CRISPR-Cas13 effectors are conditionally active RNases that provide antiphage immunity in microbes. RNase activity by its HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) nuclease is initiated after Cas13 sequence-specifically recognises phage transcripts using complementary gRNA. Widespread RNA cleavage by active Cas13 is reported to drive immunity through abortive infection. However, various HEPN-containing RNases across nature have adapted bespoke RNA cleavage signatures – from highly specific to indiscriminate. Emerging evidence suggests that some Cas13 enzymes may have specific activity, challenging the prevailing paradigm of non-specificity. Given the importance of Cas13 effectors in precision RNA knockdown and infectious disease diagnostics, there is a critical need for a rigorous understanding of Cas13 function.
We investigated how Cas13 carries out RNA turnover to shed light on enzyme’s biochemical mechanisms of action. Substrate preferences of diverse Cas13 homologs were explored by carrying out RNA-sequencing of cleaved heterogeneous RNA samples. We find homolog-dependent RNA cleavage sequence motifs with a clear bias towards tRNA and with varying degrees of specificity. RNA cleavage at the HEPN nuclease was characterised in various tRNA-bound cryo-EM structures of Leptotrichia buccalis Cas13a at high-resolution (2.3 – 2.6 Å). We establish how substrates are initially captured, recognised, and committed to catalysis by both the conserved active site and previously overlooked auxiliary residues. Additionally, the HEPN catalytic mechanism is revealed, showcasing a novel mechanism of efficient RNA turnover involving induced substrate fitting into distorted conformations. Our work provides the foundation that Cas13 antiphage responses in microbes are diverse but may converge on tRNA as central regulatory modules using conserved HEPN-mediated cleavage. Finally, we lay the foundation to engineer Cas13 constructs with novel substrate preferences suitable for biotechnological application.