CRISPR-Cas13 bacterial immune systems are programmable ribonucleases (RNases) that utilise guide RNA (gRNA) to base-pair with defined RNA targets. Following gRNA-target pairing, Cas13 conformationally transitions to an activated state with catalytically capable Higher Eukaryotic and Prokaryotic Nucleotide-binding (HEPN) domains. Activated HEPN-nucleases in Cas13 cleave both target RNA in cis and freely diffusing RNA in trans. Notably, phosphodiesters adjacent to U or A nucleotides are susceptible to cleavage by Cas13 depending on the homolog. Besides purely sequence, however, RNases often exhibit remarkable substrate specificity in terms of RNA structure. Here we describe our work to holistically answer which RNA are preferred substrates for Cas13 HEPN-nucleases using RNA-sequencing. Concomitantly, we also describe our investigation into the molecular mechanisms underpinning HEPN-nuclease function, including substrate binding and catalysis, using cryo-EM. These approaches from both the substrate and enzyme perspective may challenge the notion that Cas13 are indiscriminate RNases. Since Cas13 is widely used for RNA manipulations in biotechnology, our results will help to develop strategies for enzyme or substrate engineering.