One-step generation of mice by Transposon-Enhanced Multi-Plex Orchestration Editing(TEMPO-Editing)

Genome-edited mice are widely used to elucidate molecular mechanisms in vivo and are an indispensable tool, particularly for studies aimed at clarifying gene function at the organismal level. Currently, there is an increasing demand for mice in which multiple genes are simultaneously modified in order to investigate interactions among multiple genes. Notably, generating conditional multiple-gene knockout mice with temporal and spatial specificity requires extensive crossing between multiple Cre-driver mice and floxed mice, resulting in a prolonged time frame for line establishment. To address this limitation, we developed Transposon-Enhanced Multi-Plex Orchestration Editing (TEMPO-editing), a single-step strategy for generating multiple-gene-edited mice. TEMPO-editing enables simultaneous modification of multiple genes through the integration of transposon, Cre-loxP, and CRISPR/Cas9 systems. Using the DNA transposon piggyBac, we constructed a single cassette harboring gRNAs targeting genes of interest together with a conditionally expressed Cas9 (lsl-Cas9). By injecting this cassette into fertilized eggs of Cre mice, we enabled the generation of temporally and spatially specific genome edited mice in the F0 generation. In this study, we generated double-gene-edited mice targeting Hoxa13 and Hoxd13, which are key regulators of embryonic body patterning and are essential for autopod development. The phenotype observed in these mice was consistent with the incomplete autopod phenotype previously reported in mice generated by crossing Hoxa13 knockout and Hoxd13 knockout mice. These results demonstrate the utility of TEMPO-editing for the generation of multiple-gene-edited mice in the F0 generation. Notably, this study establishes a simplified strategy for producing conditional multi-gene-edited mice, a process that has traditionally required substantial time and labor using conventional methods.