CRISPR‐Mediated Gene Targeting of Human Induced Pluripotent Stem Cells

Susan M. Byrne1, George M. Church1

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.8
DOI:  10.1002/9780470151808.sc05a08s35
Online Posting Date:  November, 2015
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Abstract

CRISPR/Cas9 nuclease systems can create double‐stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single‐stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1‐25% and gene insertion/replacement efficiencies of 0.5‐10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single‐cell FACS sorting, and genotyping successfully edited cells. © 2015 by John Wiley & Sons, Inc.

Keywords: gene targeting; CRISPR/Cas9 nuclease; human induced pluripotent stem cells; transfection; genome engineering

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Transfection of Plasmids into Human Induced Pluripotent Stem Cells
  • Basic Protocol 2: Assessment of Cutting Efficiency
  • Basic Protocol 3: Cloning Human iPSCs by Single‐Cell FACS
  • Basic Protocol 4: Purification of Genomic DNA in 96‐Well Tissue Culture Plates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Transfection of Plasmids into Human Induced Pluripotent Stem Cells

  Materials
  • Human induced pluripotent stem cells (e.g., PGP1 iPSCs, Coriell, cat. no. GM 23338)
  • mTeSR1 defined feeder‐free medium (StemCell Technologies, cat. no. 05850)
  • Matrigel human ES cell−qualified matrix (Corning, cat. no. 354277), aliquoted according to manufacturer's instructions
  • Knockout DMEM/F12 medium (Life Technologies, cat. no. 12660‐012)
  • 10 mM Y‐27632 ROCK inhibitor (see recipe)
  • P3 Primary Cell 4D‐Nucleofection Kit (Lonza, V4XP‐3032) containing:
    • Nucleofector solution
    • Supplement
    • pMaxGFP control vector
    • 100‐μl Nucleocuvettes or 16‐well Nucleocuvette Strips
  • Endotoxin‐free plasmid DNA expressing Cas9 nuclease
  • Endotoxin‐free plasmid DNA expressing single guide RNA (sgRNA)
  • Endotoxin‐free plasmid DNA (or HPLC‐purified ssODN) donor construct
  • Accutase (e.g., EMD Millipore, cat. no. SF006, or Stem Cell Technologies, cat. no. 07920)
  • 0.4% (w/v) trypan blue solution (Life Technologies, cat. no. 15250)
  • Sterile 10‐cm and 24‐well tissue culture plates
  • Humidified 37°C incubator with 5% CO 2
  • Sterile conical tubes
  • Cell counter: automated system (e.g., Countess Cell Counter, Life Technologies) or hemacytometer chamber slide
  • 4D‐Nucleofector System with Core and X units (Lonza, cat. nos. AAF‐1002B and AAF‐1002X)

Basic Protocol 2: Assessment of Cutting Efficiency

  Materials
  • PCR kit with a high‐fidelity DNA polymerase (e.g., KAPA HiFi HotStart, cat. no. KK2501)
  • CRISPR gene‐targeted hiPSCs (see protocol 1)
  • Genomic DNA purification kit for mammalian cell culture (e.g., Sigma, cat. no. G1N70, or Qiagen, cat. no. 13323)
  • 1:500 working dilution of SYBR Green I Nucleic Acid Stain (Life Technologies, cat. no. S‐7563) in ddH 2O
  • Gel extraction kit (e.g., Qiagen QIAquick, cat. no. 28704)
  • NEBNext dual index primers for Illumina (New England Biolabs, cat. no. E7600S)
  • Quantitative real‐time PCR machine capable of detecting SYBR Green
  • PCR plates or tubes with optically clear films or lids
  • Nanodrop spectrophotometer or other system for measuring DNA concentration
  • Access to MiSeq sequencing facility
  • Computer for primer design and CRISPR analysis of MiSeq results
  • Additional reagents and equipment for agarose gel electrophoresis

Basic Protocol 3: Cloning Human iPSCs by Single‐Cell FACS

  Materials
  • 0.1% gelatin solution (Millipore, cat. no. ES‐006‐B)
  • Irradiated CF‐1 mouse embryonic fibroblasts (MEFs; Global Stem, cat. no. 6001G)
  • DMEM‐10 (see recipe)
  • Gene‐targeted hiPSCs (see protocol 1)
  • mTeSR1 defined feeder‐free medium (StemCell Technologies, cat. no. 05850)
  • SMC4 inhibitors (see recipe)
  • hES cell medium (see recipe)
  • 1 mg/ml fibronectin (optional; Sigma, cat. no. F1141; do not vortex or agitate stock)
  • Accutase (e.g., EMD Millipore, cat. no. SF006; StemCell Technologies, cat. no. 07920)
  • Fluorescently conjugated antibody (optional: e.g., mouse anti−human Thy1 clone 5E10, BD Biosciences, eBioscience)
  • Fetal calf serum (FCS, ES‐cell‐qualified, e.g., Life Technologies, cat. no. 10439), heat‐inactivated
  • 10 mM Y‐27632 ROCK inhibitor (see recipe)
  • 96‐well flat‐bottom plates, sterile, tissue culture treated
  • 37°C water bath
  • Sterile conical tubes
  • Tabletop centrifuge with a swinging‐bucket rotor for conical tubes and 96‐well plates
  • Humidified 37°C incubator with 5% CO 2
  • Cell strainer (Corning, cat. no. 352350) or 70‐μm nylon mesh, cut into squares and autoclaved (Spectrum Labs, cat. no. 146490)
  • Cell viability dye (e.g., propidium iodide, DAPI, YoPro from Life Technologies, BD Biosciences, eBioscience)
  • FACS sorter capable of sterile sorting into 96‐well plates (e.g., Beckman Coulter MoFlo Astrios or BD Biosciences FACS Aria)
NOTE: All centrifugations are carried out at room temperature, and all cultures are incubated at 37°C unless otherwise indicated.

Basic Protocol 4: Purification of Genomic DNA in 96‐Well Tissue Culture Plates

  Materials
  • 96‐well tissue culture plate containing hiPSC colonies (see protocol 3)
  • Cell lysis buffer (see recipe)
  • NaCl‐saturated ethanol (see recipe)
  • 70% ethanol (molecular biology grade)
  • 0.5× TE buffer (see recipe)
  • 96‐well tissue culture plates
  • Humidified 55°C incubator
  • Multichannel pipetter
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Figures

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Literature Cited

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