Architecture of a DNA-guided Cas12a

CRISPR–Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-electron microscopy structure of Acidaminococcus sp. Cas12a bound to a pseudo-DNA (ΨDNA) guide and RNA target, revealing how Cas12a can accomplish 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 recognition lobe. This provides a structural framework for its activity and provides a potential blueprint for future engineering. Ocampo and Orosco et al. present the cryo-electron microscopy structure of Acidaminococcus sp. Cas12a bound to a pseudo-DNA guide and RNA target, showing how structural mimicry enables DNA-guided RNA recognition, providing a blueprint for engineering CRISPR–Cas systems.