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

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• IscB is an OMEGA RNA (ωRNA)-guided endonuclease and an evolutionary ancestor of Cas9.
• IscB offers a compact size and mechanistic similarity to Cas9, making it attractive for in vivo genome editing.
• Wild-type IscB-ωRNA systems exhibit limited activity in human cells, necessitating improvements for effective genome editing.

Purpose of the Study:

• To enhance the activity and efficiency of the IscB-ωRNA system for human genome editing.
• To engineer a more potent and specific genome editing tool based on the IscB endonuclease.

Main Methods:

• Introduced eight amino acid substitutions into IscB to create the enhanced OgeuIscB variant.
• Engineered ωRNA using structure-guided approaches.
• Tested the OgeuIscB-ωRNA system across 26 target sites in human cells.
• Analyzed DNA-binding affinity, insertion-deletion (indel) formation, base-editing frequencies, and off-target effects.

Main Results:

• OgeuIscB demonstrated a fourfold increase in in vitro DNA-binding affinity compared to wild-type IscB.
• Achieved a 30.4-fold improvement in indel formation efficiency in human cells.
• Attained up to 87.3% indel and 62.2% base-editing frequencies across multiple target sites.
• Identified an NARR target-adjacent motif (TAM) and specific R-loop nucleotides as key determinants for target selection.

Conclusions:

• The engineered OgeuIscB-ωRNA system exhibits significantly enhanced potency and efficiency for human genome editing.
• The system demonstrates programmability and sufficient specificity for therapeutic and research applications.
• OgeuIscB-ωRNA represents a promising alternative to existing genome editing technologies.