University of Colorado School of Medicine, Anschutz Medical Campus

2 ARTICLES PUBLISHED IN JoVE

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.

Genetics

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Jeffrey Inen^1,2, Chann Makara Han^1,2, David M. Farrel³, 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.