Jayshen Arudkumar

Duchenne Muscular Dystrophy:
This inherited X-linked genetic disorder is caused by mutations in the Dmd gene encoding dystrophin protein on the X-chromosome. The structural implications of dysfunctional dystrophin are severe, with approx. 1 in 35,000 male births burdened with this progressive muscle weakness and shortened life span. This cytoskeletal protein is integral for the structural maintenance of the muscle cell membrane (sarcolemma) integrity in cardiac, respiratory and skeletal muscle tissue. in association with the dystrophin-glycoprotein complex (DGC).

CRISPR-Cas9 as a technological forefront can be manipulated from its biological purpose (bacterial adaptive immunity) to be used to induce targeted double-stranded breaks (DSBs) at crucial sites in the genome, where the error-prone non-homologous end joining (NHEJ) is the primary repair pathway in absence of a homologous template sequence. All we need is a single gRNA (made from covalently linked crRNA + tracrRNA) and a Cas9 nuclease to let it cut! Microhomology-mediated end joining (MMEJ) is an alternative repair outcome that can also play a role in generation of large deletions:
1) Enable specific insertion of gene-edited sequence through 5'-3' resection on coding and non-coding strands, where microhomologous sequences (25bp) on either ends can function to allow deletion after annealing.

Theory and Experimental Plan:
The Dmd gene spans 79 exons of around 2500kb, where there are over 4000 characterised mutations in the vicinity of critical coding residues of exon 45-55 amenable to the majority of those diagnosed. We aim to utilise the innovative CRISPR-Cas9 as a gene therapy in targeting Exons 51 - 55 of the Dmd gene for the purpose of re-framing changes made from the single exon deletion and restoring the correct reading frame as a viable outcome of gene therapy. Through the testing of multiple re-framing strategies across an exon deletion model, the most efficient sgRNA candidates that induce favourable re-framing mutations can be discovered and tested in vitro and in a mouse disease model. Thus, there can be advancement of this proof-of-concept towards clinical viability.

Cool tools!
- Aran and colleagues (2019) have developed CRISPR-CHIP - a graphene-based field effect transistor that can be used as a dCas9-guided biosensor to screen clinical samples for the DMD-edited alleles without the need for isolation and PCR-amplification.
- David Liu and his lab (2019) have developed an efficient PRIME Editing system that conssists of a Cas9 H840A Nickase fused to a Reverse Transcriptase, in combination with an elongated gRNA to introduce desired mutations in human cells without the need for DSBs!