Fig. 5: Development of a dual-target synergistic screening strategy.

a Schematic illustrating the construction of a synergetic library, where SlA represents the single-target library and SlB denotes the synergistic library designed for simultaneous inhibition of two target genes. b Expression and inhibition of gfp and mCherry genes in S. roseosporus determined by laser confocal microscopy. WT^gfp-mCherry: WT expressing both gfp and mCherry. WT^{gfpi-mCherryi}: CRISPRi-mediated inhibition of gfp and mCherry in the WT^gfp-mCherry. The excitation wavelengths of GFP and mCherry are 488 nm and 587 nm, respectively. c GFP (green) and mCherry (red) fluorescence curves of WT^gfp-mCherry and WT^{gfpi-mCherryi} during a 6-day culture period. d Indigoidine level of 171 dual-target combinations correspond to 600 single colonies. The brown circle indicates the mean distribution, and the black dash indicates the standard deviation. The blue line indicates the indigoidine levels in the control strain Srdi^-. e Daptomycin titer in strains (SrdiC1 through SrdiC5) exhibiting the five highest levels of indigoidine, as well as in control strains Srdi and Srdi^-. [p = 0.982953367, 0.006952983, 0.00115138, 0.001932551, 0.003069223, 0.002012816 (left to right)]. f Daptomycin titer in strains (SrdiC6 through SrdiC10) with lower indigoidine levels than the control strains Srdi and Srdi^-. [p = 0.982953367, 0.000820299, 0.000797634, 0.000673364, 0.003788098, 0.00092792 (left to right)]. Data in (c, e, f) are shown as the mean ± SD (n = 3 biological replicates). Two-tailed Student’s t test was used in (e, f) to analyze the statistical significance (n.s. not significant (p >  0.05); **p  <  0.01; ***p  <  0.001).