Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools

Area of Science:

• Plant molecular biology and genetics
• Gene editing technologies
• CRISPR/Cas9 applications

Background:

• Cytosine base editors (CBEs) and adenine base editors (ABEs) enable specific base substitutions in plants.
• Achieving base transversions (e.g., C-to-G, A-to-T) remains a significant challenge in plant gene editing.
• Limited research exists on base transversion editing in plants.

Purpose of the Study:

• To construct and characterize novel C-to-G base editors (CGBEs) for use in rice.
• To explore the potential for A-to-Y (A-to-T or A-to-C) base editing in plants.
• To develop and evaluate new base editing tools for plant genetic engineering.

Main Methods:

• Construction of CGBE vectors by fusing evoFENRY deaminase with a PAM-relaxed Cas9-nickase (Cas9n-NG) and uracil DNA N-glycosylase (rUNG or hUNG).
• Development of ABE vectors by fusing TadA8e deaminase with Cas9-nickase variants (SpGn) and DNA glycosylases (EndoV or hAAG).
• Assessment of editing efficiency and byproducts at target sites in rice (T0 generation).

Main Results:

• CGBEs achieved C-to-G conversions with monoallelic editing efficiencies up to 27.3% in rice, with insertion/deletion mutations as major byproducts.
• ABE vectors (ABE8e-EndoV, ABE8e-hAAG) produced A-to-G substitutions but did not yield detectable A-to-Y editing.
• ABE8e-EndoV demonstrated potential for generating precise small fragment deletions within the editing window.

Conclusions:

• The study successfully evaluated the performance of novel CGBEs in rice, demonstrating their capability for C-to-G base editing.
• The exploration of A-to-Y editing revealed limitations with current constructs but highlighted the potential of ABE8e-EndoV for generating specific deletions.
• The developed CGBE and ABE8e-EndoV tools offer valuable additions to the plant base editing toolbox.