Efficient and heritable A-to-K base editing in rice and tomato

Area of Science:

• Plant Molecular Biology
• Gene Editing Technologies
• Crop Science

Background:

• Cytosine and adenosine base editors (CBE and ABE) are crucial tools for plant gene function research and crop improvement.
• Existing base editors efficiently perform A-to-G and C-to-T/G/A edits, but efficient and heritable A-to-Y (A-to-T/C) editing in plants is underdeveloped.

Purpose of the Study:

• To construct and evaluate novel A-to-K base editor (AKBE) systems for both monocot and dicot plants.
• To expand the targeting scope of base editors by incorporating a PAM-less Cas9 variant (nSpRY).
• To assess the efficiency and heritability of A-to-T and A-to-C base editing in plants.

Main Methods:

• Development of a series of A-to-K base editor (AKBE) systems.
• Integration of the PAM-less Cas9 variant (nSpRY) to broaden target accessibility.
• Application of AKBEs in rice and tomato, followed by analysis of editing outcomes in T0 and T1 generations across multiple endogenous loci.

Main Results:

• Plant AKBEs demonstrated high efficiency in A-to-G substitution (41.0% average).
• Achieved significant A-to-T conversion efficiencies (up to 25.9% in rice, 10.5% in tomato).
• Rice-optimized AKBE successfully generated A-to-C conversions (1.8% average), enhancing genetic diversity.
• Introduced early stop codons by targeting tyrosine or cysteine residues, showing potential for gene disruption.
• While A-to-T and A-to-C edits were often chimeric in T0, they were heritably transmitted to T1 offspring.

Conclusions:

• The developed AKBE systems are effective for precise A-to-T and A-to-C base editing in plants.
• These novel editors significantly expand the capabilities of base editing in monocots and dicots, facilitating crop genetic diversity.
• AKBEs offer a valuable tool for gene function studies and crop breeding, including targeted gene disruption.