Engineering hypercompact IscB nucleases for efficient and versatile genome editing in rice

Area of Science:

• Molecular Biology
• Biotechnology
• Plant Science

Background:

• IscB (Insertion sequences Cas9-like OrfB) are novel, small RNA-guided nucleases.
• Engineered IscB variants show potential for mammalian genome editing.
• Natural IscB efficiency in eukaryotes is limited, necessitating optimization.

Purpose of the Study:

• To screen and identify high-activity IscB variants for efficient genome editing in rice.
• To develop versatile base editors (CBEs and ABEs) from IscB scaffolds.
• To enhance the utility of IscB systems for crop improvement.

Main Methods:

• Screening of IscB variants, including enOgeuIscB and ωRNA-v13, for rice genome editing.
• Development and testing of IscB-based cytosine base editors (CBEs) and adenine base editors (ABEs).
• Evaluation of deaminase activity (Sdd7 vs. APOBEC3A) and TadA8e fusions for base editing.

Main Results:

• The pIscB-v3 system achieved an average editing efficiency of 17.61% across ten endogenous rice targets.
• High editing efficiency (up to 83.33% in T0 generation) with substantial homozygous and bi-allelic mutations was observed.
• IscB-CBEs utilizing Sdd7 deaminase showed effective C-to-T conversions (up to 47.92%), outperforming human APOBEC3A.
• Enhanced adenine base editor activity was achieved through TadA8e fusion strategies.

Conclusions:

• IscB systems offer robust capabilities for developing miniature plant genome editing toolkits.
• The engineered IscB variants significantly facilitate efficient and versatile crop breeding.
• This study establishes IscB as a powerful tool for advancing agricultural biotechnology.