The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

Freudenberg R, Wittemeier L, Einhaus A, Baier T, Kruse O (2022)
Cells 11(5): 837.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Abstract / Bemerkung
Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas9 allows directed exploration of new endogenous selectable markers. Since editing frequencies with CRISPR/Cas9 techniques are often low, the Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, increasing editing frequencies from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was targeted to either the cytosol or the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.
Stichworte
microalgae; CRISPR/Cas9; gene editing; spermidine; SPD1; auxotrophy; selectable marker
Erscheinungsjahr
2022
Zeitschriftentitel
Cells
Band
11
Ausgabe
5
Art.-Nr.
837
eISSN
2073-4409
Finanzierungs-Informationen
Open-Access-Publikationskosten wurden durch die Universität Bielefeld gefördert.
Page URI
https://pub.uni-bielefeld.de/record/2960644

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Freudenberg R, Wittemeier L, Einhaus A, Baier T, Kruse O. The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing. Cells. 2022;11(5): 837.
Freudenberg, R., Wittemeier, L., Einhaus, A., Baier, T., & Kruse, O. (2022). The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing. Cells, 11(5), 837. https://doi.org/10.3390/cells11050837
Freudenberg, R., Wittemeier, L., Einhaus, A., Baier, T., and Kruse, O. (2022). The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing. Cells 11:837.
Freudenberg, R., et al., 2022. The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing. Cells, 11(5): 837.
R. Freudenberg, et al., “The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing”, Cells, vol. 11, 2022, : 837.
Freudenberg, R., Wittemeier, L., Einhaus, A., Baier, T., Kruse, O.: The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing. Cells. 11, : 837 (2022).
Freudenberg, Robert, Wittemeier, Luisa, Einhaus, Alexander, Baier, Thomas, and Kruse, Olaf. “The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing”. Cells 11.5 (2022): 837.
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2022-03-01T12:24:54Z
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Preprint: 10.20944/preprints202201.0211.v1

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