Fig. 3.

Direct observation of sequential double-stranded DNA cleavage by AsCas12a RNP. Representative time traces of fluorescence intensity (top) and FRET efficiency (bottom) for WT DNA with catalytically deactivated dAsCas12a (E993A) (a), TS (target strand)-nicked DNA with WT enzyme (b), NTS (non-target strand)-nicked DNA with WT enzyme (c), NTS-nicked DNA with dAsCas12a (E993A) (d) in the presence of 20 nM enzyme and 20 nM crRNA. FRET histograms of individual reaction steps were added to allow direct comparison of each state. To obtain the FRET histograms, all data points for each state were collected from at least more than 46 time trajectogires (125 for a; 50 for b; 46 for c; and 113 for d). Experimental schemes to show the state assignment of FRET intermediates were added below each time trace