#apaperaday: Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy
In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy.
A paper from the international journal of molecular sciences by Happi Mbakam et al on prime editing in the dystrophin gene. Doi 10.3390/ijms23116160.
Genome editing with cas9 creates double stranded breaks at a target location . The more recently developed prime editing allows conversion of a nucleotide into another at a target location. This relies on a one strand nick.
Here authors tested if they could generate mutations in the dystrophin gene in HEK293 cells. They were able to generate known Duchenne point mutations in exon 9, 35, 36, 20, 43, 55 and 61 but efficiency was low.
Efficiency could be increased by treating with two guide RNAs or treating multiple times. For some targets optimizing the PAM sequence worked as well. Using multiple guides and treatments and optimized PAM sequence authors managed to get 38% editing in vitro.
They prime edited myoblasts from a Duchenne patient with an exon 6 nonsense mutation. This restored dystrophin production in vitro. Authors discuss editing efficiency is low and so far they only used proliferating cells while muscle is post mitotic.
Studies in myotubes and mouse models will have to assess efficiency and feasibility in post mitotic cells. Authors speculate that perhaps a low efficiency will still be efficient as 1 nuclear domain that is edited can produce dystrophin for a larger area of muscle.
Hopefully our own study with mdx/Xist mice with 3% of dystrophin expressing nuclei suggest otherwise: Dystrophin does not migrate far from where it is produced. So prime editing is feasible in principle but work is needed to improve efficiency and assess feasibility in vivo.