Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores

Sequence-specific interactions between nucleic acids and proteins are fundamental to many critical biological processes. Despite the ubiquitous nature of protein-DNA binding, versatile methods to probe the specificity of these events remain elusive. In particular, single-molecule methods that enable the quantification of these processes are essential towards understanding and manipulating protein binding. To this end, we report a system which leverages solid state nanopores with diameters of ~10 nm to identify binding events between DNA and CRISPR associated (Cas) probes – specifically catalytically inactive or dead Cas9 (dCas9), which binds to DNA but does not cleave it. The rational design of DNA nanostructures allows for the incorporation of user-defined binding sequences, enabling a systematic study of how mismatch position and identity impacts the binding efficiency. These experiments reveal the relationship between sequence and binding at the single nucleotide level, exemplifying the utility of both nanopore measurements and DNA nanotechnology towards the next generation of biosensing assays.S.E.S. acknowledges funding from Oxford Nanopore Technologies, Engineering and Physical Sciences Research Council (EPSRC) and Cambridge Trust. N.E.W. acknowledges funding from Oxford Nanopore Technologies, the Canada UK Foundation, and the University of Cambridge Office of Postdoctoral Affairs. S.Y. acknowledges funding from the EPSRC grant EP/S022953/1 and A.D. acknowledges funding from the EPSRC grant EP/L015889/1. U.F.K and K.C. acknowledge funding through a ERC-2019-POC PoreDetect 899538