Efficient Multiplex Genome Editing of the Cyanobacterium Synechocystis sp. PCC6803 via CRISPR‐Cas12a
Open accessAbstract
into biochemical products, but efficient genetic engineering tools, including CRISPR-Cas systems, remain limited. This is primarily due to the polyploid nature of cyanobacteria, which hinders their effectiveness. Here, we address the latter by specifically (i) modifying the RSF1010-based replicative plasmid to simplify cloning efforts while maintaining high conjugation efficiency; (ii) improving the design of the guide RNA (gRNA) to facilitate chromosomal cleavage; (iii) introducing template DNA fragments as pure plasmids via natural transformation; and (iv) using sacB to facilitate replicative plasmid curing. With this system, the replicative plasmid containing both Cas12a and gRNA is introduced to Synechocystis sp. PCC6803 cells via conjugation to cleave the circular chromosomes. Template DNA plasmid that has meanwhile been assimilated will then repair it achieving the desired genetic modifications. This system was validated by successfully deleting various "neutral" chromosomal loci, both individually and collectively, as well as targeting an essential gene, sll1797. With the sacB-sucrose counter-selection, all deletions were simultaneously made markerless in < 4 weeks. Moreover, we also integrate YFP with various protein degradation tags into the chromosome, allowing for their characterization at the chromosomal level. We foresee this system will greatly facilitate future genome engineering in cyanobacteria.
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Marker-free multiplex genome editing via optimised CRISPR-Cas12a has been demonstrated in Synechocystis sp. PCC 6803 and the endogenous Type I-D CRISPR-Cas system of Synechococcus sp. PCC 7002 has been repurposed for scarless gene deletions using native cyanobacterial machinery.
“Marker-free multiplex genome editing via optimised CRISPR-Cas12a has been demonstrated in Synechocystis sp. PCC 6803 [122] and the endogenous Type I-D CRISPR-Cas system of Synechococcus sp. PCC 7002 has been repurposed for scarless gene deletions using native cyanobacterial machinery [123].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [122]
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- Authors
- Du, W., Meister, L. L., van Grinsven, T., & Branco dos Santos, F.
- Affiliations
- University of Amsterdam (NL)
- Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, Faculty of Science University of Amsterdam Amsterdam The Netherlands
- Venue
- Biotechnology and Bioengineering
- Publisher
- Wiley
- Published
- 2024-12-19
- Volume
- 122
- Issue
- 3
- Pages
- 736-743
- ISSN
- 0006-3592 (ISSN-L)
- 1097-0290
- PMC
- PMC11808434
- OpenAlex
- W4405654692
- Cited by
- 7
- References
- 25
- Citations by year
- 2026: 6 · 2025: 1
- FWCI
- 0.95
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Biotechnology and Bioengineering (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- UvA-DARE (University of Amsterdam) (publishedVersion, cc-by-nc-nd)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- Synechocystis sp. PCC 6803, CRISPR/Cas12a, multiplex genome editing, conjugation, guide RNA design, natural transformation, plasmid curing, polyploidy
- MeSH terms
- Gene Editing, Bacterial Proteins, Endodeoxyribonucleases, Plasmids, Genome, Bacterial, Synechocystis, CRISPR-Cas Systems, CRISPR-Associated Proteins
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