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. 2022 Jul 13;50(14):8377–8391. doi: 10.1093/nar/gkac578

Figure 3.

Figure 3.

Kinetic analysis of LbCas12a trans-cleavage activity. (A) Time-dependent trans-cleavage activity of activated LbCas12a on FRET-labelled DNA reporters (250 nM). Substrate (e.g. DNA reporters, S) and degraded products (P) were resolved by polyacrylamide gel electrophoresis (PAGE). The intrinsic fluorescence background of the DNA reporters (lines 1) and signal change over time is consistent with the increased amount of degraded oligonucleotides over time (lines 3 to 8). (B) Fraction of DNA reporters cleaved by the active LbCas12a/crRNA/dsDNA complex (RNP complex, final concentration 20 nM/20 nM/1 nM) vs time. Fluorescence assays were performed at 37°C by adding 4.5 μl of a 10x concentrated RNP complex to a solution containing the DNA reporter (final concentration 100 nM). (C) Representative Michaelis-Menten plots for LbCas12a trans-cleavage activity on the different linear/hairpin DNA reporters using a dsDNA activator. Measured Km, kcat and kcat/Km values reported as mean ± SD, where n = 3.