CRISPR-Cas13 systems are powerful tools for RNA biotechnology with applications spanning RNA knockdown, precision RNA editing, and sensitive RNA detection for use in molecular diagnostics [1]. Despite their widespread use, and in contrast to CRISPR-Cas9 and CRISPR-Cas12, the field lacks a complete picture describing CRISPR-Cas13 effector activity in the context of microbial adaptive immunity. In biology, Cas13 proteins (including Cas13a, Cas13b, and Cas13d) are CRISPR RNA (crRNA) guided effectors that base pair with target RNA to license the activation of conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) nuclease domains. The specific activation of the Cas13 HEPN RNase then drives prokaryotic immunity to infection through what is believed to be widespread RNA degradation driving dormancy [2].
Recent evidence suggests that Cas13 HEPN RNase activity converges on the cleavage of tRNA to drive immunity to infection [3]. To shed light on the HEPN RNase activity of CRISPR-Cas13 in a biological context, we investigated the substrate preferences of representative Cas13 orthologs. By integrating computational and experimental structural biology with biochemistry, cell biology, and microbiology, we reveal that diverse Cas13 enzymes exist on spectrum of non-specific to highly specific tRNase activity [4]. These findings have fundamental implications on our understanding of how Cas13 systems function in adaptive immunity and inform on the systems applied potential across the CRISPR toolbox.