RNA-targeting by Cas13a effector and DNA-proficient spacer acquisition in Type VI-A CRISPR-Cas system: How to encounter enemy, not die and prevent infection spreading

Among evolutionary and mechanistically diverse CRISPR-Cas systems, Type VI systems are unique as they exclusively target RNA. Type VI single-protein effector Cas13 exhibits a dual activity: Cas13 is responsible for crRNA biogenesis as well as for target RNA destruction. Significant progress has been made in understanding the mechanism of Type VI crRNA biogenesis. In contrast, little is known about interference and adaptation in these systems. Interference by Cas13 has been mostly studied using in vitro assays, which revealed that target RNA recognition activates collateral damage of non-complementary RNAs. Cas13 collateral RNase activity has been adopted for developing of highly sensitive diagnostic platforms. Observation of growth defect exhibited by cells undergoing Type VI interference with non-essential targets led to a hypothesis of programmed cell death/dormancy induced by Cas13 collateral damage activity.

To investigate a mechanism of cell growth inhibition we study Cas13a system from Leptotrichia shahii transplanted into Escherichia coli cells. Live microscopy and pulse-labeling experiments showed a cessation of cell division probably due to inhibition of protein synthesis upon Cas13a RNA-targeting. We suggest that Cas13a system likely switches to cell dormancy rather than programmed cell death. RNA sequencing and primer extension assay revealed numerous RNA cleavage sites in mRNAs, rRNAs, and tRNAs. The analysis also detected the Cas13a-mediated activation of type II toxin-antitoxin systems. Using strains deficient in toxin RNases we aim to explore a predominate mechanism for induction of dormancy state.

Another intriguing questions are how do the cells that carry type VI CRISPR-Cas system can memorize phage infection and how can spacer acquisition be compatible with induced cell dormancy. I will present our results on adaptation in Type VI-A system and discuss why cell dormancy would be more beneficial for the population than cell death to retain immunity status against invaders.