Herbicide-resistant plants produced by precision adenine base editing in plastid DNA

Abstract

CRISPR-free, protein-only cytosine base editors (CBEs) or adenine base editors, composed of DNA-binding proteins such as zinc finger proteins or transcription activator-like effectors (TALEs) and nucleobase cytosine or adenine deaminases, respectively, enable organellar DNA editing in cultured cells, animals and plants1–4. TALE-linked double-stranded DNA deaminase toxin A (DddAtox)-derived CBEs (DdCBEs) and TALE-linked adenine deaminases (TALEDs) install C-to-T and A-to-G single-nucleotide conversions, respectively, in mitochondria and chloroplasts5–9. Interestingly, whereas TALEDs exclusively induce A-to-G conversions without C-to-T conversions in mammalian mitochondrial DNA10, they often install unwanted C-to-T edits in addition to intended A-to-G edits in plastid DNA7,9,11,12. Here we show that uracil DNA glycosylase (UDG)-fused TALEDs (UDG-TALEDs) minimize C-to-T conversions without reducing the A-to-G editing efficiency and install a mutation in the chloroplast psbA gene that encodes a single-amino-acid substitution (S264G), which confers herbicide resistance in the resulting plants. Uracil DNA glycosylase-fused TALE-linked deaminases achieve precision A-to-G base editing without bystander C-to-T editing in chloroplast DNA to create herbicide-resistant plants with a heritable homoplasmic mutation in the psbA gene.