Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120
Open accessAbstract
High Resolution Image Download MS PowerPoint Slide In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli . Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.
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Conjugation is particularly advantageous for transferring large plasmids or complex genetic constructs that are challenging to introduce via other transformation methods.
“This approach is particularly advantageous for transferring large plasmids or complex genetic constructs that are challenging to introduce via other transformation methods [82].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [82]
The Abstract (page not provided) reports that comparatively large plasmids containing CAST and an engineered transposon were successfully transferred into Anabaena by conjugation. The paper does not establish that conjugation is particularly advantageous over other transformation methods for constructs that those methods struggle to introduce. (LLM, conf=0.85)
Experimental — accuracy not yet measured
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- Authors
- Arévalo, S., Pérez Rico, D., Abarca, D., Dijkhuizen, L. W., Sarasa-Buisan, C., Lindblad, P., Flores, E., Nierzwicki-Bauer, S., & Schluepmann, H.
- Affiliations
- Uppsala University (SE)
- Biology Department, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
- Utrecht University (NL)
- Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Avenida Americo Vespucio 49, Sevilla 41092, Spain
- Instituto de Bioquímica Vegetal y Fotosíntesis (ES)
- Department of Biological Sciences, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180-3590, United States
- Corresponding author
- Schluepmann, Henriette
- Venue
- ACS Synthetic Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2024-03-06
- Volume
- 13
- Issue
- 3
- Pages
- 901-912
- ISSN
- 2161-5063 (ISSN-L)
- PMC
- PMC10949235
- OpenAlex
- W4392519069
- Cited by
- 13
- References
- 48
- Citations by year
- 2026: 6 · 2025: 3 · 2024: 4
- FWCI
- 1.77
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- ACS Synthetic Biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Uppsala University Publications (Uppsala University) (submittedVersion, other-oa)
- DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Funders
- Gordon and Betty Moore Foundation (9355)
- Universiteit Utrecht
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO-ALWGS.2016.020)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Keywords
- CRISPR-associated transposases, CAST, Anabaena sp. PCC 7120, filamentous cyanobacteria, genome editing, conjugation, suicide plasmid, single guide RNA, transposon insertion, plasmid curing, DNA integration
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Cyanobacteria, DNA, DNA Transposable Elements, Escherichia coli, Humans, Plasmids, RNA, Anabaena
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