A Simple and Fast Method for Transcriptional Inactivation in Synechocystis PCC6803 Using CRISPR-Interference from a Single Plasmid

Synechocystis PCC6803 is an important organism for its biotechnological applications and in studying photosynthesis. However, due to the dynamic nature of its chromosome number, creating a desired mutant is a tedious process. Recently, CRISPR-interference using deadCas9 was applied to create mutants in Synechocystis PCC6803 to speed up the process, however, as this method includes genomic integration of deadCas9-cassette, it is not lesion free [1]. Here, we show a simple, fast and lesion free method to apply CRISPR-interference for transcriptional inactivation in Synechocystis PCC6803 using the single shuttle-plasmid (pCRPB1010). The technique is easier to execute for gene silencing in Synechocystis PCC6803 as it circumvents the genome integration and segregation steps and thereby significantly shortens the construction of the mutant strains.

To validate the technique, we targeted well characterized genes involved in photosynthesis to get a clear phenotype. First, we targeted the non-template DNA strand of psbO using a single spacer. 70% reduction in PsbO expression and about 50% reduction of oxygen evolution were observed compared to the wild type. Further, targeting the common span of the template DNA strand of psbA2 and psbA3 genes using a single spacer resulted in a complete abolition of D1 protein expression, and complete loss of both oxygen evolution and photo-autotrophic growth was observed. When the spacers were combined could achieve significant extent of silencing of multiple genes stated above using one common plasmid construct.

This is a first report of a method developed for applying CRISPR-interference in a completely lesion free single-plasmid based episomal manner in Synechocystis PCC603.

1. Yao, L., et al., Multiple Gene Repression in Cyanobacteria Using CRISPRi. ACS Synth Biol, 2016. 5(3): p. 207-12.