Impact of heparin on kinetic parameters of DNA endonuclease Cas9Public Deposited
The bacterial CRISPR/Cas9 system shows promise as a genome-editing tool to treat human disease. It is integral to understand the binding and unbinding kinetics of Cas9 to its target DNA to study specificity of the Cas9 protein. Standard practices involve the use of negatively charged polymer Heparin to reduce unspecific binding of Cas9 to non-target DNA. This study proposes a novel method for study of Cas9, while also demonstrating the negative impact Heparin has on the Cas9-DNA interaction. By using short electrically switchable DNA layers tethered to a gold surface, the change in DNA movement through solution after binding of Cas9 is studied. We found that (1) Heparin changes the on and off rates that characterize Cas9 (2) It is vital to see some minimal level of unspecific binding of Cas9, to understand the risk of Cas9 cutting functional DNA in vivo, yet Heparin removes the ability to do this. We demonstrate the benefit of studying Cas9 without Heparin by using the switchSENSE method, which does not require Heparin to obtain accurate on and off rates that describe the Cas9-DNA interaction.
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