SNAP output for PDB entry 7lys [SNAP web server]

PDB-id

7lys; SNAP-derived features in text and JSON formats; DNAproDB

Class

viral protein-RNA-DNA

Method

cryo-EM (3.05 Å)

Summary

cryo-EM structure of casphi-2 (cas12j) bound to crrna and DNA

Reference

Pausch P, Soczek KM, Herbst DA, Tsuchida CA, Al-Shayeb B, Banfield JF, Nogales E, Doudna JA (2021): "DNA interference states of the hypercompact CRISPR-Cas Phi effector." Nat.Struct.Mol.Biol., 28, 652-661. doi: 10.1038/s41594-021-00632-3.

Abstract

CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.