Figure 4.

CRISPR/Cas10-mediated single-gene interference in M. tuberculosis

A. Schematic for the CRISPR interference strategy in which the gRNA is flanked by a 5′-GAAAC-3′ tag at its 5′-end. This tag is recognized by the CRISPR/Cas10 complex, which stops the DNase activity of Cas10. The target RNA is cleaved by Csm3 without any DNA cleavage. B. Sequence and position of the gRNA designed for katG interference. The 40-bp region (1233–1272 bp) along with the pentanucleotide motif at 1228–1232 bp on the non-coding strand of katG was selected as the gRNA. C.–E. Inhibition of gene expression in the katG (C), dcD (D), and esxT (E) interference strains compared with the wild-type and control (empty vector) strains. F. Sequences and positions of the gRNAs designed for lpqN interference. G.lpqN expression in the S1 gRNA and S2 gRNA strains compared with that in the wild-type and control (empty vector) stains. Student’s t-test (***, P < 0.001; ****, P < 0.0001; ns, not significant).