Targeted base editing in the mitochondrial genome of Arabidopsis thaliana

Abstract

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.