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Application of CRISPR-Cas9 genome editing by microinjection of gametophytes of Saccharina japonica (Laminariales, Phaeophyceae)

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing has been used for reverse genetics studies in many organisms. However, application to commercially important species of seaweeds is still limited. The genetics and breeding technologies for species of the Laminariales will be advanced by developing a genome editing tool. In this study, we attempted to edit a counter-selectable marker, the adenine phosphoribosyl transferase (APT) gene using CRISPR-Cas9 ribonucleoprotein (RNP) complexes delivered by microinjection into gametophytes of Saccharina japonica. After injection of CRISPR-Cas9 RNP, 2-fluoroadenine (2-FA) was added to the medium (10–40 μM) to select APT mutants. Twenty-three female and 12 male 2-FA resistant gametophytes had mutations in their APT genes. Genome editing efficiency for the injection trials was 8.64% and 4.46% for the female and male gametophytes, respectively. The apt mutant gametophytes were able to produce sperm or eggs. Sporophytes derived by crossing sperm and eggs from apt mutant gametophytes were resistant to 40 μM 2-FA. Sporophytes derived from crosses with one wild type and one mutant parent also showed resistance to high concentrations (20–40 μM) of 2-FA, while wild-type sporophytes died in 10 μM 2-FA. This is the first report showing the validity of gene editing of S. japonica using the CRISPR-Cas9 RNP complex and microinjection method.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgement

We would like to thank Dr. J Mark Cock, Station Biologique de Roscoff for his critical reading of the manuscript and helpful suggestions. We also would like to express our gratitude to Mr. Minori Harada (Department of Algal Development and Evolution Max Planck Institute for Biology Tübingen) for giving a useful technical suggestion for microinjection and genome editing.

Funding

This paper is based on results obtained from a project JPNP14004, commissioned by the New Energy and Industrial Technology Development Organization (NEDO).

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Authors and Affiliations

  1. Muroran Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, Muroran, 051-0013, Japan

    Yuan Shen, Taizo Motomura, Kensuke Ichihara & Chikako Nagasato

  2. Department of Bioscience, School of Biological and Environmental Sciences, Kwansei Gakuin University, Sanda, Hyogo, 669-1330, Japan

    Yusuke Matsuda

  3. Advanced Technology Research Laboratories, Nippon Steel, Futtsu, 293-8511, Japan

    Ko Yoshimura & Chika Kosugi

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  1. Yuan Shen
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Contributions

Yuan Shen performed design of crRNA, microinjection, and selection by 2-FA and crossing experiments. Taizo Motomura isolated and maintained the cultured S. japonica strain. Kensuke Ichihara, Yusuke Matsuda and Ko Yoshimura designed methodology. Chika Kosugi and Chikako Nagasato designed this project and methodology, and critically reviewed the manuscript. All authors have written and edited the manuscript.

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Correspondence to Chikako Nagasato.

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Supplementary Information

Supplementary Figure 1

The APT gene (locusID: SJ15126) of S. japonica. a Coding sequence. b Deduced amino acid sequence (PNG 446 kb)

High Resolution Image (TIF 18580 kb)

Supplementary Figure 2

Delivery of CRISPR-Cas9 RNP into a male gametophyte using microinjection. a–c Just after injection. Bright-field image of the injected gametophyte (a), fluorescence of FITC-dextran loaded into two cells indicating an arrow (b), and merged image of a and b (c). d–h Development of a 2-FA-resistant cell. Five days after injection (d), 3 days (e), 10 days (f), 20 days (g), 30 days (h) from the beginning of 2-FA treatment. Bars, 100 μm (PNG 887 kb)

High Resolution Image (TIF 25697 kb)

Supplementary Figure 3

Sanger sequencing chromatographs of PCR products from 2-FA-resistant gametophytes. The range of the chromatograph and individual names correspond to Table 1. Green for adenine (A), red for thymine (T), blue for cytosine (C) and black for guanine (G) (PNG 3156 kb)

High Resolution Image (TIF 76002 kb)

Supplementary Figure 4

Crossing experiment with Wild type (WT) and apt mutant gametophytes. a–d One-month old thalli derived from the following crosses: WT-female × WT-male (a), WT-female × Δapt-male (b), Δapt-female × WT-male (c), Δapt-female × Δapt-male (d). Bar, 1 mm (PNG 1393 kb)

High Resolution Image (TIF 24069 kb)

Supplementary Figure 5

Sequence data of PCR products amplified by female-specific primers. Capital letters are regions of the exon, the primers are shown by underlines. (PNG 58 kb)

High Resolution Image (TIF 12826 kb)

Supplementary Table S1

(PNG 551 kb)

High Resolution Image (TIF 13707 kb)

Supplementary Table S2

(PNG 454 kb)

High Resolution Image (TIF 21502 kb)

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Shen, Y., Motomura, T., Ichihara, K. et al. Application of CRISPR-Cas9 genome editing by microinjection of gametophytes of Saccharina japonica (Laminariales, Phaeophyceae). J Appl Phycol 35, 1431–1441 (2023). https://doi.org/10.1007/s10811-023-02940-1

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