Chapter 18, Problem 35P

Recently, scientists have used a mouse model for Duchenne muscular dystrophy (called the mdx mouse) to test whether Cas9 and an sgRNA could be an effective therapy for this disease. The cause of muscular dystrophy is homozygosity or hemizygosity for loss-of-function mutations in the X-linked Dmd gene. The mdx mouse has a nonsense mutation in exon 23 (of 79 exons) of Dmd. Researchers tested a technique called exon skipping; their idea was to use CRISPR/Cas9 to delete exon 23 in the mutant Dmd gene in the mdx mouse. AAV vectors with genes that express Cas9 and two different sgRNAs were injected into the muscles of adult mice. In about 10% of the muscle cells, exon 23 was deleted, and functional dystrophin protein was detected. Some muscle function was restored, although only to a small extent.

a.	Draw a diagram of exon 23 and the introns that flank it. On your diagram, draw the locations where the sgRNAs would hybridize with the genomic DNA. Explain how the deletion of exon 23 would take place.
b.	Design a PCR assay to determine if exon 23 is deleted from the genomic DNA of cell clones. Where would the PCR primers hybridize, and how would you be able to tell if the exon was deleted?
c.	Skipping exon 23 restored dystrophin protein function at least partially. What does this say about the amino acids encoded by exon 23?
d.	Considering your answer to part (c), would the exon-skipping strategy work for all Dmd point mutations that cause muscular dystrophy?
e.	What must be true about exons 22, 23, and 24 that would allow the researchers to consider this exon-skipping strategy? (Hint: See Problems 28 and 29.)