CRISPR/Cas9 System-Mediated Gene Editing in the Fujian Oysters (Crassostrea angulate) by Electroporation

Kaidi Jin,Yaqiong Liu,Zhongqiang Cai,Yitao Qi,Baoyu Huang,Xiaotong Wang,Baolu Zhang,Qianqian Jin,Meiwei Zhang,Xiaomei Wang,Yanxin Zheng,Lei Wei

doi:10.3389/fmars.2021.763470

Abstract

The Fujian oyster (Crassostrea angulate) is an important marine bivalve mollusk with high economic value. Gene function research and gene editing techniques have broad application prospects in oyster. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for genome engineering in many species. CRISPR-mediated gene editing has also been used successfully in the Pacific oyster through direct delivery of the CRISPR/Cas9 components into oyster embryos by microinjection. However, the low throughput and operational difficulties associated with microinjection is one of the factors limiting the widespread application of CRISPR/Cas9 in oysters. In this study, we attempted to deliver the CRISPR/Cas9-system into the embryos of C. angulate by electroporation. An all-in-one CRISPR/Cas9 vector plasmid was used as CRISPR/Cas9 system in this study. Electroporation was carried out using both eggs and blastula larvae. A large number of larvae became malformed or die after electroporation. A single base substitution mutation was detected in the D-larvae developed from electroporated eggs. Our results demonstrate that the CRISPR/Cas9 system can be delivered into embryos of C. angulate for gene editing by electroporation, which provides a reference and will further contribute to the future application of electroporation in mollusks.

Highlights

In the past few decades, the marine mollusk farming industry has been developing rapidly
The pYSY-CRISPR-associated protein 9 (Cas9)-gRNA-GFP vector plasmid, carrying a gRNA scaffold driven by a U6 promoter, a Cas9 expression cassette driven by a CMV promoter, and a GFP reporter gene driven by an EF1α promoter, was used as the express component of the allin-one clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system
The Hox domain of the C. angulata Pitx gene was used for gRNA design. sgRNACaPitx was fused into the pYSY-Cas-gRNA-GFP vector, which encodes a functional CRISPR/Cas9 system

Summary

Introduction

In the past few decades, the marine mollusk farming industry has been developing rapidly. Various types of economic mollusks, including oysters, have been farmed on a large scale. Cultivating stable molluscan strains with fast growth, good quality, and strong stress resistance through artificial selection breeding is required. The development of genomic techniques, including CRISPR/Cas system-mediated genomic editing technology, can improve selection of breeding techniques for mollusks (Gutierrez et al, 2018; Hollenbeck and Johnston, 2018). The discovery and better comprehension of the gene function will help in understanding the mechanism of oyster growth, development, and response to stress at the molecular level. It can lay a foundation for genome research and genetic engineering breeding of mollusks

Methods

Results

Conclusion