Skip to main content
SpringerLink
Log in

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

  • Short Communication
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K (2015) Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol 16:87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bono JM, Olesnicky EC, Matzkin LM (2015) Connecting genotypes, phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing. Mol Ecol 24:3810–3822

    Article  CAS  PubMed  Google Scholar 

  • Burger A, Lindsay H, Felker A, Hess C, Anders C, Chiavacci E, Zaugg J, Weber LM, Catena R, Jinek M, Robinson MD, Mosimann C (2016) Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development 143:2025–2037

    Article  CAS  PubMed  Google Scholar 

  • Cai M, Si Y, Zhang J, Tian Z, Du S (2018) Zebrafish embryonic slow muscle is a rapid system for genetic analysis of sarcomere organization by CRISPR/Cas9, but not NgAgo. Mar Biotechnol 20:168–181

    Article  CAS  Google Scholar 

  • Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096

    Article  CAS  PubMed  Google Scholar 

  • Fabioux C, Corporeau C, Quillien V, Favrel P, Huvet A (2009) In vivo RNA interference in oyster-vasa silencing inhibits germ cell development. FEBS J 276:2566–2573

    Article  CAS  PubMed  Google Scholar 

  • FAO (2018) The state of world fisheries and aquaculture 2016. Food and Agriculture Organization of the United Nations, Rome

    Google Scholar 

  • Hirsinger E, Carvalho JE, Chevalier C, Lutfalla G, Nicolas JF, Peyriéras N, Schubert M (2015) Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes. J Vis Exp 95:52042

    Google Scholar 

  • Hiruta C, Kakui K, Tollefsen KE, Iguchi T (2018) Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex. Genes Cells 23:494–502

    Article  CAS  PubMed  Google Scholar 

  • Huang X, Huan P, Liu B (2017) A comparative proteomic analysis reveals important proteins for the fertilization and early embryonic development of the oyster Crassostrea gigas. Proteomics 17:1600251

    Article  CAS  Google Scholar 

  • Huvet A, Béguel JP, Cavaleiro NP, Thomas Y, Quillien V, Boudry P, Alunno-Bruscia M, Fabioux C (2015) Disruption of amylase genes by RNA interference affects reproduction in the Pacific oyster Crassostrea gigas. J Exp Biol 218:1740–1747

    Article  PubMed  Google Scholar 

  • Kim S, Kim D, Cho SW, Kim J, Kim JS (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li Q, Wang Q, Liu S, Kong L (2011) Selection response and realized heritability for growth in three stocks of the Pacific oyster Crassostrea gigas. Fish Sci 77:643–648

    Article  CAS  Google Scholar 

  • Lin CY, Su YH (2016) Genome editing in sea urchin embryos by using a CRISPR/Cas9 system. Dev Biol 409:420–428

    Article  CAS  PubMed  Google Scholar 

  • Liu C, Zhang L, Liu H, Cheng K (2017) Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release 266:17–26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mehravar M, Shirazi A, Nazari M, Banan M (2018) Mosaicism in CRISPR/Cas9-mediated genome editing. Dev Biol 445:156–162

    Article  CAS  PubMed  Google Scholar 

  • Midic U, Hung PH, Vincent KA, Goheen B, Schupp PG, Chen DD, Bauer DE, VandeVoort CA, Latham KE (2017) Quantitative assessment of timing, efficiency, specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesus monkey embryos. Hum Mol Genet 26:2678–2689

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Momose T, Concordet JP (2016) Diving into marine genomics with CRISPR/Cas9 systems. Mar Genomics 30:55–65

    Article  PubMed  Google Scholar 

  • Perry KJ, Henry JQ (2015) CRISPR/Cas9-mediated genome modification in the mollusc, Crepidula fornicate. Genesis 53:237–244

    Article  CAS  PubMed  Google Scholar 

  • Riviere G, He Y, Tecchio S, Crowell E, Gras M, Sourdaine P, Guo X, Favrel P (2017) Dynamics of DNA methylomes underlie oyster development. PLoS Genet 13:e1006807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song K, Li Y, Huang B, Li L, Zhang G (2017) Genetic and evolutionary patterns of innate immune genes in the Pacific oyster Crassostrea gigas. Dev Comp Immunol 77:17–22

    Article  CAS  PubMed  Google Scholar 

  • Stepicheva NA, Song JL (2014) High throughput microinjections of sea urchin zygotes. J Vis Exp 83:e50841

    Google Scholar 

  • Taning CNT, Van Eynde B, Yu N, Ma S, Smagghe G (2017) CRISPR/Cas9 in insects: applications, best practices and biosafety concerns. J Insect Physiol 98:245–257

    Article  CAS  PubMed  Google Scholar 

  • Walles, de Paiva JS, van Prooijen BC, Ysebaert T, Smaal AC (2015) The ecosystem engineer Crassostrea gigas affects tidal flat morphology beyond the boundary of their reef structures. Estuar Coasts 38:941–950

    Article  Google Scholar 

  • Wang Q, Li Q, Kong L, Yu R (2012) Response to selection for fast growth in the second generation of Pacific oyster (Crassostrea gigas). J Ocean Univ China 11:413–418

    Article  Google Scholar 

  • Wang Q, Cao R, Ning X, You L, Mu C, Wang C, Wei L, Cong M, Wu H, Zhao J (2016) Effects of ocean acidification on immune responses of the Pacific oyster Crassostrea gigas. Fish Shellfish Immunol 49:24–33

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Cobine PA, Coleman JJ (2018) Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes. Fungal Genet Biol 117:21–29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yue C, Li Q, Yu H (2018) Gonad transcriptome analysis of the Pacific oyster Crassostrea gigas identifies potential genes regulating the sex determination and differentiation process. Mar Biotechnol 20:206–219

    Article  CAS  Google Scholar 

  • Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, Xiong Z, Que H, Xie Y, Holland PW, Paps J, Zhu Y, Wu F, Chen Y, Wang J, Peng C, Meng J, Yang L, Liu J, Wen B, Zhang N, Huang Z, Zhu Q, Feng Y, Mount A, Hedgecock D, Xu Z, Liu Y, Domazet-Lošo T, Du Y, Sun X, Zhang S, Liu B, Cheng P, Jiang X, Li J, Fan D, Wang W, Fu W, Wang T, Wang B, Zhang J, Peng Z, Li Y, Li N, Wang J, Chen M, He Y, Tan F, Song X, Zheng Q, Huang R, Yang H, Du X, Chen L, Yang M, Gaffney PM, Wang S, Luo L, She Z, Ming Y, Huang W, Zhang S, Huang B, Zhang Y, Qu T, Ni P, Miao G, Wang J, Wang Q, Steinberg CE, Wang H, Li N, Qian L, Zhang G, Li Y, Yang H, Liu X, Wang J, Yin Y, Wang J (2012) The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490:49–54

    Article  CAS  PubMed  Google Scholar 

  • Zhao X, Yu H, Kong L, Liu S, Li Q (2016) High throughput sequencing of small RNAs transcriptomes in two Crassostrea oysters identifies microRNAs involved in osmotic stress response. Sci Rep 6:22687

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao X, Li Q, Meng Q, Yue C, Xu C (2017) Identification and expression of cysteine sulfinate decarboxylase, possible regulation of taurine biosynthesis in Crassostrea gigas in response to low salinity. Sci Rep 7:5505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zuo E, Cai YJ, Li K, Wei Y, Wang BA, Sun Y, Liu Z, Liu J, Hu X, Wei W, Huo X, Shi L, Tang C, Liang D, Wang Y, Nie YH, Zhang CC, Yao X, Wang X, Zhou C, Ying W, Wang Q, Chen RC, Shen Q, Xu GL, Li J, Sun Q, Xiong ZQ, Yang H (2017) One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Res 27:933–945

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Author notes

  1. Hong Yu and Huijuan Li contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, 266003, China

    Hong Yu, Huijuan Li, Qi Li, Rui Xu & Chenyang Yue

  2. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China

    Hong Yu & Qi Li

  3. Institute of Marine and Environmental Technology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, 21202, USA

    Shaojun Du

Authors
  1. Hong Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Huijuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rui Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chenyang Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shaojun Du
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

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.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-019-09885-y

Keywords

Search

Navigation