Pentatricopeptide repeat (PPR) proteins are considered a potential tool for manipulating organelle gene expression in plants because they can recognise a wide range of different RNA sequences, and the molecular basis for this sequence recognition is partially known and understood. A library of redesigned PPR proteins related to restorer-of-fertility proteins was created and transformed into plants in order to target mitochondrial transcripts. Ninety different variants tested in vivo showed a wide range of phenotypes. One of these lines, which displayed slow growth and downward curled leaves, showed a clear reduction in complex V. The phenotype was due to a specific cleavage of atp1 transcripts induced by a modified PPR protein from the library, validating the use of this library as a source of mitochondrial ‘mutants’. This study is a step towards developing specific RNA targeting tools using PPR proteins that can be aimed at desired targets.