Delivery of CRISPR/Cas9 into Blood Cells of Zebrafish: Potential for Genome Editing in Somatic Cells

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Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair ... continued below

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Schneider, Sara Jane August 2017.

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  • Schneider, Sara Jane

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Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.

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  • August 2017

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  • Oct. 9, 2017, 11:44 a.m.

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Schneider, Sara Jane. Delivery of CRISPR/Cas9 into Blood Cells of Zebrafish: Potential for Genome Editing in Somatic Cells, thesis, August 2017; Denton, Texas. (digital.library.unt.edu/ark:/67531/metadc1011754/: accessed October 24, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; .