Project Summary/Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRIPSR associated (Cas) proteins are components of a bacterial immune system with memory. Cas proteins acquire segments of the genomes of invading pathogens and place them in the CRISPR array. Upon reinfection, Cas9 or Cas12a are mobilized and loaded with guide RNAs transcribed from the CRISPR array. They then cleave invading DNA strands that contain sequences matching the guide RNA after the creation of a 20-base pair RNA-DNA heteroduplex called an R-loop. R-loop formation initiates a complex set of conformational shifts in both enzymes, with each proceeding through distinct checkpoints on the pathway to cleavage competency. Importantly, these conformational shifts differ between the two enzymes, indicating mechanistic differences. The programmability and specificity imparted by R-loop induced cleavage make Cas9 and Cas12a excellent biophysical tools. However, both enzymes can bind to and cleave sites that possess mismatches in the R-loop, leading to potentially hazardous off-target activity. A full understanding of the effect of mismatches and target DNA topology on R-loop formation and cleavage is needed to optimize usage and engineering of Cas9 and Cas12a. DNA in cells is globally underwound and locally under constant flux to due processes that mechanically deform DNA. In this proposal, high-resolution single-molecule methods developed in the Bryant lab will be used to observe Cas9/Cas12a R-loop formation and conformational changes simultaneously on supercoiled DNA. Recently, these methods were used to develop a model for Cas9 R-loop formation in which R-loop mismatches and DNA supercoiling alter the shape of the Cas9 R-loop formation energy landscape. The resolution of the methods allowed identification of a discrete R-loop intermediate. Currently, a similar model is being produced for Cas12a, which also has a discrete R-loop intermediate. The central hypothesis of this proposal is that R-loop mismatches and DNA supercoiling modulate kinetic transitions between Cas9/Cas12a R-loop and conformational checkpoints. In aim 1, Cas9 FRET and R-loop states will be simultaneously observed, correlating conformational and R-loop checkpoints. This will require technical updates to microscopy methods to increase resolution. Preliminary experiments indicate the feasibility of these measurements, showing coincident R-loop and FRET signals. In aim 2, similar measurements will be performed using Cas12a. Current data show that Cas12a has different R-loop checkpoints and is highly sensitive to supercoiling. Data acquired in these aims will build a complete picture describing the effect of R-loop mismatches and DNA supercoiling on Cas9 and Cas12a activity and specificity. The models developed from these measurements will reveal links between Cas9/Cas12a mechanistic and specificity differences. This information will assist in designing mutations and perturbations to ...