Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools

Type 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. [Display omitted] •Type I-A Cascade and Cas3 form an integral effector complex•Cas3 nuclease is allosterically activated by Cascade upon full R-loop formation•Type I-A is repurposed to a heat-activated streamlined nucleic acid detection platform•Type I-A CRISPR-Cas3 is highly efficient in bi-directional DNA deletion in human cells Hu et al. show that type I-A CRISPR-Cas3 uses an allosterically controlled mechanism to selectively cleave the DNA target, which is very different from six other type I systems. They further developed a highly sensitive nucleic acid detection platform and an efficient deletion-editing method from type I-A CRISPR-Cas3.