Abstract
Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about 2/5 the size of Cas9 but shares similar domain organization. The associated ωRNA plays the combined role of crRNA and tracrRNA to guide dsDNA cleavage. Here we report a 2.78 Å cryo-EM structure of IscB-ωRNA bound to dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 RNPs. 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.
