Kinetic dissection of pre-crRNA binding and processing by CRISPR–Cas12a

Binding and dissociation kinetics for truncated pre-crRNA variants. (A–C) Binding kinetics measurements for (A) pre-crRNA_−23:+24, (B) pre-crRNA_−34:0, (C) pre-crRNA_−23:0. Binding rate constants (average ± SEM) were determined from the results of fits of each of four independent data sets to a hyperbolic equation. Rate constants were 2.6 (±0.2) × 10⁶ M⁻¹ sec⁻¹ for pre-crRNA_−23:+24, 6.7 (±0.3) × 10⁵ M⁻¹ sec⁻¹ for pre-crRNA_−34:0, and 6.2 (±1.2) × 10⁵ M⁻¹ sec⁻¹ for pre-crRNA_−23:0. (D–F) Representative dissociation kinetics measurements for the Cas12a variants shown in A–C, respectively. Rate constants were calculated as the average and SEM from at least three independent measurements, with each data set fit by a first-order rate equation. Values of k_off were 3.6 (±0.3) × 10⁻⁶ sec⁻¹ for pre-crRNA_−23:+24, 5.3 (±0.7) × 10⁻⁶ sec⁻¹ for pre-crRNA_−34:0, and 6.7 (±0.5) × 10⁻⁶ sec⁻¹ for pre-crRNA_−23:0.