Importance of nuclear localization signal-fused Cas9 in the production of genome-edited mice via embryo electroporation

Abstract Previously, to generate genome-edited animals by introducing CRISPR-associated protein 9 (Cas9) into embryos, we developed the Technique for Animal Knockout system by Electroporation (TAKE). Additionally, by fluorescently labeling Cas9, we successfully visualized the Cas9 introduced into the pronuclei of embryos; however, whether Cas9 was introduced directly into the pronuclei by electric pulse or transferred from the cytoplasm by nuclear localization signal (NLS) remained unknown. Herein, we evaluated the localization of Cas9 with (Cas9-NLS) or without NLS (Cas9-noNLS) in mice embryos following electroporation by fusing them with GFP. Furthermore, we visually studied their effects on genome-editing rates in offspring by targeting tyrosinase gene. Fluorescence intensity in pronuclei of Cas9-NLS-electroporated embryos and genome-editing rates of offspring were significantly higher than those of Cas9-noNLS-electroporated embryos. Furthermore, fluorescence in Cas9-NLS-electroporated embryos in which pronuclei had not yet appeared 2.5 h after insemination was observed in the pronuclei of embryos appearing 3.5 h after electroporation. We demonstrated the effective transportation of Cas9 from the cytoplasm to pronuclei by the NLS following TAKE, which resulted in increased genome-editing rates in offspring. The TAKE along with fluorescently labeled nucleases can be used to verify nuclease delivery into individual embryos prior to embryo transfer for efficiently producing genome-edited animals.