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TitleStructural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9.
Journal, issue, pagesScience, Vol. 376, Issue 6600, Page 1476-1481, Year 2022
Publish dateJun 24, 2022
AuthorsGabriel Schuler / Chunyi Hu / Ailong Ke /
PubMed AbstractClass 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ...Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.
External linksScience / PubMed:35617371 / PubMed Central
MethodsEM (single particle)
Resolution2.74 - 3.2 Å
Structure data

26782

7utn

EMDB-26782, PDB-7utn:
IscB and wRNA bound to Target DNA
Method: EM (single particle) / Resolution: 2.74 Å

26976

8csz

EMDB-26976, PDB-8csz:
IscB and wRNA bound to Target DNA
Method: EM (single particle) / Resolution: 3.2 Å

26994

8ctl

EMDB-26994, PDB-8ctl:
IscB and wRNA bound to Target DNA (locked state)
Method: EM (single particle) / Resolution: 3.1 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-ZN:
Unknown entry

Source
  • synthetic construct (others)
  • unidentified (others)
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / IscB / HEARO RNA / omega RNA / RNA BINDING PROTEIN-RNA-DNA complex

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