Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Biotechnology

Background:

• CRISPR-Cas systems enable small genomic modifications, but large, targeted deletions in plants remain challenging.
• Developing precise tools for large DNA deletion is crucial for crop improvement and functional genomics.

Purpose of the Study:

• To engineer novel APOBEC-Cas9 fusion-induced deletion systems (AFIDs) for efficient and predictable large deletions in plant genomes.
• To evaluate the efficiency and predictability of AFID-3 and eAFID-3 systems in rice and wheat.

Main Methods:

• Fusion of Cas9 with human APOBEC3A (A3A) or truncated APOBEC3B (A3Bctd) to create AFID systems.
• Application of AFIDs in rice and wheat protoplasts and regenerated plants to induce deletions.
• Analysis of deletion size, location, and predictability relative to Cas9 cleavage sites and deamination events.

Main Results:

• AFID-3 system generated deletions in rice and wheat, with approximately one-third being predictable.
• The eAFID-3 system, utilizing A3Bctd, produced more uniform deletions from a preferred TC motif to the double-strand break site.
• Successful induction of deletions in both protoplasts and regenerated plant tissues.

Conclusions:

• AFIDs represent a novel strategy for generating predictable, large deletions in plant genomes.
• These systems offer potential applications in studying regulatory elements and protein domains for crop trait enhancement.
• AFIDs advance genome editing tools for functional genomics and crop breeding.