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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

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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NOV 15, 2018 8:00 AM PST

Precise Gene Editing of Human Pluripotent Stem Cells


Specialty

Molecular Biology

Cell Biology

Genetics

Cancer Research

Cell Line Development

Clinical Research

Medicine

Assay Development

Biology

Biotechnology

Stem Cell Technologies

Cell Culture

Systems Biology

Dna

Biochemistry

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