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NOV 15, 2018 8:00 AM PST

Precise Gene Editing of Human Pluripotent Stem Cells

Speaker
  • Assistant Professor, Department of Biomedical Engineering, Wisconsin Institute for Discovery, University of Wisconsin-Madison
    Biography
      Krishanu Saha is an Assistant Professor in the Department of Biomedical Engineering at the University of Wisconsin-Madison. He is also a member of the Wisconsin Institute for Discovery, Carbone Cancer Center, and Stem Cell and Regenerative Medicine Center as well as the National Academies' Forum on Regenerative Medicine. Prior to his arrival in Madison, Dr. Saha studied Chemical Engineering at Cornell University and at the University of California in Berkeley. He was a Society in Science: Branco-Weiss fellow at the Whitehead Institute for Biomedical Research at MIT and in the Science and Technology Studies program at Harvard University. Major thrusts of his lab involve gene editing and cell engineering of human cells found in the retina, central nervous system and blood.

    Abstract

    CRISPR ribonucleoproteins (RNPs) can generate programmable gene edits, however imprecise editing and efficient delivery to human stem cells are key challenges. Here we describe novel biochemical techniques to assemble various biomolecules and coatings with nanoscale precision around a RNP. First, by modifying the sgRNA with a short S1m RNA aptamer, we developed a modular strategy, termed an “S1mplex,” to assemble Cas9 RNPs with biotinylated moieties. Using S1mplexes with biotinylated short oligonucleotides improves the ratio of precise to imprecise editing up to 18-fold over conventional methods approaching a ratio of 4 precise edits to every imprecise mutation, while assembly with fluorescent molecules allows selection and enrichment for cells with multiplexed gene edits. Second, we developed synthetic coatings for nonviral delivery of RNPs to human pluripotent stem cells. Combined, these strategies, which utilize chemically-defined, off-the-shelf reagents, have significant promise for gene editing applications in vitro (e.g., drug discovery, disease modeling) with human stem cells.


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