Competition experiment for Cas12a binding by pre-crRNA−34:+24 and pre-crRNA−23:0. (A) Denaturing gel image showing the substrates and products for both pre-crRNAs. The reaction included 50 nM of each pre-crRNA and 30 nM Cas12a. (B) Progress curves depicting cleavage of each pre-crRNA variant. Because Cas12a binding is rate-limiting for cleavage, the relative amplitudes of the “burst” phase of product accumulation correspond to the relative rate constants for binding of the two pre-crRNA variants. Data are fit by an equation with an exponential term, reflecting cleavage of the first round of pre-crRNA, and a linear term, reflecting slow additional rounds of processing that are limited in rate by crRNA release from Cas12a.
