DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity

Cell Rep. 2018 Jan 9;22(2):359-371. doi: 10.1016/j.celrep.2017.12.041.

Abstract

Bacterial adaptive immunity utilizes RNA-guided surveillance complexes comprising 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 the RuvC nuclease domains cleave opposing strands. 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, and R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. 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.

Keywords: CRISPR; Cas9; DNA cleavage; R-loop; kinetics; sgRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • DNA Cleavage
  • Humans