Hu, ChunyiNi, DongchunNam, Ki HyunMajumdar, SonaliMcLean, JustinStahlberg, HenningTerns, Michael P.Ke, Ailong2022-08-292022-08-292022-08-292022-08-0410.1016/j.molcel.2022.06.007https://infoscience.epfl.ch/handle/20.500.14299/190397WOS:000841899600006Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.Biochemistry & Molecular BiologyCell Biologyguided surveillance complexr-loop formationcas systemsvitro reconstitutioncrystal-structureforeign dnacascadedegradationinterferencemechanismtext::journal::journal article::research article