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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.

The biological functions of human acyl-CoA thioesterase III (ACTEIII/PTE-1), initially identified as an HIV-1 Nef binding protein, have remained unclear. We report herein that the stable overexpression of ACTEIII/PTE-1 in human and murine T-cell lines resulted in an increase in both peroxisome number and lipid droplet formation in a manner dependent on the amount of the protein. Peroxisome proliferation was evidenced by immunofluorescence staining for catalase, a peroxisome marker protein, as well as by direct peroxisome enumeration on electron micrographs. Consistently, the amount of catalase was elevated as the amount of ACTEIII/PTE-1 was increased. ACTEIII/PTE-1 mutants with reduced enzymatic activity or with the defect in peroxisome localization did not induce peroxisome proliferation, indicating that peroxisome proliferation was mediated by metabolites generated by ACTEIII/PTE-1 within peroxisomes. Finally, thymocytes isolated from a T-cell-specific ACTEIII/PTE-1 transgenic mouse as well as human and murine cell lines of lymphoid and non-lymphoid origins exhibited a similar proliferation of peroxisomes. Thus, ACTEIII/PTE-1 may be involved in the metabolic regulation of peroxisome proliferation.