The compact Casπ (Cas12l) ‘bracelet’ provides a unique structural platform for DNA manipulation

CRISPR-Cas modules serve as the adaptive nucleic-acid immune systems for prokaryotes, and provide versatile tools for nucleic-acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (∼860 aa), from the environmental metagenome. Complexed with a large guide-RNA (∼170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5’ C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a ‘bracelet’ architecture of Casπ effector encircling the DNA target at 3.4-Å resolution, substantially different from the canonical ‘two-lobe’ architectures of Cas12 and Cas9 nucleases. And the large guide-RNA serves as a ‘two-arm’ scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulations.