Translational enhancement of target endogenous mRNA in mammalian cells using programmable RNA-binding pentatricopeptide repeat proteins

Construction of designer PPR protein gene

The designer PPR protein gene vector was constructed using the golden gate assembly method and PPR3.0 scaffold as described by Yagi et al²⁹. Briefly, an intermediate plasmid containing two PPR motifs was chemically synthesized (Azenta Life Science, Chelmsford, MA, USA). The respective PPR scaffolds comprised 144 plasmids and were divided into nine groups (Tw-1 to Tw-9). The respective Tw groups contained 16 plasmids for all the 16 dinucleotide combinations recognized by the two PPR motifs. The designer PPR gene with 18 PPR motifs was assembled using…