CRISPR‐Cas9‐Based Genome Editing of Human Induced Pluripotent Stem Cells

Joseph C. Giacalone1, Tasneem P. Sharma1, Erin R. Burnight1, John F. Fingert1, Robert F. Mullins1, Edwin M. Stone1, Budd A. Tucker1

1 Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5B.7
DOI:  10.1002/cpsc.46
Online Posting Date:  February, 2018
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Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease‐causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR‐Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR‐based genome editing of patient‐specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR‐based genome editing of patient‐ specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre‐lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.

Keywords: autologous cell replacement; CRISPR‐Cas9; genome editing; hiPSC

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Electroporation of hiPSCs to Deliver CRISPR‐Cas9 Plasmids
  • Alternate Protocol 1: Reagent‐Based Transfection of hiPSCs to Deliver CRISPR‐Cas9 Plasmids
  • Basic Protocol 2: Positive Selection of Genomically Modified hiPSCs
  • Basic Protocol 3: Assessment of Genomic Modifications
  • Basic Protocol 4: Removal of Selection Cassette from Genomically Modified hiPSCs and Negative Selection
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Electroporation of hiPSCs to Deliver CRISPR‐Cas9 Plasmids

  • Recombinant human laminin‐521 (rhLaminin‐521; Corning Life Sciences, cat. no. 354222)
  • Dulbecco's phosphate‐buffered saline (DPBS; Thermo Fisher Scientific, cat. no. 15040066)
  • Complete E8 medium:
    • Essential 8 medium (Thermo Fisher Scientific, cat. no. A1517001)
    • 10 ng/ml rhFGF2 (Waisman Biomanufacturing, cat. no. rhFGF)
    • 100 μg/ml Primocin (InvivoGen, cat. no. ant‐pm‐1)
  • 100× RevitaCell Supplement (Thermo Fisher Scientific, cat. no. A2644501)
  • pX330‐U6‐Chimeric_BB‐CBh‐hSpCas9 (Addgene, plasmid no. 42230)
  • Homology‐directed repair plasmid (pHDR; optional)
  • Versene solution (Thermo Fisher Scientific, cat. no. 15040066)
  • Sterile six‐well tissue culture plates
  • 37°C, 5% CO 2 incubator
  • 1.5‐ml microcentrifuge tubes
  • Sterile conical tubes
  • Cell counter or hemocytometer
  • NEON Transfection System (Thermo Fisher Scientific, cat. no. MPK5000) with 100‐μl kit (cat. no. MPK10096)

Alternate Protocol 1: Reagent‐Based Transfection of hiPSCs to Deliver CRISPR‐Cas9 Plasmids

  Additional Materials (also see protocol 1)
  • Opti‐MEM I Reduced Serum Medium (Thermo Fisher Scientific, cat. no. 31985070)
  • Lipofectamine Stem Transfection Reagent (Thermo Fisher Scientific, cat. no. STEM00001)

Basic Protocol 2: Positive Selection of Genomically Modified hiPSCs

  Additional Materials (also see protocol 1)
  • Genomically modified hiPSCs (see protocol 1 or protocol 2Alternate Protocol)
  • 10 mg/ml puromycin dihydrochloride (Thermo Fisher Scientific, cat. no. A1113802)
  • Sterile twelve‐well tissue culture plates
  • StemPro EZPassage Disposable Stem Cell Passaging Tool (Thermo Fisher Scientific, cat. no. 23181010)

Basic Protocol 3: Assessment of Genomic Modifications

  • Genomically modified hiPSCs (see protocol 1Basic Protocols 1 and protocol 32 and Alternate Protocol)
  • NucleoSpin RNA kit (Macherey‐Nagel/Clontech, cat. no. 740955)
  • NucleoSpin Tissue kit (Macherey‐Nagel/Clontech, cat. no. 740952)
  • SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, cat. no. 11754‐050)
  • AccuPrime Taq DNA Polymerase, high fidelity (Thermo Fisher Scientific, cat. no. 12346086), including 10× Buffer II
  • E‐Gel Agarose Gels Starter Pak, 2% (Thermo Fisher Scientific, cat. no. G600002)
  • MinElute PCR Purification Kit (Qiagen, cat. no. 28004)
  • T7 Endonuclease I (New England BioLabs, cat. no. M0302), including NEB Buffer 2
  • NanoDrop One Microvolume UV‐Vis Spectrophotometer (Thermo Fisher Scientific, cat. no. ND‐ONE‐W)
  • Thermocycler (e.g., T100 Thermal Cycler, Bio‐Rad, cat. no. 1861096)
  • Additional reagents and equipment for collecting hiPSCs (see protocol 1)

Basic Protocol 4: Removal of Selection Cassette from Genomically Modified hiPSCs and Negative Selection

  Additional Materials (also see protocol 1)
  • Positively selected genomically modified hiPSCs generated using a pHDR (see protocol 3)
  • Opti‐MEM I Reduced Serum Medium (Thermo Fisher Scientific, cat. no. 31985070)
  • Lipofectamine Stem Transfection Reagent (Thermo Fisher Scientific, cat. no. STEM00001)
  • pCSCre2 (Addgene, plasmid no. 31308)
  • Ganciclovir (InvivoGen, cat. no. sud‐gcv)
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