Biophysical analysis of Cas9-DNA interactions and enzymatic activity with the switchSENSE® Biosensor Platform I Poster
The analysis of the biophysical properties of Cas9 – DNA interactions (binding kinetics, affinity, endonuclease activity) is of fundamental importance for the development of new CRISPR/Cas9 applications. So far, the majority of published data is based on electro mobility shift assays. Interest in sensing technologies like SPR or BLI systems has developed. However, these conventional approaches suffer from inherent limitations and are prone to measurement artifacts. We present an assay to extract binding kinetic rate constants of Cas9 and dCas9 as well as their cutting efficiency in a single experiment on target and off-target DNA. Additionally we present a standardized workflow which allows efficient screening of sequence-variations with by hybridization of unlabeled target DNAs to a standard anchor DNA. We compare different mismatch influences on binding of gRNA programmed Cas9 to immobilized DNA sequences and discuss effects on the association and dissociation rate constants, respectively.
Author: Andreas Langer (January 2016)