Engineering Cas9-fusion proteins that enable safe and precise gene therapies

Genetic disorders cause a serious public health burden. Targeted repair of disease-causing mutations has the potential to cure many single-gene genetic disorders. CRISPR-Cas9 can be used to introduce double-strand breaks (DSBs) in disease-causing genes.

A donor template that encodes a corrected version of the mutated gene can be used by the cell for homology-directed repair (HDR) of the DSB.

While successful HDR can restore the non-pathogenic sequence of a mutated gene, it directly competes with an alternative DSB repair mechanism, non-homologous end joining (NHEJ), which results in random, undesirable insertions and deletions (INDELs). NHEJ-caused INDELs create safety concerns that slow the translation of CRISPR-based therapies to the clinic.

The aim of this project is to engineer Cas9-fusion proteins that prevent NHEJ at targeted sites and enhance the efficiency of HDR.Cas9 fusion proteins will be designed with several approaches that use the latest developments in protein engineering, AI, and directed evolution.

These best-performing fusion proteins that emerge from these approaches will be characterized for their efficiency and safety in correcting different disease-causing mutations.

Elucidation of the mechanism of the discovered Cas9-fusion protein variants may uncover unexpected aspects of the DNA repair process.

The best Cas9-fusion proteins have the potential to accelerate the advancement of curative gene correction therapies to the clinic.