Fig. 1 Configuration of CRISPRa in PCC7942(a) Components of the CRISPRa system. (b) The expression cassette encoding dCas9 anda transcriptional activator fusion protein was inserted into the NSⅢ site of PCC7942, and the report genes sfgfp and sgRNA were integrated into the NSⅡ site of PCC7942. (c) The fluorescence intensities of strains G03, G09, G10, G11, G12, and G13 with different transcriptional activators. (d) The fluorescence signal level of G01(control strain without sgRNA) and G02(control strain with an off target sgRNA 106).

Fig. 2 Optimization of the CRISPRa system(a)Scheme of different targeting sites for CRISPRa. (b) The fluorescence intensities of strains G09, G14, G15,G16, and G17 with different targeting sites. (c) The fluorescence intensity of G19 strain with genetic knockout of endogenous rpoZ. (d) Effect of different orientations of the dCas9 and RpoZ fusion on CRISPRa performance. (e) Expression of dCas9-RpoZ was increased in G45 strain by replacing the promoter Pendo with PJ23108. (f) The fluorescence intensity of G45 strain.

Fig. 3 Activation of nudⅠ by CRISPRa(a) Scheme of nudⅠ activation by CRISPRa. The sgRNA H4 and nudⅠ were inserted into the NSⅡ site. The DNA fragment encoding dCas9-RpoZ was inserted into the NSⅢ site or to replace endogenous rpoZ, generating strains S03 and S04, respectively. The control strain S01 lacked dCas9-RpoZ. (b) Isopentenol production by strains S01, S03, and S04. Cells were cultivated for 96 h, and isopentenol in the culture supernatant was detected by a colorimetric assay. (c) Transcriptional level of nudⅠ in strains S01, S03, and S04. RNA was isolated from cellscultivated for 72 h.

Fig. 4 Simultaneous activation of nudⅠ and dxs by CRISPRa(a) 1-Deoxy-D-xylulose 5-phosphate synthase encoded by dxs catalyzes the first reaction of the MEP pathway. (b)Scheme of nudⅠ and dxs activation by CRISPRa. The sgRNA H4, nudⅠ, and dxs were inserted into the NSⅡ site. The DNA fragment encoding dCas9-RpoZ was inserted into the NSⅢ site or to replace endogenous rpoZ, generating strains S05 and S06, respectively. S02 (without dCas9-RpoZ) and S07 (with an off target sgRNA 106) are the control strains. (c) Isopentenol production by strains S02, S05, S06, and S07.Cells were cultivated for 96 h, and isopentenol in the culture supernatant was detected by a colorimetric assay. (f) Transcriptional levels of nudI and dxs in strains S02, S05, S06, and S07. RNA was isolated from cells cultivated for 72 h.

Fig. 5 Simultaneous activation of nudⅠ and dxs and repression of gpps by CRISPRa(a) gpps catalyzes a key competing reaction for isopentenol biosynthesis; (b) Location of the gpps gene and the targeting sites g1 and g2 on the PCC 7942 genome; (c) The g1 and g2 sites of gpps in strain S06 were targeted, generating strains S12 and S13, respectively. S14 (with an off target sgRNA 106) is a control strain. (d) Transcriptional levels of gpps, nudⅠ and dxs in strains S02, S06, S12, S13, and S14. RNA was isolated from cells grown to OD730 of about 0.7. (e) Isopentenol production by strains S02, S06, S13, and S14. Isopentenol in the culture supernatant was detected by a colorimetric assay. (f) Isopentenol production by strains S02, S06, and S13. Culture supernatants were collected at OD730 of about 2.0, and isopentenol was detected by gas chromatography.