PT  - JOURNAL ARTICLE
AU  - Shanzhong Gong
AU  - Helen Hong Yu
AU  - Kenneth A. Johnson
AU  - David W. Taylor
TI  - DNA unwinding is the primary determinant of CRISPR-Cas9 activity
AID  - 10.1101/205823
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 205823
4099  - http://biorxiv.org/content/early/2017/10/19/205823.short
4100  - http://biorxiv.org/content/early/2017/10/19/205823.full
AB  - Bacterial adaptive immunity utilizes RNA-guided surveillance complexes composed of CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single guide RNA that base-pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and RuvC nuclease domains can cleave opposing strands. Cas9 has been repurposed for a variety of important genome engineering applications. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. Although the HNH nuclease activity is stimulated by Mg2+ with a single measureable Kd, the RuvC activity requires two distinct Mg2+ binding events. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.