Exon-skipping with CRISPR-Cas9

The Gersbach lab focuses on developing innovative methods in molecular and genetic engineering for applications in regenerative medicine, treating genetic disease, and enhancing understanding of fundamental biological processes. More specifically, many members of the lab aim to treat Duchenne’s muscular dystrophy with CRISPR-Cas9 genetic engineering tools by creating an insertion or deletion mutation that restores the reading frame of the DMD gene.

So far, their methods have been successful in skipping an exon to allow the production of dystrophin, though the deletion efficiency has been close to 2%. Though this is satisfactory for restoring muscle strength in mice, 2% is too low for humans and the methods would not be applicable to other genetic diseases. This inefficiency may be caused by the choice in guide RNAs that lead the Cas9 to the desired deletion site, as some gRNAs have been shown to have higher mutation rates than other. However, there are thousands of possible gRNAs and gRNA combinations for each exon and testing each one is tedious and time-consuming. My mentor’s project is developing a high-throughput strategy for screening pairs of gRNAs for hDMD exon deletions. For my project this summer, I will be testing 5 gRNAs on each side of exon 51 to determine which is most efficient to use as a positive control for my mentor’s screening techniques. By targeting both sides, my designed Cas9 will delete the exon and restore the reading frame shift that was caused by a mutation in an earlier exon. I can then sequence the DNA to see how many of the cells I transfected have exon 51 deleted and how well each gRNA worked.

In the future, this will hopefully lead to more frequent deletions with the use of CRISPR and Cas9 so that these genetic engineering tools can be used to treat those born with genetic diseases.

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