Matvei Kolesnik1*, Ishita Jain2, Ekaterina Semenova2 and Konstantin Severinov2
1 Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Moscow, Russia
2 Waksman Institute, Rutgers, The State University of New Jersey, NJ, USA
matvei.kolesnik [at] skoltech.ru
Abstract
Type VI CRISPR-Cas systems exclusively recognize and cleave RNA molecules. A distinct feature of Type VI systems is collateral RNA damage. Specifically, the binding of a target transcript by Cas13a, charged with cognate CRISPR RNA (crRNA), activates the Cas13a enzyme, turning it into an active ribonuclease that mediates the cleavage of non-complementary RNA molecules. Previously, Cas13a-mediated collateral RNA cleavage was observed in in vitro experiments and was described to be nonspecific. In Escherichia coli, targeting of nonessential transcripts by heterologously expressed Leptotrichia shahii Cas13a enzyme (LshCas13a) leads to cell growth retardation, which was proposed to be a consequence of collateral degradation of essential cellular transcripts. However, the direct link between collateral RNA cleavage and cell growth retardation was not established. Specifically, the products of collateral RNA cleavage mediated by target-activated Cas13a enzyme were not identified in living cells.
To detect RNA cleavage sites associated with collateral Cas13a activity, a specific approach based on high-throughput RNA sequencing was developed. This approach was successfully applied to detect RNA cleavage sites introduced by target-activated LshCas13a enzyme in both in vivo and in vitro experiments. In E. coli cells, the target-activated LshCas13a enzyme cleaves tRNA molecules within anticodon loops, leading to protein synthesis inhibition and slowing down cell growth. Additionally, LshCas13a-mediated collateral tRNA cleavage indirectly activates cellular ribonucleases encoded by Type II toxin-antitoxin systems.
Together, the results suggest that the L. shahii Type VI CRISPR-Cas system mediates the immune response by inhibiting translation through collateral tRNA cleavage.
Keywords: CRISPR-Cas systems, RNA-Seq, Cas13
Acknowledgement: I want to thank Prof. Pavel Mazin for his comments on the RNA-Seq data analysis.