Development of cell labeling and gene editing tools in urochordate Ciona.

Urochordate Ciona spp. are ideal marine model organisms for studying embryogenesis and developmental and evolutionary biology. However, the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging. This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp. The destination vector sets contained two Gateway cassettes flanked by Minos sites, allowing the N- or C-terminal tagging of a protein of interest with various fluorescent markers. In addition, we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry, a fluorescent indicator for Cas9 expression at cellular resolution. We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs. Furthermore, we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues. The genetic tools developed in this study offer a robust method for gene expression, cell tracking, and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-025-00300-1.