AsCas12a Exhibits Intrinsic, DNA-Independent ATPase Activity

Abstract

Cas12a (previously Cpf1) is a class 2 CRISPR-Cas effector protein with RNA-guided DNA endonuclease activity and is widely used for genome editing. While its DNA cleavage and target recognition mechanisms have been studied extensively, the possibility of auxiliary enzymatic functions remains underexplored. Here, I report that Acidaminococcus sp. Cas12a (AsCas12a) possesses intrinsic ATPase activity, despite lacking canonical nucleotide-binding or hydrolysis motifs. Using a radiometric thin-layer chromatography (TLC) assay, I demonstrate that AsCas12a hydrolyzes ATP in a concentration and time-dependent manner. Importantly, this activity occurs independently of DNA cofactors, as neither single-stranded nor double-stranded DNA could influence the rate or extent of ATP hydrolysis. Bioinformatic analyses using NsitePred and SwissDock, as well as ATPbind, suggests potential ATP-binding residues with predicted favorable binding energies. This preliminary finding uncovers a previously unrecognized biochemical property of AsCas12a.