Efficient Microinjection and CRISPR/Cas9 Mediated Genome Editing in Urechis unicinctus.

Urechis unicinctus is an economically important aquaculture species and an emerging model for developmental and evolutionary research; however, progress in functional genetic studies in this species has remained limited by the absence of effective genome editing approaches. Here, we establish a CRISPR/Cas9-mediated genome editing platform in U. unicinctus based on an optimized microinjection procedure. Targeting the cilia-associated gene Caveolin-1, Cas9/sgRNA ribonucleoprotein complexes were delivered into oocytes, generating mosaic G0 larvae harboring insertion-deletion mutations, as confirmed by ICE analysis and cloning-based Sanger sequencing with mutation detection rates of 68.51% and 64.86%, respectively. Ciliary defects were frequently observed in edited larvae, including shortening of the circumoral ciliary ring and reduction of apical ciliary tufts, accompanied by impaired swimming performance. Whole-mount in situ hybridization further revealed altered spatial expression patterns of Caveolin-1 in edited embryos, with 78% of individuals showing detectable changes in expression patterns. To independently assess the robustness of the platform, the conserved cytoskeletal gene α-tubulin was additionally targeted, resulting in successful mutagenesis and associated ciliary abnormalities. Together, these findings establish the first CRISPR/Cas9 genome editing workflow for U. unicinctus, providing a technical framework for functional genomics in echiurans and facilitating future studies of developmental mechanisms and molecular breeding in this species.