Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression. The structure of the Cas13a RNase from the purple bacterium R. capsulatus in complex with its crRNA is reported here. The study reveals that the enzyme combines shape and sequence-specific readout to recognize its crRNA.

Automated summary

Cas13a is a single-molecule effector of the CRISPR-Cas system that plays a role in bacterial and archaeal defense. It has the ability to cleave target RNAs and bystander RNAs, providing protection to the bacterial population. This study focuses on the Cas13a enzyme from the purple bacteria Rhodobacter capsulatus, and reveals its structural and functional characteristics. The research provides insights into the molecular mechanisms and function of this family of RNA-dependent RNA endonucleases.