+Search query
-Structure paper
Title | DNA interference states of the hypercompact CRISPR-CasΦ effector. |
---|---|
Journal, issue, pages | Nat Struct Mol Biol, Vol. 28, Issue 8, Page 652-661, Year 2021 |
Publish date | Aug 11, 2021 |
Authors | Patrick Pausch / Katarzyna M Soczek / Dominik A Herbst / Connor A Tsuchida / Basem Al-Shayeb / Jillian F Banfield / Eva Nogales / Jennifer A Doudna / |
PubMed 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 ...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 links | Nat Struct Mol Biol / PubMed:34381246 / PubMed Central |
Methods | EM (single particle) |
Resolution | 2.9 - 3.54 Å |
Structure data | EMDB-23600, PDB-7lys: EMDB-23601, PDB-7lyt: EMDB-23678, PDB-7m5o: |
Chemicals | ChemComp-ZN: ChemComp-MG: |
Source |
|
Keywords | VIRAL 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 |