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Hydrobiologia

, 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
ROTIFERA XV

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.

Keywords

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

Notes

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.

Supplementary material

10750_2018_3854_MOESM1_ESM.docx (341 kb)
Supplementary material 1 (DOCX 341 kb)

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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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