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Targeted Gene Disruption in Pacific Oyster Based on CRISPR/Cas9 Ribonucleoprotein Complexes

Abstract

The Pacific oyster (Crassostrea gigas) is a representative bivalve mollusc that is widely cultured in the world. In recent years, it has become an important model species for ecological, evolutionary, and developmental studies because of its ability to survive in extreme environmental conditions as a sessile filter feeder and its classical mosaic pattern of development. Although the complete genome sequence of C. gigas is available and omics data have been rapidly generated for the past few years, the genetic tools for gene functional studies have thus far been limited to RNA interference technology. In this study, we developed a gene editing system for C. gigas based on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 ribonucleoprotein complexes. Two candidate genes, myostatin (MSTN) and Twist, were selected as targets. After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, CRISPR-induced indel mutations were detected in the target genes. The CRISPR/Cas9-induced mutations were predominantly small indel mutations ranging in size from 1 to 24 bp in these two target genes. These results demonstrate that CRISPR/Cas9 can be successfully used as an effective targeted gene editing system in C. gigas. The method reported here provides a powerful tool for gene functional studies in oysters and other marine bivalves, and potentially as a new technology for genetic engineering to improve oyster traits for aquaculture.

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Funding

This research was supported by grants from National Natural Science Foundation of China (31672649), Shandong Province (2017LZGC009), the Fundamental Research Funds for the Central Universities (201762014), and Taishan Scholars Seed Project of Shandong.

Author information

Conceived and designed the experiments: QL and SD. Performed the experiments: HL, RX, CY, and HY. Analyzed the data: HL, HY, and SD. Wrote and revised the paper: HY, QL, and SD.

Correspondence to Qi Li.

Ethics declarations

All animal studies were carried out according to the guideline for the Care and Use of Laboratory Animals of Ocean University of China. The protocol was approved by the Institutional Animal Care and Use Committee of Ocean University of China.

Competing Interest

The authors declare that there is no conflict of interest.

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Cite this article

Yu, H., Li, H., Li, Q. et al. Targeted Gene Disruption in Pacific Oyster Based on CRISPR/Cas9 Ribonucleoprotein Complexes. Mar Biotechnol 21, 301–309 (2019). https://doi.org/10.1007/s10126-019-09885-y

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Keywords

  • CRISPR
  • Cas9
  • Crassostrea gigas
  • Gene editing