Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.
Keywords: Tol2; CRISPR/Cas9; CreER; LSL-Cas9; conditional knockout mouse; genome editing; one-step; tamoxifen; transposon; tyrosinase gRNA.
© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.