Fig. 1
Establishment of CRISPRi in PCC 7942. a Map of pSdCas9-CY’ harboring CmR, dCas9 under Psmt and eyfp under PconII, which was flanked by homology arms targeting the NSI site. Trrnb, transcriptional terminator. Psmt consists of the smtA promoter and the smtB repressor. b Map of psgRNA harboring the cassette expressing KmR and sgRNA under the PJ23119 promoter, which was flanked by NSII-targeting homology arms. The sgRNAs were designed to target no sequence on the PCC 7942 chromosome (Φ) or the eyfp cassette at the non-template strand of promoter (P1) or coding regions near the transcription start site (NT1 and NT2). The numbers indicate the position relative to the transcription start site. c Confocal micrographs of cells. d Flow cytometry analysis data. pSdCas9-CY’ was first transformed into PCC 7942 for cassette integration into NSI site, re-streaked, then individual psgRNA was transformed into the recombinant cells for integration into NSII site. The transformants colonies were transferred to shake flasks and cultured to OD730 = 1–1.5 in BG-11 medium containing appropriate antibiotics