Single-molecule studies of CRISPR/Cas9 off-target activity

Abstract

The discovery of CRISPR/Cas9 as an easily re-programmable endonuclease has heralded a new era of genetic manipulation. With it comes the prospect of novel gene therapy approaches, and the potential to cure previously un-treatable genetic diseases. However, reports of unintended off-target editing by CRISPR/Cas9 pose a significant hurdle to realising this potential. In this thesis I use single-molecule techniques to investigate this off-target activity of CRISPR/Cas9. First I demonstrate how specificity can be improved by modulating the hybridisation energy of guide and target DNA - through incorporation of DNA bases into the RNA guide molecules. Next I investigate how force applied to target DNA alters Cas9 specificity, demonstrating that disruption of the canonical DNA double-helix structure dramatically increases Cas9 promiscuity, allowing cutting at sites with as many as 50 % of bases mismatched. Finally, I extend this finding by showing that the presence of supercoiling in target DNA also has a significant impact of specificity, with negative supercoiling resulting in the induction of thousands of potential off-target sites across human genomic DNA sequences. Together this work suggests that in vivo processes which destabilise the DNA structure have the potential to induce off-target Cas9 activity at previously overlooked sites. I propose that rather than considering Cas9 targeting specificity as an intrinsic property of the protein complex, it is vital that we consider the structural state of DNA it is targeting. These findings will be important for on-going efforts to engineer Cas9 variants with higher specificity, in the endeavour to develop safe and efficient gene therapies.