The rapid evolutionary arms race between bacteria and mobile genetic elements has generated a large repertoire of bacterial defense strategies. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR associated) systems are adaptive immune systems that protect hosts from invading nucleic acids. Cas proteins use RNA guides that target them to foreign sequences, leading to their destructions. A diverse set of Cas effector proteins exist, which differ in the molecular mechanisms behind target recognition, and nucleic acid cleavage. The programmable cleavage of a target DNA by CRISPR-Cas systems has greatly advanced the field of genome editing, both as a research tool and a potential therapeutic. While most Cas single effectors proteins target DNA, the discovery of RNA targeting Type VI CRISPR systems (Cas13) opened the door for programmable engineering of the transcriptome. Nevertheless, the collateral cleavage induced by the Higher Eukaryote Prokaryote Nucleotide binding domain (HEPN) of Cas13 enzymes leads to a high degree of toxicity in certain cell types, and the molecular mechanisms behind target recognition and binding remain unclear. In this work, we first profiled the ability of various catalytically inactive Cas13 enzymes to inhibit translation of an mRNA in E. coli when targeted to the proximity of a ribosome binding site (RBS). While Cas13d enzymes possessed the ability to interfere with the translation machinery, all Cas13a enzymes tested showed a lack of translational repression. To this end, we performed a high throughput directed evolution screen on Cas13a and identified a series of stabilizing mutations that greatly increased both protein expression and translational repression. Stabilization of Cas13a led to altered cleavage kinetics in vitro, and altered binding properties, shedding light into the kinetic mechanism of Cas13a.
To further investigate the molecular mechanism behind Cas13 activator binding, we performed high throughput mismatch profiling for three different Lbu Cas13a mutants isolated above and show that these stabilizing mutations provide insight into the coupling of Cas13a RNA-binding and HEPN nuclease activity. Combining sets of mutations together led to a complete abolishment of single mismatch sensitivity with respect to binding but had no effect on mismatch sensitivity for HEPN activation, suggesting a link between protein stabilization and mismatch sensitivity. We propose a model in which initial seed binding, followed by duplex propagation, leads to an unstable intermediate, lacking stabilizing binary and/or ternary contacts and displaying a high activator off-rate. Once duplex propagation has completed, ternary contacts can fully stabilize the complex, and the HEPN senses proper base pairing, which leads to nuclease activation.
Finally, we sought to increase the detection limit of conventional Cas13 based RNA detection assay using feedforward loops encoded by RNA structures, in a process termed Nuclease Chain Reaction. We present our initial trials in developing efficient feedback systems driven by Cas13 nuclease activity. We attempted to design various “caged” activator sequences that are ineffective at switching on the nuclease domain, unless cleaved by an upstream RNAse. Such substrates can be supplemented to normal Cas13 detection at a high concentration, along with a secondary Cas13 RNP which targets the uncaged activator, in hopes of increasing the time to detection, as well as the limit of detection. Testing these various caged RNA structures also shed light into non-specific RNA association mediated by Cas13, as well as requirements for HEPN activation.
