, Volume 844, Issue 1, pp 117–128 | Cite as

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

  • Duck-Hyun Kim
  • Jihyeon Yu
  • Jun Chul Park
  • Chang-Bum Jeong
  • Sangsu BaeEmail author
  • Jae-Seong LeeEmail author


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.


Electroporation CRISPR-Cas9 Marine rotifer Brachionus koreanus Bk-CYP3045C1 gene 



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.

Supplementary material

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Supplementary material 1 (DOCX 341 kb)


  1. Bae, S., J. Kweon, H. S. Kim & J.-S. Kim, 2014a. Microhomology-based choice of Cas9 nuclease target sites. Nat Methods 11: 705–706.PubMedGoogle Scholar
  2. 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.PubMedPubMedCentralGoogle Scholar
  3. 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.PubMedPubMedCentralGoogle Scholar
  4. 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.PubMedGoogle Scholar
  5. 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
  6. Dahms, H.-U., A. Hagiwara & J.-S. Lee, 2011. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat Toxicol 101: 1–12.PubMedGoogle Scholar
  7. 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.Google Scholar
  8. 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
  9. 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.PubMedGoogle Scholar
  10. 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.PubMedGoogle Scholar
  11. 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.Google Scholar
  12. 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
  13. 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.PubMedGoogle Scholar
  14. 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.PubMedGoogle Scholar
  15. 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.Google Scholar
  16. 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.Google Scholar
  17. 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
  18. 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.PubMedGoogle Scholar
  19. 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.Google Scholar
  20. 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.PubMedGoogle Scholar
  21. 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.PubMedGoogle Scholar
  22. 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.Google Scholar
  23. 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.PubMedPubMedCentralGoogle Scholar
  24. 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
  25. 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.PubMedGoogle Scholar
  26. 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.PubMedPubMedCentralGoogle Scholar
  27. 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.PubMedGoogle Scholar
  28. 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.PubMedGoogle Scholar
  29. 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.PubMedGoogle Scholar
  30. Lin, C. Y. & Y. H. Su, 2016. Genome editing in sea urchin by using a CRISPR/Cas9 system. Dev Biol 409: 420–428.PubMedGoogle Scholar
  31. 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.PubMedGoogle Scholar
  32. 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.Google Scholar
  33. 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.PubMedPubMedCentralGoogle Scholar
  34. Momose, T. & J. P. Concordet, 2016. Diving into marine genomics with CRISPR/Cas9 systems. Mar Genom 30: 55–65.PubMedGoogle Scholar
  35. 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.PubMedPubMedCentralGoogle Scholar
  36. Nakanishi, T., Y. Kato, T. Matsuura & H. Watanabe, 2014. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna. PLoS ONE 9: e98363.PubMedPubMedCentralGoogle Scholar
  37. 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.PubMedPubMedCentralGoogle Scholar
  38. 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.PubMedGoogle Scholar
  39. 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.PubMedGoogle Scholar
  40. 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.PubMedGoogle Scholar
  41. 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.PubMedGoogle Scholar
  42. 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
  43. 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.PubMedGoogle Scholar
  44. 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.PubMedPubMedCentralGoogle Scholar
  45. 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.PubMedGoogle Scholar
  46. Shearer, T. L. & T. W. Snell, 2007. Transfection of siRNA into Brachionus plicatilis (Rotifera). Hydrobiologia 593: 141–150.Google Scholar
  47. Snell, T. W., 2011. A review of the molecular mechanisms of monogonont rotifer reproduction. Hydrobiologia 662: 89–97.Google Scholar
  48. 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.PubMedPubMedCentralGoogle Scholar
  49. 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.PubMedGoogle Scholar
  50. Stolfi, A., S. Gandhi, F. Salek & L. Christiaen, 2014. Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9. Development 141: 4115–4120.PubMedPubMedCentralGoogle Scholar
  51. 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.PubMedGoogle Scholar
  52. 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.Google Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Biological Science, College of ScienceSungkyunkwan UniversitySuwonSouth Korea
  2. 2.Department of Chemistry and Research Institute for Convergence of Basic SciencesHanyang UniversitySeoulSouth Korea

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