Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis

Abstract

In many bacteria and archaea, an adaptive immune system (CRISPR–Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR–Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR–Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3′ half of the CRISPR repeat for recognition and processing.