Efficient CRISPR/Cas9‐Based Genome Engineering in Human Pluripotent Stem Cells

Cody Kime1, Mohammad A. Mandegar2, Deepak Srivastava3, Shinya Yamanaka4, Bruce R. Conklin5, Tim A. Rand2

1 Present address: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, 2 Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, California, 3 Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, 4 Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 5 Department of Medicine, University of California, San Francisco, San Francisco
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.4
DOI:  10.1002/0471142905.hg2104s88
Online Posting Date:  January, 2016
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Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)–cell‐like properties has led to hPS cells with disease‐specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site‐specific double‐strand break (DSB)–mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA. © 2016 by John Wiley & Sons, Inc.

Keywords: human pluripotent stem cells; genomic engineering; CRISPR; Cas9

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Design and Preparation of gRNA Plasmids
  • Basic Protocol 2: Preparation of Recombinant DNA with Homology ARMS
  • Basic Protocol 3: Human Pluripotent Stem Cell Culture and Nucleofection
  • Basic Protocol 4: Colony Picking and Subclone Selection
  • Basic Protocol 5: Cryopreservation and Genomic DNA Preparation
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Design and Preparation of gRNA Plasmids

  • pX330‐U6‐Chimeric_BB‐CBh‐hSpCas9 (pX330; Addgene, cat. no. 42230)
  • 10 U/μl BbsI nuclease (New England Biolabs, cat. no. R0539S)
  • 10 U/μl calf intestine alkaline phosphatase (CIP; New England Biolabs, cat. no. M0290S)
  • Nuclease‐free H 2O (e.g., DEPC‐treated; appendix 2D)
  • 1 M dithiothreitol (DTT)
  • T4 DNA Ligase Kit (New England Biolabs cat. no. M0202S)
  • 10 U/μl T4 Polynucleotide Kinase (PNK; New England Biolabs cat. no. M0201S)
  • Competent cells (choice depends on transformation method)
  • LB agar plates ( appendix 2D) containing 100 μg/ml ampicillin
  • EndoFree Maxiprep Kit (Qiagen, cat. no. 12362) or NucleoBond Xtra Midi EF Kit (Macherey‐Nagel, cat. no. 740420.10)
  • CRISPR tool (http://crispr.mit.edu)
  • Oligonucleotide synthesis facility
  • PCR tube strips
  • Programmable thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ), transformation of cells (Ausubel et al., , Chapter 9), DNA miniprep (Wilson, ), and DNA sequencing (Ausubel et al., , Chapter 7)

Basic Protocol 2: Preparation of Recombinant DNA with Homology ARMS

  • Gateway pENTR1 A Dual Selection Vector (optional; Life Technologies, cat. no. A10462)
  • Cold Fusion Cloning Kit (suggested; Systems Biosciences cat. no. MC100B‐1)
  • High‐fidelity DNA polymerase kit of choice
  • LB agar plates ( appendix 2D) containing 50 μg/ml kanamycin
  • QIAGEN EndoFree Maxiprep Kit (Qiagen, cat. no. 12362)
  • Additional reagents and equipment for standard molecular biology procedures (construct design, high‐fidelity PCR and DNA cloning techniques, sequencing, DNA preparation; Ausubel et al., )
NOTE: pENTR vectors are desirable for cloning since uncut vector contains a cell‐death signal for standard bacteria and will not survive in standard selection. These vectors will not be used for Gateway cloning. The purpose is to readily generate donor plasmid DNA with the large homology arms flanking transgenes, so researchers may opt for their own plasmids instead. TOPO vectors can also be used for insertion of homology arms. Alternative donor vectors can be used, but be sure to prepare the appropriate antibiotic in LB agar plates for your vector of choice.

Basic Protocol 3: Human Pluripotent Stem Cell Culture and Nucleofection

  • Human pluripotent stem (hPS) cell stock culture
  • Matrigel substrate (see recipe)
  • ROCK inhibitor (Y‐27632, stocked at 10 mM; SelleckChem, cat. no. S1049)
  • hES cell medium: we prefer mTeSR1 (StemCell Technologies, cat. no. 05850); an alternative is Essential 8 medium (Life Technologies, cat. no. A1517001); store either medium up to 1 week at 4°C
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS)
  • Accutase (Millipore, cat. no. SCR005)
  • Human Stem Cell Nucleofector Kit 1 (Lonza/Amaxa, cat. no. VPH‐5012)
  • pX330 gRNA/spCas9 plasmids ( protocol 1)
  • Donor vector(s) ( protocol 2; optional)
  • 100‐mm, 150‐mm, and 6‐well plates as needed
  • Amaxa Nucleofector 2 b with program A‐023 (mouse ES cell program) and nucleofection cuvettes
  • Centrifuge and microcentrifuge
  • Additional reagents and equipment for cell culture techniques including counting cells ( appendix 3G; Phelan, )

Basic Protocol 4: Colony Picking and Subclone Selection

  • Nucleofected cells ( protocol 3)
  • Plating medium: hES cell medium supplemented with 10 μM ROCK inhibitor (see protocol 3)
  • Matrigel substrate (see recipe)
  • 24‐well culture plates
  • 96‐well flat‐bottom plate (for manual colony dispersion)
  • 200‐μl (P‐200) aerosol‐barrier pipet tips
  • Picking microscope with sufficient overhead space to pick colonies (e.g., EVOS XL)

Basic Protocol 5: Cryopreservation and Genomic DNA Preparation

  • 24‐well plates of 70% to 90% confluent hPS cells ( protocol 4)
  • Accutase (Millipore, cat. no. SCR005)
  • Cryopreservation solution: combine 90 ml fetal bovine serum (FBS; filter sterilized using 0.22‐μm filter) with 10 ml dimethylsulfoxide (DMSO)
  • Mineral oil, sterile filtered (Sigma, cat. no. M5310‐100 ml)
  • Lysis buffer (see recipe)
  • 75 mM NaCl in ethanol, ice‐cold
  • 70% ethanol in nuclease‐free water
  • 96‐well flat‐bottom plates
  • Multichannel pipettor
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