High-efficiency and multiplex adenine base editing in plants using new TadA variants

Area of Science:

• Plant biotechnology
• Molecular biology
• Genetics

Background:

• Adenine base editors (ABEs) are powerful tools for gene editing, but their efficiency varies across target sites.
• Developing ABEs with improved efficiency and broader applicability is crucial for gene function studies and crop breeding.

Purpose of the Study:

• To investigate and compare the editing activities of evolved adenosine deaminase TadA variants in rice.
• To develop novel ABE variants with enhanced editing capabilities and broader compatibility for plant applications.

Main Methods:

• Transgenic rice lines were generated to test three evolved E. coli TadA variants (TadA8e, TadA8.17, TadA8.20).
• A new variant, TadA9, was engineered by incorporating specific mutations into TadA8e.
• TadA9's compatibility with various CRISPR systems (SpCas9, SpCas9-NG, SpRY, ScCas9 nickase) was assessed.
• Simultaneous SNP installation in endogenous herbicide target genes in rice was performed using TadA9.

Main Results:

• TadA8e showed higher editing efficiency than TadA8.17 and TadA8.20 in rice, editing previously inaccessible sites.
• TadA9 exhibited broad compatibility with multiple CRISPR systems and a larger editing window compared to TadA8e.
• TadA9 successfully introduced novel SNPs into four herbicide target genes in a commercial rice cultivar.

Conclusions:

• Novel ABEs, TadA8e and TadA9, were successfully developed for efficient adenosine editing in the rice genome.
• TadA9 shows significant potential as a plant base editor for crop molecular breeding and developing herbicide resistance traits.