Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome

CRISPR/Cas9 and Cas12a belonging to the Class II CRISPR system are characterized by a single-component effector protein. Despite unique features of Cas12a like DNA cleavage with 5′ staggered ends and a single crRNA, Cas12a has not been adopted in biotechnological applications to the similar extent as Cas9. To better understand the CRISPR/Cas12 systems, we selected two candidates, designated mgCas12a-1 and mgCas12a-2, from an analysis of the human microbiome metagenome (mg) and provided biochemical characterization. These new Cas12a proteins shared about 37% identity in amino acid sequences and shared the same direct repeat sequences in the crRNA with FnCas12a from Francisella novicida . The purification yield of the recombinant proteins was up to 3.6-fold greater than that of FnCas12a. In cell-free DNA cleavage assays, both mgCas12a proteins showed the higher cleavage efficiencies when Mn 2+ was provided with KCl (