻单细胞菌株。PCC 6803。该毒株携带三个CRISPR-Cas系统。其中I-D亚型和III-D亚型。第三种是III-B变异体(III-BV)，其特征是Cmr6_CMR1融合，替换的Cmr5蛋白，可能参与多肽酶的参与，以及相关的Cas6成熟内切核酸酶的缺失。生物信息学预测表明，类似的变异系统也存在于其他原核生物中。而不是Cas6，我们发现宿主编码的内切核酸酶E被招募为主要的成熟酶，这对涉及的分子和进化机制提出了疑问。这直接表明，还有其他宿主和CRISPR编码的因素需要确定，例如，在将成熟的crRNAs加载到监视复合体中，在执行crRNAs的3‘端成熟过程中，在调节RNase特异性以及CRISPR-Cas复合体靶向的核酸类型方面。我们将通过下拉分析、干扰分析中各个候选基因缺失的表征和分子分析、RNase E-CRISPR-Repeat-RNA共晶体结构的阐明和整个CRISPR3复合体结构的确定来解决这些问题。然而，CRISPR相关的RNase与调控转录本的成熟和稳定的过程之间可能的相互作用似乎比先前预期的更加复杂，对于依赖Cas6的另外两个CRISPR-Cas系统也是如此。因此，我们还将通过宿主编码的RpaB-NBLS双组分系统来解决对宿主转录组以及氧化还原控制的可能影响。在这两种情况下，都将遵循额外的酶的参与以及对基因表达的转录后调控的可能后果。项目工作将得到蓝藻群体发育的元基因组分析的补充，以了解所研究类型的CRISPR-CAS系统的演变架构及其与细胞调控装置的整合和相互作用。

With this proposal we aim at analyzing the CRISPR-Cas systems in cyanobacteria with a focus on their interaction with the regulatory machinery of the host. As model organism we have chosen the unicellular strain Synechocystis sp. PCC 6803. This strain carries three CRISPR-Cas systems. Two of them were classified as subtype I-D and subtype III-D. The third constitutes a III-B variant (III-Bv), characterized by a Cmr6_Cmr1 fusion, a replaced Cmr5 protein, the possible involvement of a peptidase and the lack of an associated Cas6 maturation endoribonuclease. Bioinformatic predictions suggest similar variant systems to exist also in other prokaryotes. Instead of Cas6 we found that the host-encoded endoribonuclease E was recruited as the major maturation enzyme, which raises questions about the involved molecular and evolutionary mechanisms. It directly suggests that there are additional host- and CRISPR-encoded factors that need to be identified, e.g., in loading mature crRNAs into the surveillance complexes, in performing the 3’ end maturation of crRNAs, in modulating RNase specificity as well as the type of nucleic acid targeted by the CRISPR-Cas complexes. We will address these questions using pull-down assays, the characterization of deletions for respective candidate genes in interference assays and by molecular analyses, the elucidation of an RNase E-CRISPR-repeat-RNA cocrystal structure and determination of the entire CRISPR3 complex structure. However, the possible interplay between CRISPR-associated RNases and the processes governing the maturation and stability of transcripts appears more complex than previously anticipated also with regard to the Cas6-dependent other two CRISPR-Cas systems. Therefore, we will also address the possible impact on the host transcriptome as well as redox control through the host-encoded RpaB-NblS two-component system. In both cases, the involvement of additional enzymes and the possible consequences for the post-transcriptional regulation of gene expression will be followed. The project work will be complemented by the metagenomic analysis of cyanobacterial mass developments to understand the evolving architecture of the studied types of CRISPR-Cas systems and their integration into and interaction with the cellular regulatory apparatus.