Synthetic gRNA-mediated multiplex CRISPR enables the generation of translucent F0 Xenopus laevis for in vivo imaging

Abstract Transparent model organisms are invaluable for live imaging, yet generating them remains challenging. Here, we present a robust strategy to produce translucent Xenopus laevis , enabling non-invasive, deep-tissue imaging in intact organisms. Using CRISPR/Cas9 technology, we generated quadruple knockout animals targeting slc2a7 , hps6 , and the two tyrosinase homeologs. Instead of in vitro-transcribed single guide RNAs, we employed chemically modified, commercially synthesized two-part guide RNAs, which enabled efficient multiplex genome editing. We produced a high proportion of translucent frogs directly in F0 founder tadpoles, eliminating the need for multi-generational breeding. We validated in vivo live imaging using two transgenic reporter lines with GFP expression in the eye, brain, and heart. Loss of both eumelanin and iridescent pigments in tyr ; hps6 knockouts markedly improved optical clarity and fluorescence visibility. Overall, this multiplexed genome-editing strategy enables the rapid generation of translucent transgenic X. laevis suitable for live imaging, while also providing a simple and efficient protocol for simultaneous multi-gene targeting in X. laevis .