Nature chemical biologyJul 8, 2024

Studying and improving the IscB-ωRNA system for targeted genome editing

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 28, 2026

Abstract

Enhanced OgeuIscB shows a 30.4-fold improvement in insertion-deletion formation efficiency in human cells.

OgeuIscB with eight amino acid substitutions significantly increased DNA-binding affinity by fourfold.
The engineered OgeuIscB-ωRNA systems achieved up to 87.3% indel and 62.2% base-editing frequencies across 26 target sites.
Both wild-type and enhanced OgeuIscB-ωRNA exhibited moderate fidelity in genome editing.
Off-target analysis identified key factors affecting target selection, including specific motifs adjacent to the target site.

Simplified

OMEGA RNA (ωRNA)-guided endonuclease IscB, the evolutionary ancestor of Cas9, is an attractive system for in vivo genome editing because of its compact size and mechanistic resemblance to Cas9. However, wild-type IscB-ωRNA systems show limited activity in human cells. Here we report enhanced OgeuIscB, which, with eight amino acid substitutions, displayed a fourfold increase in in vitro DNA-binding affinity and a 30.4-fold improvement in insertion-deletion (indel) formation efficiency in human cells. Paired with structure-guided ωRNA engineering, the enhanced OgeuIscB-ωRNA systems efficiently edited the human genome across 26 target sites, attaining up to 87.3% indel and 62.2% base-editing frequencies. Both wild-type and engineered OgeuIscB-ωRNA showed moderate fidelity in editing the human genome, with off-target profiles revealing key determinants of target selection including an NARR target-adjacent motif (TAM) and the TAM-proximal 14 nucleotides in the R-loop. Collectively, our engineered OgeuIscB-ωRNA systems are programmable, potent and sufficiently specific for human genome editing.

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Competing interests

Competing interests: The University of Chicago and Cornell University filed provisional patent applications (63/523,862 and 63/523,884) on the engineered IscB–ωRNA systems and their applications for programmed genome editing. These patent applications are pending and the inventors are W.T., H.Y. and A.K. The other authors declare no competing interests.

PubMed

Studying and improving the IscB-ωRNA system for targeted genome editing

Abstract

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Abstract

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