Relative Cas12a assembly rate constants measured by direct competition. Each pre-crRNA variant tested is shown in the cartoon at the left, and the corresponding bar at the right shows the rate constant relative to the reference pre-crRNA. The pre-crRNA−34:+24 (top) is assigned a relative rate constant of 1 by definition. Binding of the second variant, pre-crRNA−23:+24, was measured and found to be equivalent in rate, as indicated by the absence of a bar to its right. All of the variants below were measured using one of these two pre-crRNAs as a reference, whichever one differed in length of the 5′ leader sequence from the variant being tested and therefore could be distinguished by the length of the labeled cleavage product. Bars reflect the average ± SEM from three independent measurements.
