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TitleAllosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.
Journal, issue, pagesMol Cell, Vol. 82, Issue 15, Page 2754-22768.e5, Year 2022
Publish dateAug 4, 2022
AuthorsChunyi Hu / Dongchun Ni / Ki Hyun Nam / Sonali Majumdar / Justin McLean / Henning Stahlberg / Michael P Terns / Ailong Ke /
PubMed AbstractType I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme ...Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.
External linksMol Cell / PubMed:35835111 / PubMed Central
MethodsEM (single particle)
Resolution3.3 - 6.6 Å
Structure data

EMDB-26081, PDB-7tr6:
Cascade complex from type I-A CRISPR-Cas system
Method: EM (single particle) / Resolution: 3.4 Å

EMDB-26082, PDB-7tr8:
Cascade complex from type I-A CRISPR-Cas system
Method: EM (single particle) / Resolution: 3.6 Å

EMDB-26083, PDB-7tr9:
Cascade complex from type I-A CRISPR-Cas system
Method: EM (single particle) / Resolution: 3.9 Å

EMDB-26084, PDB-7tra:
Cascade complex from type I-A CRISPR-Cas system
Method: EM (single particle) / Resolution: 3.3 Å

EMDB-26097: Cas3-Cas8 complex from type I-A CRISPR-Cas system
Method: EM (single particle) / Resolution: 6.6 Å

Chemicals

ChemComp-NI:
NICKEL (II) ION

Source
  • Geobacter sulfurreducens (bacteria)
  • pyrococcus furiosus dsm 3638 (archaea)
  • escherichia coli (E. coli)
KeywordsRNA BINDING PROTEIN / CRISPR / cascade / type I-A / genome editing / Cas3 / DNA targeting

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