Universal conditional knockout approach to multiple species, from fish to human induced pluripotent stem cells

Investigating essential gene function in vertebrate development and disease is challenging due to associated lethality, necessitating precise conditional inactivation. While existing conditional knockout methods can be inefficient and demanding, especially in models like zebrafish and human induced pluripotent stem cells (iPSCs), a widely applicable system has remained elusive. Here, we demonstrate the expanded utility and versatility of a Short Conditional intrON (SCON) knockout cassette, now validated for efficient conditional loss-of-function mutation generation in diverse vertebrate models, including zebrafish, human iPSCs, and intestinal organoids. Building on this validated broad applicability, we establish a comprehensive, user-friendly web-based GenPos-SCON database (https://genpos.org/) to streamline conditional knockout design for over 300 vertebrate species. This powerful resource and proven methodology significantly accelerate systematic gene functional studies across the vertebrate tree, providing an unprecedented tool for dissecting fundamental biological processes and disease mechanisms in relevant contexts.