The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.
Animal cultured cell DNA-binding protein Genome editing HEK293T cell Translational activation Pentatricopeptide repeat PPR RNA-binding protein
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This work was in part supported by Cross-ministerial Strategic Innovation Promotion Program (SIP) of Council for Science, Technology and Innovation (CSTI) (T.N.) and Grants-in-Aid 26117718 and 25292219 (T.N.), and 26870430 (to Y.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology. Y.Y. is supported by JSPS fellowship.
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