The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing
Abstract
:1. Introduction
2. Materials and Methods
2.1. C. reinhardtii Cultivation and Nuclear Transformation
2.2. CRISPR/Cas9-Based Gene Editing
2.3. Vector Design and Cloning
2.4. Fluorescence Screening and Imaging
2.5. Starch Assay and Calculation of Editing Frequency
2.6. Polyamine Extraction and Quantification
3. Results and Discussion
3.1. Nitrogen Starvation of the Pre-Culture Substantially Increases Editing Frequency of CRISPR/Cas9-Based Gene Editing
3.2. High Throughput Confirmation of Successful Gene Edits by Colony PCR
3.3. SPD1 Is Essential for C. reinhardtii and Its Knockout Creates a Novel Spermidine Auxotrophy
3.4. Complementation of the ΔSPD1 Mutant Has Versatile Biotechnological Utility
3.5. A Marker-Less ΔSPD1 Mutant Strain
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Freudenberg, R.A.; 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, 837. https://doi.org/10.3390/cells11050837
Freudenberg RA, 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. https://doi.org/10.3390/cells11050837
Chicago/Turabian StyleFreudenberg, Robert A., Luisa Wittemeier, Alexander Einhaus, Thomas Baier, and Olaf Kruse. 2022. "The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing" Cells 11, no. 5: 837. https://doi.org/10.3390/cells11050837
APA StyleFreudenberg, R. A., 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