CRISPR/Cas9‐Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells

Arun Sharma1, Christopher N. Toepfer2, Tarsha Ward1, Lauren Wasson3, Radhika Agarwal1, David A. Conner3, Johnny H. Hu1, Christine E. Seidman3

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Radcliffe Department of Medicine, University of Oxford, Oxford, 3 Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.11
DOI:  10.1002/cphg.52
Online Posting Date:  January, 2018
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Abstract

Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology‐directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length. These fluorescently tagged hiPSCs can be used to visualize protein function and dynamics in real time as cells proliferate and differentiate. Given that nearly any intracellular protein can be fluorescently tagged, this system serves as a powerful tool to facilitate new discoveries across many biological disciplines. In this unit, we present protocols for the design, generation, and monoclonal expansion of genetically customized hiPSCs encoding fluorescently tagged endogenous proteins. © 2018 by John Wiley & Sons, Inc.

Keywords: CRISPR; fluorescent reporters; genome editing; pluripotent stem cells; synthetic biology

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Design and Preparation of gRNA, Cas9, and HDR Template Plasmids
  • Basic Protocol 2: Transfection of hiPSCs with Cas9, gRNA, and HDR Template Plasmids
  • Basic Protocol 3: Monoclonal Expansion and Genetic Validation of hiPSCs Encoding Fluorescently Tagged Endogenous Proteins
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Design and Preparation of gRNA, Cas9, and HDR Template Plasmids

  Materials
  • pSpCas9(BB)‐2A‐Puro (PX459) V2.0 (PX459 V2.0; Addgene, cat. no. 62988) bacterial stock
  • LB agar plate containing 100 µg/ml ampicillin
  • LB liquid medium containing 100 µg/ml ampicillin
  • Plasmid Midi Kit (Qiagen)
  • Nuclease‐free water
  • Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800‐20), containing:
    • Salt solution
    • pCR‐Blunt II‐TOPO vector
    • M13 forward (5′‐GTAAAACGACGGCCAG‐3′) and reverse (5′‐CAGGAAACAGCTATGAC‐3′) sequencing primers
    • Sterile water, PCR grade and deionized
  • One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, cat. no. C404010), pre‐thawed on ice, with S.O.C. medium
  • LB agar plates containing 50 μg/ml kanamycin
  • LB liquid medium containing 50 μg/ml kanamycin
  • Plasmid Mini Kit (Qiagen)
  • L‐shaped bacterial spreaders
  • 37°C bacterial incubator shaker
  • Erlenmeyer flask
  • Sterile pipette tips
  • NanoDrop microspectrophotometer (Thermo Scientific) or equivalent
  • Benchling bioinformatics software or equivalent
  • 1.5‐ml Eppendorf tubes
  • 45°C bacterial incubator
  • 10‐ml bacterial culture tubes
  • DNA sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR Lasergene Suite)
  • Additional reagents and equipment for Sanger sequencing (unit 7.1; Shendure et al., )
NOTE: Experiments involving PCR and RNA require extremely careful technique to prevent contamination and RNA degradation; see appendix 2D.

Basic Protocol 2: Transfection of hiPSCs with Cas9, gRNA, and HDR Template Plasmids

  Materials
  • Matrigel (hESC qualified; BD Biosciences, cat. no. 354277; store stock at –20°C)
  • DMEM/F‐12 medium (Thermo Fisher Scientific, cat. no. 11320033), 4°C
  • Wild‐type, low‐passage hiPSCs adapted for feeder cell–free growth on Matrigel
  • mTeSR1 feeder‐free pluripotent stem cell maintenance medium (StemCell Technologies, cat. no. 05850), 37°C
  • Rho kinase inhibitor (ROCKi; Tocris, cat. no. 1254)
  • Human Stem Cell Nucleofector Kit 2 (Lonza, cat. no. VPH‐5022):
    • Nucleofector Solution
    • Supplement
    • pmaxGFP Vector
    • Nucleocuvettes
  • Cas9 plasmid DNA (see protocol 1, steps 1 to 3)
  • gRNA expression vector (see protocol 1, steps 4 to 14)
  • HDR template vector (see protocol 1, steps 4 to 14)
  • 0.5 M EDTA (Thermo Fisher Scientific, cat. no. 15575020)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 20012‐050)
  • Amaxa Nucleofector II Device (Lonza)
  • 6‐well tissue culture–treated plates
  • Pipette tips, pre‐cooled to 4°C
  • 37°C cell culture incubator
  • 1000‐μl manual pipettor
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Hemocytometer
  • Tabletop centrifuge with plate adapter
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper sterile technique should be used accordingly.

Basic Protocol 3: Monoclonal Expansion and Genetic Validation of hiPSCs Encoding Fluorescently Tagged Endogenous Proteins

  Materials
  • hiPSCs transfected with gRNA, Cas9, and HDR template plasmids (see protocol 2)
  • mTeSR1 feeder‐free pluripotent stem cell maintenance medium (StemCell Technologies, cat. no. 05850), 37°C
  • Puromycin dihydrochloride (Thermo Fisher Scientific, cat. no. A1113803)
  • Matrigel‐coated 6‐, 24‐, and 96‐well plates (see protocol 2, step 1)
  • 0.5 M EDTA (Thermo Fisher Scientific, cat. no. 15575020)
  • PBS (Life Technologies, cat. no. 20012‐050)
  • ROCKi (Tocris, cat. no. 1254)
  • prepGEM Tissue Kit (VWR, cat. no. 95044‐034), containing:
    • prepGEM 10X Buffer
    • prepGEM tissue protease enzyme
  • KOD Xtreme Hot Start DNA Polymerase (Millipore, cat. no. 71975‐3) kit, containing:
    • 2X Xtreme Buffer
    • dNTPs
    • KOD Xtreme Hot Start DNA Polymerase
  • Bambanker cell freezing medium (Fisher Scientific, cat. no. NC9582225)
  • Inverted microscope
  • 96‐well thermocycler‐compatible plate
  • Tabletop centrifuge with plate adapter
  • Standard thermocycler for PCR
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 1.8‐ml cryotubes (e.g., Nunc Biobanking and Cell Culture Cryogenic Tubes, Fisher Scientific, cat. no. 375418)
  • CoolCell LX Freezing Container (Sigma‐Aldrich, cat. no. BCS‐405)
  • Additional reagents and equipment for Sanger sequencing (unit 7.1; Shendure et al., ) and agarose gel electrophoresis (unit 7.2; Armstrong & Schulz, )
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper sterile technique should be used accordingly.NOTE: Experiments involving PCR require extremely careful technique to prevent contamination; see appendix 2D.
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Figures

Videos

Literature Cited

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