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

ABSTRACT CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro , and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a ( K d = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the affinities of both the precursor and mature forms of the crRNA, while deletion of an upstream sequence had no significant effect on affinity of the pre-crRNA. After processing, the mature crRNA remains very tightly bound to Cas12a, with a half-life of ∼1 day and a K d value of 60 pM. Addition of a 5’-phosphoryl group, which is normally lost during the processing reaction as the scissile phosphate, tightens binding of the mature crRNA by ∼10-fold by accelerating binding and slowing dissociation. Using a direct competition assay, we found that pre-crRNA binding specificity is robust to other changes in RNA sequence, including tested changes in the guide sequence, addition of a 3’ extension, and secondary structure within the guide region. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in some genome editing applications.