Modification of a Chlamydomonas reinhardtii CRISPR/Cas9 Transformation Protocol for Use with Widely Available Electroporation Equipment
Open accessAbstract
A recently reported protocol demonstrates efficient CRISPR/Cas9 gene editing of Chlamydomonas reinhardtii [1]. The published protocol demonstrates transformation and editing of a wall-less strain of C. reinhardtii using plasmid encoded Cas9 and sgRNA. However, the published protocol utilizes a complex electroporation waveform that cannot be generated by most electroporation systems. It is unknown whether transformation via this complex electroporation waveform is essential for high efficiency of Cas9 edits, perhaps by optimizing Cas9 or guide RNA gene expression or incorporation into the genome. We demonstrate that a simple electroporation waveform can deliver plasmid encoded CRISPR/Cas9 into and edit the genome of a wall-less strain of C. reinhardtii as efficiently as the more complex waveform. Our modified electroporation protocol makes the plasmid based CRISPR/Cas9 genome editing method accessible to a greater number of Chlamydomonas researchers. •Our protocol uses a simple electroporation waveform to replace a complex waveform used to achieve efficient CRISPR/Cas9 gene editing in a wall-less strain of Chlamydomonas reinhardtii. •We also increased concentration of plasmids to maintain high gene editing efficiency. •We minimized modifications to other steps of the original protocol.
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Algal strains such as Chlamydomonas reinhardtii and Synechocystis sp. have been modified to improve resistance to environmental stressors and boost growth rates by manipulating pathways involved in light harvesting.
“Algal strains such as Chlamydomonas reinhardtii and Synechocystis sp. have been modified to improve resistance to environmental stressors and boost growth rates by manipulating pathways involved in light harvesting [119-123].”
From A Review of Algae-Based Carbon Capture, Utilization, and Storage (Algae-Based CCUS) (2024) · cites this paper as [119]
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Direct visualization at the single-cell level of electrically mediated gene delivery
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Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii
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DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins
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A Review of Algae-Based Carbon Capture, Utilization, and Storage (Algae-Based CCUS)
Li, G., & Yao, J. (2024) · Gases · doi:10.3390/gases4040024 · Cited by 80 · see citing works
Recent Progress and Challenges in CRISPR-Cas9 Engineered Algae and Cyanobacteria
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- Authors
- Park, R. V., Asbury, H., & Miller, S. M.
- Affiliations
- University of Maryland, Baltimore County (US)
- Corresponding author
- Miller, Stephen M.
- Venue
- MethodsX
- Publisher
- Elsevier BV
- Published
- 2020
- Volume
- 7
- Pages
- 100855
- ISSN
- 2215-0161 (ISSN-L)
- PMC
- PMC7139109
- OpenAlex
- W3011834118
- Cited by
- 21
- References
- 9
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 5 · 2023: 5 · 2022: 5 · 2021: 2 · 2020: 2
- FWCI
- 1.14
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- MethodsX (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Science Foundation (NSF-EFRI-1332344, PA-4000)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
- Keywords
- Chlamydomonas reinhardtii, CRISPR/Cas9 genome editing, plasmid transformation, guide RNA, Cas9 expression, protoplast transformation, genome editing efficiency
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