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. 2022 Nov;20(11):2042-2044.
doi: 10.1111/pbi.13904. Epub 2022 Sep 8.

CRISPR/Cas9-mediated generation of non-motile mutants to improve the harvesting efficiency of mass-cultivated Euglena gracilis

Marumi Ishikawa  1 , Toshihisa Nomura  1   2 , Shun Tamaki  1 , Kazunari Ozasa  3 , Tomoko Suzuki  4   5 , Kiminori Toyooka  4 , Kikue Hirota  1 , Koji Yamada  1   6 , Kengo Suzuki  1   6 , Keiichi Mochida  1   2   7   8   9
Affiliations

CRISPR/Cas9-mediated generation of non-motile mutants to improve the harvesting efficiency of mass-cultivated Euglena gracilis

Marumi Ishikawa et al. Plant Biotechnol J. 2022 Nov.
No abstract available

Keywords: Euglena gracilis; Bardet-Biedl syndrome (BBS) genes; Cas9 ribonucleoprotein (RNP)-based genome editing; harvesting efficiency.

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Conflict of interest statement

This study was partially supported by a matching fund‐based research programme between RIKEN and euglena Co., Ltd.

Figures

Figure 1
Figure 1
Generating non‐motile Euglena gracilis mutants by creating CRISPR/Cas9‐targeted deletions in EgBBS7 and EgBBS8. (a, b) Detection of the truncated PCR fragments of EgBBS7 (a) and EgBBS8 (b). (c, d) Alignment of representative mutated sequences in truncated PCR fragments of EgBBS7 (c) and EgBBS8 (d) vs. WT. (e) Colony formation by isolated bbs7 and bbs8 cells. (f, g) Micrographs of the bbs7 and bbs8 mutants cultured in KH medium for 4 days taken by light microscopy (f, Scale bars, 10 μm) and scanning electron microscopy (g, Scale bars, 5 μm). Arrowheads indicate the flagellum. (h, i) Trace momentum assay. Green dots and light green lines indicate cells and their trajectory, respectively. (h) Trace momentum was calculated based on the total area of light green lines obtained at 450 time points in 10 min (i). (j–m) Sedimentation analysis. Time‐lapse observation of gravitational sedimentation (j) and 2D kymographs representing sedimentation speed (k, scale bars, 5 mm). Transparent areas were analysed in images obtained at each time point (l). Sedimentation rates of each experiment (m). (n–q) Growth of bbs cells cultured in KH medium for 7 days (n), biomass on Day 7. (o) Lipid (p) and paramylon contents (q). Error bars in all graphs show standard errors (n = 3). Significant differences were tested by one‐way ANOVA, and all statistical values were calculated by Dunnett's test; P‐values are shown.
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