Application of CRISPR Interference for Metabolic Engineering of the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120.

摘要: Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This has been extensively studied as model for multicellularity in prokaryotic cells. We have interested photosynthetic production of nitrogenous compounds using A. 7120. However, the lack efficient gene repression tools limited its usefulness. originally developed an artificial endogenous method this small antisense RNA. narrow dynamic range needs to be improved. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) technology was and successfully applied some unicellular cyanobacteria. The requires expression nuclease-deficient CRISPR-associated protein 9 (dCas9) single guide RNA (sgRNA) complementary target sequence, repress gene. In study, we employed CRISPRi ammonium through glnA, only encoding glutamine synthetase essential assimilation By strictly regulating dCas9 TetR induction system, succeeded fine-tuning GlnA addition level glnA transcripts. Expression sgRNA by heterocyst-specific nifB promoter led heterocysts, well vegetative Finally, showed excreted into medium when inducers were added. conclusion, enables temporal control desired products will useful tool basic science.