A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept
Open accessAbstract
We developed a simple method to apply CRISPR interference by modifying an existing plasmid pCRISPathBrick containing the native S. pyogenes CRISPR assembly for Synechocystis PCC6803 and named it pCRPB1010. The technique presented here using deadCas9 is easier to implement for gene silencing in Synechocystis PCC6803 than other existing techniques as it circumvents the genome integration and segregation steps thereby significantly shortens the construction of the mutant strains. We executed CRISPR interference against well characterized photosynthetic genes to get a clear phenotype to validate the potential of pCRPB1010 and presented the work as a "proof of concept". Targeting the non-template strand of psbO gene resulted in decreased amount of PsbO and 50% decrease in oxygen evolution rate. Targeting the template strand of psbA2 and psbA3 genes encoding the D1 subunit of photosystem II (PSII) using a single spacer against the common sequence span of the two genes, resulted in full inhibition of both genes, complete abolition of D1 protein synthesis, complete loss of oxygen evolution as well as photoautotrophic growth arrest. This is the first report of a single plasmid based, completely lesion free and episomal expression and execution of CRISPR interference in Synechocystis PCC6803.
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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 [123]
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- Authors
- Kirtania, P., Hódi, B., Mallick, I., Vass, I. Z., Fehér, T., Vass, I., & Kós, P. B.
- Affiliations
- University of Szeged (HU)
- HUN-REN Szegedi Biológiai Kutatóközpont (HU)
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2019-11-26
- Volume
- 14
- Issue
- 11
- Pages
- e0225375
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC6879144
- OpenAlex
- W2990229578
- Cited by
- 22
- References
- 38
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 3 · 2023: 4 · 2022: 5 · 2021: 4 · 2020: 2
- FWCI
- 0.91
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Repository of the Academy's Library (Library of the Hungarian Academy of Sciences) (acceptedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Nemzeti Kutatási Fejlesztési és Innovációs Hivatal (NKFIH K-116016)
- Hungarian Ministry for National Economy (GINOP-2.3.2-15-2016-00001)
- 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
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- CRISPR interference, Synechocystis sp. PCC 6803, gene silencing, photosystem II, psbA, PsbO, oxygen evolution reaction, D1 protein, photosynthetic gene expression, cyanobacteria
- MeSH terms
- Bacterial Proteins, Oxygen, Photosynthesis, Plasmids, Gene Expression Regulation, Bacterial, Photosystem II Protein Complex, Synechocystis, Clustered Regularly Interspaced Short Palindromic Repeats
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