Architecture of a DNA-guided Cas12a

Abstract CRISPR/Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-EM structure of Acidaminococcus sp. Cas12a (AsCas12a) bound to a pseudo-DNA (ΨDNA) guide and RNA target, revealing how Cas12a accomplishes DNA-guided RNA recognition. The ΨDNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the REC lobe. This provides a structural framework for its activity and provides a blueprint for future engineering. Competing Interest Statement C.O., B.H., and P.K.J., are listed as inventors on a patent application related to the content of this work. P.K.J. is a co-founder of CasNx, LLC and CRISPR, LLC. The remaining authors declare no competing interests.