Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9

Schuler, Gabriel; Hu, Chunyi; Ke, Ailong

doi:10.1126/science.abq7220

Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9

Journal Article · Fri Jun 24 04:00:00 EDT 2022 · Science

DOI:https://doi.org/10.1126/science.abq7220· OSTI ID:1982962

^[1]; Hu, Chunyi ^[1]; ^[1]

Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA.

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.

Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States). Laboratory for BioMolecular Structure (LBMS)

Sponsoring Organization:: USDOE Office of Science (SC)

DOE Contract Number:: SC0012704

OSTI ID:: 1982962

Journal Information:: Science, Journal Name: Science Journal Issue: 6600 Vol. 376; ISSN 0036-8075

Publisher:: AAAS

Country of Publication:: United States

Language:: English

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