Assessing and engineering the IscB-omegaRNA system for programmed genome editing

IscB, a transposon-associated, programmable RNA guided endonuclease, holds promise for in vivo genome editing owing to its ancient evolutionary root, functional resemblance to Cas9, and compact size. Despite these advantages, wildtype IscB proteins are minimally active in human cells. To harness the potential, we develop enhanced OgeuIscB through rational engineering. Paired with structure guided omegaRNA engineering, the enhanced OgeuIscB omegaRNA systems efficiently edit the human genome. Genome-wide, unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) analysis reveals moderate fidelity of OgeuIscB omegaRNA in the human genome, a feature inherited by the engineered OgeuIscB omegaRNA systems.