Fig. 1
CRISPRi-dCas12a-mediated gene repression in isobutanol (IB)/3-methyl-1-butanol (3M1B) producing Synechocystis strain HX11. A A scheme of the Cas12a-mediated CRISPR editing system B A scheme of the dCas12a-mediated CRISPRi system. C Base strain HX11 to test dCas12a-mediated CRISPRi system. Base strain HX11 was constructed by integrating two copies of kivdS286T into Synechocystis genome: Flag-tagged kivdS286T into the ddh locus, and His-tagged kivdS286T into the slr0168 locus. D pBB_dCas12a vector used for CRISPRi-dCas12a-mediated repression in Synechocystis. The vector is developed from a base plasmid pSL3287, harboring a kanamycin resistance cassette and a RSF1010-type replication gene. The expression of dCas12a and CRISPR RNA (crRNA) is driven by Prha rhamnose-inducible promoter. The crRNA array consists of four elements: lead sequence, L; direct repeat, R; target specific protospacer region, P; terminator, T. E Schematics of the metabolic pathways for photosynthetic IB/3M1B production in Synechocystis and selected genes for repression using dCas12a-mediated CRISPRi. Gene names in blue represent the repression targets; gene names in green represent the endogenous enzymes of 2-keto acid pathway for IB/3M1B production; gene name in red represents the heterologous enzyme of 2-keto acid pathway for IB/3M1B production. accC acetyl-CoA carboxylase, ach acetyl-CoA hydrolase, acnB aconitase, adh alcohol dehydrogenase, alsS acetolactate synthase, ccmA 3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, cpcB phycocyanin β-subunit, fbp fructose 1,6-bisphosphatase, fumC fumarase, gltA citrate synthase, icd isocitrate dehydrogenase, ilvC acetohydroxy-acid isomeroreductase, ilvD dihydroxy-acid dehydratase, ilvE branched-chain amino acid aminotransferase, kivdS286T α-ketoisovalerate decarboxylase, leuA 2-isopropylmalate synthase, leuB 3-isopropylmalate dehydrogenase, leuCD 3-isopropylmalate dehydratase, ppc phosphoenolpyruvate (PEP) carboxylase, pdh pyruvate dehydrogenase, phaA acetyl-CoA acetyltransferase, ppsA phosphoenolpyruvate synthase, pta phosphotransacetylase, sps sucrose phosphate synthase, FBP fructose-1,6-bisphosphate, F6P fructose-6-phosphate, OAA oxaloacetate, PEP phosphoenolpyruvate, PHB poly-3-hydroxybutyrate, SSA succinate semialdehyde, 2-OG 2-oxoglutarate, AP allophycocyanin, PC phycocyanin, PSII photosystem II