In Vitro Characterization of Intermediate CRISPR RNA Biogenesis in the Type III-A Staphylococcus epidermidis System.

摘要

In many bacteria and archaea, an adaptive immune system based on CRISPR- cas loci provides immunity against foreign genetic elements. The CRISPR-Cas system uses small CRISPR RNAs (crRNA) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids for destruction. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. In Types I and III CRISPR-Cas systems, a specific endoribonuclease belonging to the Cas6 family of protein cleaves the pre-crRNA within the repeat regions and can act as a single monomeric protein or in concert with other Cas proteins. Because Cas6 is responsible for crRNA biogenesis in the majority of CRISPR/cas systems, it must employ versatile mechanisms of action to recognize and process a variety of crRNA substrates. Here, I studied the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from the Staphylococcus epidermidis strain RP62a, a classic model for Type III-A CRISPR-Cas systems.;This research sought to define substrate requirements for SeCas6 activity and to elucidate a potential Cas6 CRISPR Ribo-Nucleic Protein (crRNP) complex. This study reveals that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing. Further, Cas6 appears to function as a stand-alone nuclease that mediates intermediate crRNA biogenesis in the absence of a crRNP complex.;The results suggest that Cas6 is only involved in intermediate crRNA biogenesis. Further, a separate crRNP void of a strong association with Cas6 must be responsible for loading the intermediate crRNA and mediating 3' trimming, resulting in a mature crRNA. The resulting mature crRNA substrate and effector complex are involved in downstream targeting processes.