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New Tools for improved CRISPR gene-editing in stem cells

Speaker
  • Director of Scientific Alliances, Thermo Fisher Scientific
    Biography
      James Kehler VMD, PhD is a comparative stem cell biologist who thrives on developing productive collaborations to translate scientific discoveries into transformative products. He trained at the University of Pennsylvania, where he received his VMD in 2002, and PhD in Cell and Molecular Biology in 2004. James has worked as a visiting researcher at the National Institutes of Health for over 10 years, where he and his collaborators at NEI, NCI, NINDS, NIDDK and NIAAA developed animal and stem cell-based models of human diseases. He has run workshops on reprogramming and gene-editing both at the NIH, as well as internationally. James has worked and consulted for several stem cell companies from product development and management to directing custom reprogramming and gene-editing services. In 2016, James joined MTI-GlobalStem, now part of Thermo Fisher Scientific, as Director of Scientific Alliances, to foster collaborative research projects with academic, biotech and pharmaceutical partners.

    Abstract

    The development of the CRISPR/Cas9 gene-editing platform enables the rapid generation of new genetically modified stem cell models of human diseases, as well as providing new potential therapeutic treatments. One challenge has been the efficient and consistent delivery of a range of gene-editing tools into human Pluripotent Stem Cells (PSCs) across different culture systems. We developed the Lipofectamine™ Stem transfection reagent as a complementary option to electroporation to be able to deliver large DNA plasmids, RNAs and Ribonucleoprotein (RNP) complexes into human induced Pluripotent Stem Cells and Neural Stem Cells. The ability to co-deliver Cas9 RNP complexes along with ssDNA templates enables both the disruption of genes, as well as introduction of specific basepair changes by Homology Directed Repair. Data demonstrating improved cutting activity in iPSCs with the next generation of TrueCut™ Cas9 Protein v2 with synthetic TrueGuide™ sgRNAs will be presented. In addition to Neon™ electroporation, optimized transfection and subsequent single-cell clonal isolation in a StemFlex™ Medium workflow will be reviewed. Scientists interested in gene-editing to create stem cell models for basic research, drug discovery and translational medicine will discover how these new tools can accelerate their research programs.


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