CRISPR/Cas9‐Based Safe‐Harbor Gene Editing in Rhesus iPSCs

Ravi Chandra Yada1, John W. Ostrominski1, Ilker Tunc2, So Gun Hong1, Jizhong Zou3, Cynthia E. Dunbar1

1 Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health, Bethesda, Maryland, 2 Systems Biology Core, Systems Biology Center, NHLBI, NIH, Bethesda, Maryland, 3 iPSC Core, Center for Molecular Medicine, NHLBI, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.11
DOI:  10.1002/cpsc.37
Online Posting Date:  November, 2017
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Abstract

NHP iPSCs provide a unique opportunity to test safety and efficacy of iPSC‐derived therapies in clinically relevant NHP models. To monitor these cells in vivo, there is a need for safe and efficient labeling methods. Gene insertion into genomic safe harbors (GSHs) supports reliable transgene expression while minimizing the risk the modification poses to the host genome or target cell. Specifically, this protocol demonstrates targeting of the adeno‐associated virus site 1 (AAVS1), one of the most widely used GSH loci in the human genome, with CRISPR/Cas9, allowing targeted marker or therapeutic gene insertion in rhesus macaque induced pluripotent stem cells (RhiPSCs). Furthermore, detailed instructions for screening targeted clones and a tool for assessing potential off‐target nuclease activity are provided. © 2017 by John Wiley & Sons, Inc.

Keywords: Rhesus macaque; induced pluripotent stem cells; CRISPR/Cas9; safe‐harbor; non‐human primate

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Gene Targeting and Colony Isolation
  • Basic Protocol 2: Screening and Cre‐Excision
  • Basic Protocol 3: Off‐Target Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Gene Targeting and Colony Isolation

  Materials
  • Mouse embryonic fibroblasts (MEF), 2 million cells/vial (GlobalStem, cat. no. GSC‐6201G)
  • Matrigel (BD Bioscience, cat. no. 354277)
  • RhiPSCs (see unit 4.11, Yada et al., )
  • Conditioned medium (see recipe)
  • ROCK inhibitor (Y‐27632) (Stemgent, cat. no. 04‐0012)
  • Puro MEF IRR, 2 million cells/vial (GlobalStem, cat no. GSC‐6220G)
  • Accutase (EMD Millipore, cat. no. SCR005)
  • P3 Primary Cell 4D‐Nucleofector® X Kit L (12 RCT) (Lonza, cat. no. V4XP‐3012)
  • Phosphate‐buffered saline (PBS; Lonza cat. no. 17‐516F), 1×, calcium‐ and magnesium‐free
  • RhiPSC medium (see recipe)
  • Puromycin (Sigma, cat. no. P8833)
  • DNeasy Blood & Tissue Kit (Qiagen, cat no. 69504)
  • 37°C 5%CO 2, 20%O 2 incubator
  • Hypoxia chamber (BioSpherix, cat. no. C‐274)
  • O 2 Controller (BioSpherix, cat. no. P110)
  • CO 2 Controller (BioSpherix, cat. no. P120)
  • 6‐well plates
  • 50‐ml conical tubes
  • Hemacytometer or automated cell counter
  • Centrifuge
  • FalconTM 15‐ml conical centrifuge tubes (Fisher Scientific, cat. no. 14‐959‐53A)
  • 4D‐Nucleofector™ Core Unit (Lonza, cat. no. AAF‐1002B)
  • Fluorescent microscope
  • MultiGrip™ 200 µl Tips (Optional) (Denville, cat. no. P3133‐F)
  • Cell scraper (Corning, cat. no. 3010)

Basic Protocol 2: Screening and Cre‐Excision

  Materials
  • Primers (see Table)
  • REDTaq® ReadyMix PCR Reaction Mix (Sigma, cat. no. R2523‐20RXN)
  • 1% agarose gel
  • Opti‐MEM Reduced Serum Medium (ThermoFisher, cat. no. 31985062)
  • Cre plasmid [we use the Puro‐T2A‐Cre‐GFP plasmid (Merling et al., )]
  • EditProTM Stem Transfection Reagent (MTI‐GlobalStem, cat. no. GST‐2174)
  • Mouse embryonic fibroblasts (MEF), 2 million cells/vial (GlobalStem, cat. no. GSC‐6201G)
  • Accutase (EMD Millipore, cat. no. SCR005)
  • RhiPSC medium (see recipe)
  • ROCK inhibitor (Y‐27632) (Stemgent cat. no. 04‐0012)
  • Thermal cycler
  • Spectrophotometer
  • 1.5‐ml microcentrifuge tubes
  • Fluorescent microscope
  • Centrifuge
  • Falcon® 5‐ml round‐bottom polystyrene test tube, with cell strainer snap cap (Corning, cat. no. 352235)
  • FACS machine
  • 37°C 5%CO 2, 20%O 2 incubator
  • Hypoxia Chamber (BioSpherix, cat. no. C‐274)
  • O 2 Controller (BioSpherix, cat. no. P110)
  • CO 2 Controller (BioSpherix, cat. no. P120)
  • MultiGrip™ 200‐µl Tips (Optional) (Denville, cat. no. P3133‐F)
  • Additional reagents and equipment for Southern blot (Hoopes, )

Support Protocol 1:

  Materials
  • RhiPSC medium (see recipe)
  • KnockOut DMEM (KO‐DMEM; Gibco cat. no. 10829)
  • Conjugated antibody
  • Hypoxia Chamber (BioSpherix, cat. no. C‐274)
  • O 2 Controller (BioSpherix, cat. no. P110)
  • CO 2 Controller (BioSpherix, cat. no. P120)
  • Fluorescence microscope
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Figures

Videos

Literature Cited

Literature Cited
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