Two novel mgCas12a proteins from the human gut metagenome exhibit reduced collateral DNase activity

Abstract CRISPR/Cas9 and CRISPR/Cas12a are characterized by a single-component effector protein. Despite unique features like DNA cleavage with 5′ staggered ends and a single crRNA, Cas12a has not been adopted in therapeutics to the same extent as Cas9. To address the pressing therapeutic needs for effective CRISPR/Cas12 systems, we selected two candidates, designated mgCas12a-1 and mgCas12a-2, from an analysis of the human microbiome metagenome (mg) for biochemical characterization. These new Cas12 proteins shared about 37% identity with FnCas12a from Francisella novicida and showed preference for Mn2+ and Mg2+ over Ca2+ or Fe2+ as divalent metal ions. The new Cas12 proteins also tolerated pH values from 5 to 8 and temperatures from 30°C to 50°C and had lower non-specific nuclease activity upon prolonged incubation than did FnCas12a. Considering their biochemical characteristics and greater purification yield, the novel mgCas12a proteins may expedite the application of Cas12a proteins in genomics research as well as human therapeutics.