# Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition

## TL;DR

This paper explores how a CRISPR-Cas system guides DNA transposition by revealing structural changes in proteins that help target DNA for precise insertion.

## Contribution

The study reveals dynamic structural rearrangements at the PAM-distal site that enable RNA-guided DNA transposition in type I-F3 CRISPR-Cas systems.

## Key findings

- Cryo-EM structures show how Cas8/5 recognizes the PAM and identifies a key residue for cytidine preference.
- Mismatch tolerance at the PAM-proximal site allows for flexible DNA targeting.
- Conformational changes at the PAM-distal site bend DNA to guide it toward transposition machinery.

## Abstract

Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.

Graphical Abstract