A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept


Autoři: Prithwiraj Kirtania aff001;  Barbara Hódi aff001;  Ivy Mallick aff001;  István Zoltan Vass aff001;  Tamás Fehér aff003;  Imre Vass aff001;  Peter B. Kós aff001
Působiště autorů: Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary aff001;  Doctoral School of Biology, University of Szeged, Szeged, Hungary aff002;  Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary aff003;  Department of Biotechnology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225375

Souhrn

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.

Klíčová slova:

Antibiotics – CRISPR – Genetic interference – Oxygen – Plasmid construction – Polymerase chain reaction – Sequence motif analysis – Synechocystis


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2019 Číslo 11