CRISPR-Cas9 is a game-changing gene editing tool that is likely to transform therapies for genetic disease. A programmable DNA nuclease (Cas9) is precisely guided to a specific DNA locus by a short single guide RNA (sgRNA) to elicit DNA strand breaks. Misrepair of these breaks causes gene knockouts (KOs).
However, unpredictable deletions are undesirable in the context of therapeutic gene editing, where the goal is instead to precisely correct a given mutation.Precise gene editing an be accomplished by providing a DNA template flanked by homology arms resulting in precise gene correction or knock-in (KIs). A major barrier to clinical use of precise gene correction is that KOs dominate over KIs.
We have discovered a novel property of Cas9 binding and cleavage that allows us to improve the rate of precise editing in vitro. By directly fusing unique enzyme activities onto Cas9 we can increase the number of KI vs KO events in cells.
The student will build on these findings to generate a more precise gene editing tool, through use of novel reporter assays of KO/KI, and in systems that measure correction of disease-associated alleles.
There is also an opportunity for the student to apply the technology to genes or factors associated with a genetic disorder of their own interest.
The most important outcome of this project will be a translatable application of improved gene editing for use in treatment of genetic disorders.