DNA mini-circles reveal effects of DNA topology on CRISPR-Cas9 functions.

Abstract

CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. Prior to Cas9 cleavage, the protospacer of the target DNA must be unwound to form a three-stranded R-loop with a complementary segment of the Cas9-bound guide RNA. R-loop dictates the coordinated conformational changes allosterically controlling Cas9 nuclease activities, thus is the key in target discrimination. Current studies of Cas9 specificity focus primarily on protospacer sequences (i.e., mismatch(es) between the DNA and the RNA guide), while contributions from many collective DNA duplex properties (e.g., flexibility, topological constraints), which could significantly influence DNA unwinding and R-loop structure and dynamics, are not well-understood. In this work, DNA minicircles, which exhibit distinctive features (e.g., ring constraint, curvature), were used to delineate the role of DNA topology on Cas9 function. Two hairpins containing complementary overhangs were ligated to form a single-stranded DNA circle, which was then hybridized with linear complementary strand(s) to generate the double-stranded minicircle (dsMC). With a target imbedded in a 95 base-pair dsMC, the saturating Cas9 cleavage rate, kcat, was found to be ∼1000-fold slower as compared to the corresponding linear construct, although nicking the dsMC resulted in a rate comparable to the linear DNA. Increasing the size of the dsMC to 210 base-pair also led to a significant increase in kcat. The data clearly show that topological constraints imposed by an intact dsMC affect Cas9 cleavage. Work is ongoing to correlate these observed functional effects to the impact of dsMC on the Cas9 R-loop.