Three novel Cas12a orthologs with robust DNA cleavage activity suitable for nucleic acid detection

Developing rapid and accurate pathogen detection methods is increasingly important, and CRISPR-Cas system can be optimized for this purpose. CRISPR-Cas12a is a single RNA-guided endonuclease system with the potential for nucleic acid detection. There is a broad diversity among Cas12a nucleases with robust detection capability. Herein, we characterised three Cas12a orthologs (ObCas12a, MbCas12a, and ScCas12a), including cis- and trans-cleavage activities, the identification of PAM, single-base resolution ability, and the application for nucleic acid detection. These Cas12a orthologs displayed robust cis- and trans-cleavage activities, and performed well in terms of specificity and sensitivity for nucleic acid detection. Furthermore, they have subtle differences in single-base resolution and recognised PAM sites in vitro. Therefore, these Cas12a nucleases are candidate proteins for CRISPR-based diagnostic methods. Addition of these enzymes to the nucleic acid detection toolbox will further expand the utility of this powerful technology.