FIG 5.

ddCpf1-mediated multiplex gene repression in S. coelicolor. (A, B) ddCpf1-mediated efficient block of redX transcriptional elongation. crRNAs targeting the T or NT strand of the redX coding region are illustrated. The phenotypic (A) and transcriptional (B) analyses were carried out after growth on R2YE agar for 48 h. Error bars indicate the standard deviations from three independent biological replicates. (C, D) Multiplex gene repression with ddCpf1 and a single customized crRNA array. Three target genes (cpkA, actI-orf1, and redX) were selected for simultaneous repression. The order of crRNAs targeting three genes is designated array 1 or array 2. The transcriptional levels of each gene were analyzed in the engineered strains expressing ddCpf1 with individual crRNA or crRNA arrays, and the strain expressing only ddCpf1 was used as the control. The phenotypic and transcriptional analysis was carried out for cpkA, redX, and actI-orf1 at 24, 48, and 72 h, respectively.