Fig. 5

DNA interference in the type I-F1 CRISPR-Cas system. Ribonucleoprotein Cascade complex scans DNA for a complementary protospacer, which is adjacent to the promiscuous PAM sequence. Upon DNA target recognition, Cascade complex forms an R-loop (1 and 4) where the spacer of the crRNA basepairs with the target strand (TS) of the protospacer displacing the non-target strand (NS) as an ssDNA. In a checkpoint step of DNA interference, Cascade “measures” the length of the R-loop. When R-loop reaches WT length of 32 bp or is extended above this length by Cascade complex containing longer spacer (1), DNA interference is turned on. Then, the helicase/nuclease Cas2/3 docks to the NS of the R-loop and initiates cleavage (2) at the PAM-distal end of the R-loop (22nd-25th nt from the PAM) followed by unidirectional DNA degradation upstream from the protospacer, while leaving the downstream DNA intact (3). On the contrast, truncated Cascade or mismatches at the PAM-distal end of the protospacer that mediate the formation of 26 bp length or shorter R-loops (4) prevent cleavage initiation (5); thus, DNA interference is turned off