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Increasing the accuracy of CRISPR/Cas9 gene editing for introns 44 and 55 of the DMD gene by using high fidelity Cas9 nucleases

  • Author(s): Popack, Dovber
  • Advisor(s): Spencer, Melissa J
  • et al.

Sixty percent of mutations causing Duchenne muscular dystrophy (DMD) occur within introns 44-55 of the DMD gene. The Spencer and Pyle Labs have designed a CRISPR/Cas9 platform that deletes introns 44 and 55 of the DMD gene to mimic a mild Becker muscular dystrophy (BMD) phenotype. This study compares the efficacy of two different Cas9 nucleases (either eSpCas9 or HypaCas9) in vitro to determine their respective efficiencies in comparison to wild type SpCas9. The data showed that editing efficiency of HypaCas9 was less efficient than either eSpCas9 or wild type SpCas9. Upon sequencing, it was revealed that the eSpCas9 plasmid sent from a company was actually identical to wild type SpCas9. Therefore, though we were not able to assess the efficiency of eSpCas9, the data revealed that hypaCas9 will not be a suitable substitute for wild type Cas9 due to its low efficiency.

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