SKLPT imaging: Efficient in vivo pre-evaluation of genome-editing modules using fluorescent protein with peroxisome targeting signal

Abstract

Numerous studies have used genome-editing modules such as CRISPR-Cas9 for site-directed mutagenesis; however, evaluation of the efficiency of these modules remains a time-consuming process. Here, we report the development of SKL-mediated Peroxisome Targeting Imaging (SKLPT imaging), an efficient in vivo pre-evaluation method based on the change in subcellular localization of a fluorescent protein. In this method, frameshifts resulting from successful editing cause the fusion of green fluorescent protein to the peroxisome localization signal Serine-Lysine-Leucine (SKL). Using SKLPT imaging, we pre-evaluated three CRISPR-Cas9 modules in vivo at the single-cell level, and then efficiently mutagenized the liverwort (Marchantia polymorpha) genome using a high-efficiency module.