Metagenome-Derived CRISPR-Cas12a Mining and Characterization

Metagenomic screening revealed several Cas12a proteins from cow rumen that can expand the repertoire of genomic and epigenomic effectors from this unique class of CRISPR proteins. myTXTL was used for PAM discovery of the Cas12a proteins identified bioinformatically, a process that would be much more difficult in vivo or via the purification of Cas proteins and transcription of guide RNAs in separate steps. For myTXTL PAM discovery experiments, only DNA templates/targets are needed.  From the paper: “Conveniently, cell-free in vitro assays are scalable, efficient, and bypass toxicity constraints, and enable rapid testing in vivo in E. coli for genome editing proof of concept.” This work identified five new Cas12a proteins with high diversity relative to known Cas12a enzymes and greatly expands the Cas12a toolbox for future studies, including genome editing and epigenetic regulation.