title: Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.
abstract: 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.
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan.
authors.forename: Takehito
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Yagi
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan. tnaka@agr.kyushu-u.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D014162:Transfection
chemical_list: D020121:5' Untranslated Regions; D029681:Arabidopsis Proteins; C583236:CRR4 protein, Arabidopsis; D002352:Carrier Proteins; D039603:Eukaryotic Initiation Factor-4G; D011993:Recombinant Fusion Proteins
keywords: Animal cultured cell; DNA-binding protein; Genome editing; HEK293T cell; PPR; Pentatricopeptide repeat; RNA-binding protein; Translational activation