# Establishment of CRISPR-Cas9-Mediated Gene Editing in the Swimming Crab Portunus trituberculatus

## TL;DR

Researchers successfully used CRISPR-Cas9 to edit genes in the swimming crab, opening new possibilities for genetic studies and aquaculture improvement.

## Contribution

Established CRISPR-Cas9 gene editing in the swimming crab using optimized electroporation conditions.

## Key findings

- Artificial seawater was a better buffer than PBS for electroporation in crab zygotes.
- Optimized electroporation resulted in 72.7% fluorescent zygotes and gene modifications at the myostatin locus.

## Abstract

Portunus trituberculatus is an economically important marine crustacean in East Asia’s aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in P. trituberculatus using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (mstn) gene. These results demonstrate the feasibility of Cas9-based genome editing in P. trituberculatus and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species.