Figure 4.

Cleavage and smFRET of Pre-unwound or Pre-cleaved DNAs

(A) Indiscriminate single-stranded DNA cleavage activated by 23m- or 14m- single-stranded activators, which contain 23 and 14 matched bases toward 23m-crRNA at the PAM-proximal end, respectively. Matched bases toward 23m-crRNA are shown in blue, whereas mismatched bases toward 23m-crRNA are shown in red. Non-specific ssDNA was labeled with Cy5 at the 5′ end.

(B) Cleavage of pre-unwound dsDNAs. Cas12a was guided by 23m-crRNA or 14m-crRNA, which contain 23 or 14 matched bases at their PAM-proximal ends toward 23m-dsDNA, respectively. Pre-unwound segments are plotted as bulges. Labeling sites are shown in cartoons.

(C) Cleavage of 23m-dsDNA containing pre-cleaved NTS or TS. Locations of the pre-cleaved and labeling sites are shown in cartoons. In the presence of 14m-crRNA, pre-cleaved NTS (NTS-nick) caused cleavage of TS (dsDNA #7), whereas pre-cleaved TS (TS-nick) did not lead to cleavage of NTS (dsDNA #9).

(D) Time-dependent FRET probability density and transition density plots of Cas12a complexes on NTS-nick 14m-dsDNAs.