Skip to main content
SpringerLink
Log in

Targeted cytochrome P450 3045C1 (CYP3045C1) gene mutation via CRISPR-Cas9 ribonucleoproteins in the marine rotifer Brachionus koreanus

  • ROTIFERA XV
  • Published:
Hydrobiologia Aims and scope Submit manuscript

Abstract

The CRISPR-Cas9 system has revolutionized genetic engineering and has been applied in numerous model organisms to date. To examine the capacity of the CRISPR-Cas9 system for generation of mutants in the marine rotifer Brachionus koreanus, we electroporated purified Cas9 proteins fused with GFP (Cas9-GFP) into rotifers. A dose-dependent increase in green fluorescent signal was highly detected in ovary and eggs. The purified Cas9-GFP proteins showed sustained fluorescence signals in rotifers at 24 h after electroporation, which also suggests stability of Cas9 ribonucleoproteins (RNPs) and the possibility of Cas9-mediated gene editing in rotifers. We electroporated B. koreanus with the wild-type Cas9 and single-guide RNAs targeting the endogenous Bk-CYP3045C1 gene and observed different insertion and deletion (indel) mutation profiles near the DNA cleavage sites using targeted deep sequencing. Although the indel rates were low in several salinity conditions (0.30% in 1 psu and 0.20% in 2 psu), these results confirm successful Cas9 RNP-induced mutations. Our results confirm that CRISPR-Cas9 can be applied to generate diverse mutants to demonstrate the functional roles of genes in rotifers.

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

Similar content being viewed by others

References

  • Bae, S., J. Kweon, H. S. Kim & J.-S. Kim, 2014a. Microhomology-based choice of Cas9 nuclease target sites. Nat Methods 11: 705–706.

    CAS  PubMed  Google Scholar 

  • Bae, S., J. Park & J.-S. Kim, 2014b. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30: 1473–1475.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bothmer, A., T. Phadke, L. A. Margulies, C. M. Barrera, C. S. Lee, F. Buquicchio, S. Moss, H. S. Abdulkerim, W. Selleck, H. Jayaram, V. E. Myer & C. Cotta-Ramusino, 2017. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat Commun 8: 13905.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Braglia, P., R. Percudani & G. Dieci, 2005. Sequence context effects on oligo(dT) termination signal recognition by Saccharomyces cerevisiae RNA polymerase III. J Biol Chem 280: 19551–19562.

    CAS  Google Scholar 

  • Butcher, R. W., 1959. An introductory account of the smaller algae of British coastal waters. Part I: Introduction and Chlorophyceae. Fish Investig Lond Ser IV 1: 1–74.

    Google Scholar 

  • Dahms, H.-U., A. Hagiwara & J.-S. Lee, 2011. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat Toxicol 101: 1–12.

    CAS  PubMed  Google Scholar 

  • Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C. Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, H. S. Lai, S. P. Lin, Y. Qian, H. G. Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton, B. A. Roe & R. McLaughlin, 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 98: 4658–4663.

    CAS  Google Scholar 

  • Fontaneto, D., I. Giordani, G. Melone & M. Serra, 2007. Disentangling the morphological stasis in two rotifer species of the Brachionus plicatilis species complex. Hydrobiologia 583: 297–307.

    Google Scholar 

  • Han, J., E.-J. Won, I.-C. Kim, J. H. Yim, S.-J. Lee & J.-S. Lee, 2014. Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus. Aquat Toxicol 155: 101–109.

    CAS  PubMed  Google Scholar 

  • Han, J., E.-J. Won, U.-K. Hwang, I.-C. Kim, J. H. Yim & J.-S. Lee, 2016. Triclosan (TCS) and triclocarban (TCC) cause lifespan reduction and reproductive impairment through due to oxidative stress-mediated expression of the defensome in the monogonont rotifer (Brachionus koreanus). Comp Biochem Physiol C 185(186): 131–137.

    PubMed  Google Scholar 

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

    CAS  Google Scholar 

  • Hwang, D.-S., H.-U. Dahms, H. G. Park & J.-S. Lee, 2013a. A new intertidal Brachionus and intrageneric phylogenetic relationship among Brachionus as revealed by allometry and CO1-ITS1 gene analysis. Zool Stud 52: 13.

    Google Scholar 

  • Hwang, D.-S., K. Suga, Y. Sakakura, H.-G. Park, A. Hagiwara, J.-S. Rhee & J.-S. Lee, 2013b. Complete mitochondrial genome of the monogonont rotifer, Brachionus koreanus (Rotifera, Brachionidae). Mitochondrial DNA 25: 29–30.

    PubMed  Google Scholar 

  • Ikmi, A., S. A. McKinney, K. M. Delventhal & M. C. Gibson, 2014. TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun 5: 5486.

    CAS  PubMed  Google Scholar 

  • Jeong, C.-B., E.-J. Won, H.-M. Kang, M.-C. Lee, D.-S. Hwang, U.-K. Hwang, B. Zhou, S. Souissi, S.-J. Lee & J.-S. Lee, 2016. Microplastic size-dependent toxicity, oxidative stress induction, and p-JNK and p-p38 activation in the monogonont rotifer (Brachionus koreanus). Environ Sci Technol 50: 8849–8857.

    CAS  Google Scholar 

  • Jeong, C.-B., H.-M. Kang, Y. H. Lee, M.-S. Kim, J.-S. Lee, J. Seo, M. Wang & J.-S. Lee, 2018. Nanoplastic ingestion enhances toxicity of persistent organic pollutants (POPs) in the monogonont rotifer Brachionus koreanus via multixenobiotic resistance (MXR) disruption. Environ Sci Technol 52: 11411–11418.

    CAS  Google Scholar 

  • Joly, J. S., 2017. Aquatic model organisms in neurosciences: the genome-editing revolution. In Jaenisch, R., F. Zhang & F. Gage (eds), Genome editing in neurosciences. Research and perspectives in neurosciences. Springer, New York: 21–29.

    Google Scholar 

  • Kang, H.-M., C.-B. Jeong, M.-S. Kim, J.-S. Lee, J. Zhou, Y. H. Lee, D.-H. Kim, E. Moon, H.-S. Kweon, S.-J. Lee & J.-S. Lee, 2018. The role of the p38-activated protein kinase signaling pathway-mediated autophagy in cadmium-exposed monogonont rotifer Brachious koreanus. Aquat Toxicol 194: 46–56.

    CAS  PubMed  Google Scholar 

  • Kang, H.-M., J.-S. Lee, Y.H. Lee, M.-S. Kim, H.G. Park, C.-B. Jeong & J.-S. Lee, 2019. Body size-dependent interspecific tolerance to cadmium in the marine rotifer Brachionus spp. and their molecular responses. Aquatic Toxicology, in press.

  • Kim, R.-O., J.-S. Rhee, E.-J. Won, K.-W. Lee, C.-M. Kang, Y.-M. Lee & J.-S. Lee, 2011a. Ultraviolet B retards growth, induces oxidative stress, and modulates DNA repair-related gene and heat shock protein gene expression in the monogonont rotifer, Brachionus sp. Aquat Toxicol 101: 529–539.

    CAS  PubMed  Google Scholar 

  • Kim, R.-O., J.-S. Rhee, E.-J. Won, K.-W. Lee, C.-M. Kang, Y.-M. Lee & J.-S. Lee, 2011b. Ultraviolet B retards growth, induces oxidative stress, and modulates DNA repair-related gene and heat shock protein gene expression in the monogonont rotifer, Brachionus sp. Aquat Toxicol 101: 529–539.

    CAS  PubMed  Google Scholar 

  • Kim, R.-O., B.-M. Kim, C.-B. Jeong, D. R. Nelson, J.-S. Lee & J.-S. Rhee, 2013. Expression pattern of entire cytochrome P450 genes and response of defensomes in the benzo[α]pyrene-exposed monogonont rotifer Brachionus koreanus. Environ Sci Technol 47: 13804–13812.

    CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kim, B.-M., J. W. Lee, J. S. Seo, K.-H. Shin, J.-S. Rhee & J.-S. Lee, 2015. Modulated expression and enzymatic activity of the monogonont rotifer Brachionus koreanus Cu/Zn- and Mn-superoxide dismutase (SOD) in response to environmental biocides. Chemosphere 120C: 470–478.

    Google Scholar 

  • Kim, R.-O., B.-M. Kim, C.-B. Jeong, J.-S. Lee & J.-S. Rhee, 2016. Effects of chlorpyrifos on life cycle parameters, cytochrome P450 s (CYPs) expression, and antioxidant system in the monogonont rotifer Brachionus koreanus. Environ Toxicol Chem 35: 1449–1457.

    CAS  PubMed  Google Scholar 

  • Kumagai, H., T. Nakanishi, T. Matsuura, Y. Kato & H. Watanabe, 2017. CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna. PLoS ONE 12: e0186112.

    PubMed  PubMed Central  Google Scholar 

  • Lee, J. W., H.-M. Kang, E.-J. Won, D.-S. Hwang, D.-H. Kim, S.-J. Lee & J.-S. Lee, 2016. Multi-walled carbon nanotubes (MWCNTs) lead to growth retardation, antioxidant depletion, and activation of the ERK signaling pathway but decrease copper bioavailability in the monogonont rotifer Brachionus koreanus. Aquat Toxicol 172: 67–79.

    CAS  PubMed  Google Scholar 

  • Lee, Y. H., D.-H. Kim, H.-M. Kang, C.-B. Jeong, M. Wang & J.-S. Lee, 2017. Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the rotifer Brachionus koreanus and the copepod Paracyclopina nana. Aquat Toxicol 190: 181–189.

    CAS  PubMed  Google Scholar 

  • Lee, Y. H., H.-M. Kang, M.-S. Kim, J.-S. Lee, C.-B. Jeong & J.-S. Lee, 2018. The protective role of multixenobiotic resistance (MXR)-mediated ATP-binding cassette (ABC) transporters in biocides-exposed rotifer Brachionus koreanus. Aquat Toxicol 195: 129–136.

    CAS  PubMed  Google Scholar 

  • Lin, C. Y. & Y. H. Su, 2016. Genome editing in sea urchin by using a CRISPR/Cas9 system. Dev Biol 409: 420–428.

    CAS  PubMed  Google Scholar 

  • Martin, A., J. M. Serano, E. Jarvis, H. S. Bruce, J. Wang, S. Ray, C. A. Barker, L. C. O’Connell & N. H. Patel, 2016. CRISPR/Cas9 mutagenesis reveals versatile roles of Hox genes in crustacean limb specification and evolution. Curr Biol 26: 14–26.

    CAS  PubMed  Google Scholar 

  • Mills, S., J. A. Alcántara-Rodríguez, J. Ciros-Pérez, A. Gómez, A. Hagiwara, K. H. Galindo, C. D. Jersabek, R. Malekzadeh-Viayeh, F. Leasi, J.-S. Lee, D. B. MarkWelch, S. Riss, S. Papakostas, H. Segers, M. Serra, R. Shiel, R. Smolak, T. W. Snell, C.-P. Stelzer, C. Q. Tang, R. L. Wallace, D. Fontaneto & E. Walsh, 2017. Fifteen species in one: deciphering the Brachionus plicatilis species complex (Rotifera, Monogononta) through DNA taxonomy. Hydrobiologia 796: 39–58.

    CAS  Google Scholar 

  • Mohamad Ishak, N. S., Q. D. Nong, T. Matsuura, Y. Kato & H. Watanabe, 2017. Co-option of the bZIP transcription factor Vrille as the activator of Doublesex1 in environmental sex determination of the crustacean Daphnia magna. PLoS Genet 13: e1006953.

    PubMed  PubMed Central  Google Scholar 

  • Momose, T. & J. P. Concordet, 2016. Diving into marine genomics with CRISPR/Cas9 systems. Mar Genom 30: 55–65.

    PubMed  Google Scholar 

  • Naitou, A., Y. Kato, T. Nakanishi, T. Matsuura & H. Watanabe, 2015. Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna. Biol Open 4: 364–369.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nakanishi, T., Y. Kato, T. Matsuura & H. Watanabe, 2014. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna. PLoS ONE 9: e98363.

    PubMed  PubMed Central  Google Scholar 

  • Nymark, M., A. K. Sharma, T. Sparstad, A. M. Bones & P. Winge, 2016. A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 6: 24951.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Park, J., S. Bae & J.-S. Kim, 2015. Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target site. Bioinformatics 31: 4014–4016.

    CAS  PubMed  Google Scholar 

  • Park, J., K. Lim, J.-S. Kim & S. Bae, 2017a. Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33: 286–288.

    CAS  PubMed  Google Scholar 

  • Park, J. C., J. Han, M.-C. Lee, H.-M. Kang, C.-B. Jung, D.-S. Hwang, M. Wang & J.-S. Lee, 2017b. Adverse effects of BDE-47 on life cycle parameters, antioxidant system, and activation of MAPK signaling pathway in the rotifer Brachionus koreanus. Aquat Toxicol 186: 105–112.

    CAS  PubMed  Google Scholar 

  • Park, J. C., M.-C. Lee, D.-S. Yoon, J. Han, M. Kim, U.-K. Hwang, J.-H. Jung & J.-S. Lee, 2018. Effects of bisphenol A and its analogs bisphenol F and S on life parameters, antioxidant system, and response of defensome in the marine rotifer Brachionus koreanus. Aquat Toxicol 199: 21–29.

    CAS  PubMed  Google Scholar 

  • Quiroga Artigas, G., P. Lapébie, L. Leclère, N. Takeda, R. Deguchi, G. Jékely, T. Momose & E. Houliston, 2018. A gonad-expressed opsin mediates light-induced spawning in the jellyfish Clytia. eLife 7: 29555.

    Google Scholar 

  • Rhee, J.-S., C.-B. Jeong, B.-M. Kim & J.-S. Lee, 2012. P-glycoprotein (P-gp) in the monogonont rotifer, Brachionus koreanus: molecular characterization and expression in response to pharmaceuticals. Aquat Toxicol 114(115): 104–118.

    PubMed  Google Scholar 

  • Rivetti, C., B. Campos, B. Piña, D. Raldúa, Y. Kato, H. Watanabe & C. Barata, 2018. Tryptophan hydroxylase (TRH) loss of function mutations induce growth and behavioral defects in Daphnia magna. Sci Rep 8: 1518.

    PubMed  PubMed Central  Google Scholar 

  • Sasaki, H., K. Yoshida, A. Hozumi & Y. Sasakura, 2014. CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis. Dev Growth Differ 56: 499–510.

    CAS  PubMed  Google Scholar 

  • Shearer, T. L. & T. W. Snell, 2007. Transfection of siRNA into Brachionus plicatilis (Rotifera). Hydrobiologia 593: 141–150.

    CAS  Google Scholar 

  • Snell, T. W., 2011. A review of the molecular mechanisms of monogonont rotifer reproduction. Hydrobiologia 662: 89–97.

    CAS  Google Scholar 

  • Snell, T. W., T. L. Shearer, H. A. Smith, J. Kubanek, K. E. Gribble & D. B. Mark Welch, 2009. Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera). BMC Biol 7: 60.

    PubMed  PubMed Central  Google Scholar 

  • Snell, T. W., T. L. Shearer & H. A. Smith, 2011. Exposure to dsRNA elicits RNA interference in Brachionus manjavacas (Rotifera). Mar Biotechnol 13: 264–274.

    CAS  PubMed  Google Scholar 

  • Stolfi, A., S. Gandhi, F. Salek & L. Christiaen, 2014. Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9. Development 141: 4115–4120.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang, T., J. J. Wei, D. M. Sabatini & E. S. Lander, 2014. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343: 80–84.

    CAS  PubMed  Google Scholar 

  • Won, E.-J., R.-O. Kim, H.-M. Kang, H.-S. Kim, D.-S. Hwang, J. Han, Y. H. Lee, U.-K. Hwang, B. Zhou, S.-J. Lee & J.-S. Lee, 2016. Adverse effects, expression of the Bk-CYP3045C1 gene, and activation of the ERK signaling pathway in the water accommodated fraction-exposed rotifer. Environ Sci Technol 50: 6025–6035.

    CAS  Google Scholar 

Download references

Acknowledgements

We thank two anonymous reviewers for their valuable comments on the manuscript. This work was supported by a Grant from the National Research Foundation of Korea (NRF) No. 2018M3A9H3022412 to S.B. and was also supported by a Grant from the Collaborative Genome Program of the Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) (No. 20180430) to J.-S.L.

Author information

Author notes

  1. Duck-Hyun Kim and Jihyeon Yu have contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Science, College of Science, Sungkyunkwan University, Suwon, 16419, South Korea

    Duck-Hyun Kim, Jun Chul Park, Chang-Bum Jeong & Jae-Seong Lee

  2. Department of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, 04763, South Korea

    Jihyeon Yu & Sangsu Bae

Authors
  1. Duck-Hyun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jihyeon Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jun Chul Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chang-Bum Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sangsu Bae
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jae-Seong Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Sangsu Bae or Jae-Seong Lee.

Additional information

Guest editors: Steven A. J. Declerck, Diego Fontaneto, Rick Hochberg & Terry W. Snell / Crossing Disciplinary Borders in Rotifer Research

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 341 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, DH., Yu, J., Park, J.C. et al. Targeted cytochrome P450 3045C1 (CYP3045C1) gene mutation via CRISPR-Cas9 ribonucleoproteins in the marine rotifer Brachionus koreanus. Hydrobiologia 844, 117–128 (2019). https://doi.org/10.1007/s10750-018-3854-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10750-018-3854-y

Keywords

Search

Navigation