PhD defence: A multifaceted approach to push CRISPR-induced DNA repair towards templated gene correction

Inherited diseases are written in the DNA. The type of diseases that can be passed on genetically are extremely varied. While some can be treated by conventional medication, others are more difficult to treat, and there is no cure-all medicine for this collection of diseases.

CRISPR-Cas9, often colloquially referred to as “DNA-scissors”, is a novel technology that is theoretically capable of curing most inherited diseases. Cas9 can be targeted to any part of the patient’s DNA, including the mutated part of the DNA code that is responsible for an inherited disease, and make a cut in the DNA.

Our cells are capable of repairing this cut in several ways. One is to just paste the cut back together, which does not cure the diseased DNA and even has a risk of creating new random mutations. The other is to use a similar DNA molecule as a repair template. We can use this by providing a DNA molecule which contains a fixed example of the genetic code. This mechanism can be used to encode specific cures for inherited disease by giving our cells a corrected example on a template. This genetic correction can, in theory, be used to repair DNA mutations that are responsible for more than half of the inherited diseases we know. Unfortunately, cells are far more prone to paste the DNA back together, limiting the efficiency of our template repair approach. The main goal of this thesis was therefore to increase the odds of specifically activating templated repair of the cut.

We show that the natural cell division cycle influences this process and tested drug molecules to favor genetic correction specifically. We also succeeded in clicking CRISPR-Cas9, like molecular Lego, to a drug that can in act at the same time to theoretically enhance gene correction. The discoveries in this thesis may contribute to one day curing inherited diseases.