DSSR-PyMOL cartoon-block schematics: PDB entry 8w2z

PDB-id

8w2z; DSSR-derived features in text and JSON formats

Class

immune system-RNA-DNA

Method

cryo-EM (3.37 Å)

Summary

Cas9d 6bp r-loop seed complex

Reference

Ocampo RF, Bravo JPK, Dangerfield TL, Nocedal I, Jirde SA, Alexander LM, Thomas NC, Das A, Nielson S, Johnson KA, Brown CT, Butterfield CN, Goltsman DSA, Taylor DW (2025): "DNA targeting by compact Cas9d and its resurrected ancestor." Nat Commun, 16, 457. doi: 10.1038/s41467-024-55573-4.

Abstract

Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.