Full integration requires two sequential transesterification reactions. Focusing on the CRISPR repeat, we learned that the CRISPR repeat is distorted at key conserved purine-pyrimidine steps as it passes from the first transesterification site to the second.

We next added short fragments of foreign DNA with and without a protospacer adjacent motif (PAM), to understand why the PAM blocks integration into the CRISPR array.

We learned that foreign DNA length alters the complex stability, and that adding 2 bp makes all the difference.

We next determined the structure of the complex formed by adding a short foreign DNA fragment to Cas1-2/3. Foreign DNA binding triggers dramatic conformational changes that expose new DNA binding surfaces necessary for homing the integrase to the CRISPR locus.

Intriguingly, we saw that in this DNA-unbound conformation, a short loop in the Cas3 RecA1 domain covers the nuclease active site, much like a latched gate.

Comparing Cas3 in this OFF state to a previously nuclease ON state reveals the gate swings open when the nuclease is active.

We wanted to understand how this fantastic genomic knot assembles, so we set out to determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation.

In the absence of DNA, Cas1-2/3 forms a prominent, positively charged channel on one face of the complex.

New preprint from the Wiedenheft lab!

We used #cryoEM to show how a type I-F #CRISPR integrase captures, delivers, and integrates foreign DNA.
www.biorxiv.org/content/10.1...