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University of Colorado School of Medicine, Anschutz Medical Campus

2 ARTICLES PUBLISHED IN JoVE

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Developmental Biology

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells
Patrick S. McGrath 1,2,3, Nicole Diette 1,2, Igor Kogut 1,2,3, Ganna Bilousova 1,2,3
1Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, 2Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, 3Stem Cell Biobank and Disease Modeling Core, University of Colorado School of Medicine, Anschutz Medical Campus

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

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Genetics

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Jeffrey Inen 1,2, Chann Makara Han 1,2, David M. Farrel 3, Ganna Bilousova 1,2, Igor Kogut 1,2
1Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, 2Gates Institute, University of Colorado School of Medicine, Anschutz Medical Campus, 3Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Anschutz Medical Campus

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

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