Structural analysis showed how targets single-stranded nucleic acids and guided mutations that improved RNA-targeting efficiency.
Evidence
This was a structural and mechanistic study reporting four cryo-electron microscopy structures of IscB variants bound to single-stranded nucleic acid, followed by in vitro and human-cell RNA-targeting tests.
Caveat
The work demonstrates mechanism and engineered activity in experimental systems, not therapeutic editing performance, delivery, or safety in organisms.
Simplified
Transposon-encoded was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously repurposed IscB and related Cas9s as efficient RNA editors by removing their double-stranded DNA (dsDNA) recognition module, the target-adjacent motif (TAM)/protospacer adjacent motif-interacting domain. Here, we report four cryo-electron microscopy structures of IscB, with or without TAM-interaction domain (TID), in complex with single-stranded nucleic acid () targets. Structures reveal that, regardless of TID presence, IscB engages ssNA using the same mechanism. IscB initially facilitates formation of a 10-nt seed duplex with ssNA; further base-pairing is blocked by an alternatively positioned that acts as a roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage. Only upon full duplex formation is the HNH roadblock dislodged by the duplex extension between guide RNA and ssNA. HNH and RuvC nuclease active sites become exposed as the result. A similar set of conformational rearrangements likely governs IscB activity during dsDNA target interrogation. Guided by the structural and mechanistic insights, we introduced mutations to either improve ssNA binding or ease HNH dislodging. Both approaches improved the RNA-targeting efficiency of IscB in vitro and in human cells.
Key numbers
40-fold
Increase in RNA-binding speed
Binding speed improvement of the M402A/D403A variant compared to the parental version.
2.7×
Increase in editing efficiency
Average fluorescence intensity increase per cell in splicing assays.
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