Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems

Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.