Optimization of Electroporation Parameters for Crispr-Cas9 Mediated Gene Editing in the Swimming Crab Portunus Trituberculatus

Abstract

The Portunus trituberculatus represents a significant marine economic crustacean in East Asia's aquaculture industry. Nevertheless, its precise genome modification has yet to be overcome. To overcome this challenge, we investigated the applicability of the CRISPR-Cas9 gene editing technique in P. trituberculatus using an electroporation approach. We optimized the electroporation conditions for efficient Cas9-sgRNA complex delivery into zygotes. Parameters such as voltage, capacitance, pulse times, and electroporation buffer were systematically evaluated. Eventually, we found that artificial seawater was a preferable buffer over phosphate buffer saline (PBS), and under voltage of 600 V, capacitance of 6 μF, with two pulses, yielded the best results, with approximately 72.7% fluorescent zygotes. Targeting the myostatin gene (mstn), we applied the optimized electroporation method and realized gene mutations at the targeting locus. Our results signify the successful application of CRISPR-Cas9 gene editing in P. trituberculatus, paving the way for enhanced germplasm resources, improved aquaculture production, and exploration of gene function and biological processes in this valuable species. This study establishes a foundation for the precision breeding of the non-model swimming crab through Cas9-based genome editing techniques.

Keywords: P. trituberculatus, CRISPR-Cas9, gene editing, electroporation, mstn