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CRISPR Cas9 Genome Editing in Germlines

CRISPR Cas9 Genome Editing in Germlines. PBIO 4500 December 5, 2017 Sarah Disney. What is CRISPR Cas9?. Discovered in E. coli CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats An adaptive bacterial immune defense

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CRISPR Cas9 Genome Editing in Germlines

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  1. CRISPR Cas9 Genome Editing in Germlines PBIO 4500 December 5, 2017 Sarah Disney

  2. What is CRISPR Cas9? • Discovered in E. coli • CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats • An adaptive bacterial immune defense • Cas9 = An endonuclease that uses a sgRNA to cleave a piece of DNA • NHEJ = Non-homologous end joining • HDR = homology directed repair

  3. Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA • Bassel-Duby et. Al • The objective of this study was to correct the genetic defect in the Dmd gene of mice with muscular dystrophy through CRISPR/Cas9 mediated germline editing • Did it work???

  4. Duchenne Muscular Dystrophy (DMD) • X-linked disease • 1 in 3,500 live male births • Rare in girls • Extremely debilitating • Muscle weakness and degeneration • Eventually fatal • Begins to affect heart and respiratory muscles

  5. Methodology • Developed DMD mice models • Referred to as mdx • Crossed mdx female and male to create mdx zygotes • CRISPR/Cas9 was used to treat the germlines in vitro • Zygotes were then reimplanted in a healthy mother • This lead to mice with varying levels of mocaisism • Referred to as mdx-C mice

  6. Results • Varying levels of mosaicism were effective • Direct editing of satellite cells in vivo by the CRISPR/Cas9 system represents a potentially promising alternative approach to promote muscle repair in DMD. • No significant negative effects were found in the treated mice

  7. Ethics • The ethics of human germline experimentation may be the most contentious conversation in science • At the NAS Summit 2015, the NAS ruled that “it would be irresponsible to proceed with any clinical use of germline editing”. • Specific safety and ethical criteria must be met before any use • Additionally, the baby would have to be at imminent risk of severe genetic disease with no other viable options • At what point is the use of a human embryo no longer justified? • Where do we draw the line? • Are we playing God?

  8. References • Long, C., McAnally, J. R., Shelton, J. M., Mireault, A. A., Bassel-Duby, R., & Olson, E. (2014). Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA. Science, 345(6201), 1184-1188. doi:10.1126/science.1254445 • Sharma, A., & Scott, C. (2015). The ethics of publishing human germline research. Nature Biotechnology, 33(6).

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