Type V CRISPR-Cas Cpfl endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

CRISPR-Cas9 and CRISPR-Cpfl systems have been successfully harnessed for genome editing. In the CRIS- PR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpfl system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpfl （AsCpfl） in complex with crRNA and target DNA. Structural comparison between the AsCpfl-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpfl （LbCpfl）-crRNA binary complex identifies a unique mechanism employed by Cpfl for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpfl-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpfl undergoes an ＂open-to-closed＂ conformational change upon target DNA binding, which in turn induces structural changes within Cpfl to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpfl is distinct from that reported previously for Cas9.