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TitleDNA interference states of the hypercompact CRISPR-CasΦ effector.
Journal, issue, pagesNat Struct Mol Biol, Vol. 28, Issue 8, Page 652-661, Year 2021
Publish dateAug 11, 2021
AuthorsPatrick Pausch / Katarzyna M Soczek / Dominik A Herbst / Connor A Tsuchida / Basem Al-Shayeb / Jillian F Banfield / Eva Nogales / Jennifer A Doudna /
PubMed AbstractCRISPR-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 ...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.
External linksNat Struct Mol Biol / PubMed:34381246 / PubMed Central
MethodsEM (single particle)
Resolution2.9 - 3.54 Å
Structure data

23600

7lys

EMDB-23600, PDB-7lys:
Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and DNA
Method: EM (single particle) / Resolution: 3.05 Å

23601

7lyt

EMDB-23601, PDB-7lyt:
Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and Phosphorothioate-DNA
Method: EM (single particle) / Resolution: 2.9 Å

23678

7m5o

EMDB-23678, PDB-7m5o:
Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA
Method: EM (single particle) / Resolution: 3.54 Å

Chemicals

ChemComp-ZN:
Unknown entry

ChemComp-MG:
Unknown entry

Source
  • biggievirus mos11
  • phage #d (virus)
KeywordsVIRAL PROTEIN/RNA/DNA / CRISPR / CasPhi / Cas12j / Nuclease / R-loop / crRNA / PAM / RNP / Complex / VIRAL PROTEIN-RNA-DNA complex / VIRAL PROTEIN-RNA-DNA / VIRAL PROTEIN/RNA / VIRAL PROTEIN-RNA complex

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