Self‐Cloning CRISPR

Mandana Arbab1, Richard I. Sherwood1

1 Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5B.5
DOI:  10.1002/cpsc.14
Online Posting Date:  August, 2016
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Abstract

CRISPR/Cas9‐gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self‐cloning CRISPR/Cas9 (scCRISPR) uses a self‐cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR‐amplified site‐specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR‐based addition of short homology arms, we achieve efficient site‐specific knock‐in of transgenes such as GFP without traditional plasmid cloning or genome‐integrated selection cassette (2% to 4% knock‐in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc.

Keywords: CRISPR/Cas9; embryonic stem cells; knockout; knock‐in; homologous recombination; GFP transgenesis; gene editing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: scCRISPR Site‐Specific Targeting for Non‐Homologous End‐Joining
  • Basic Protocol 2: scCRISPR Gene Insertion by Homologous Recombination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: scCRISPR Site‐Specific Targeting for Non‐Homologous End‐Joining

  Materials
  • sgRNA oligos (see steps below)
  • Primers (see steps below)
  • OneTaq 2× Master Mix with standard buffer
  • MinElute DNA purification kit (Qiagen)
  • sgPal7‐HygR plasmid maxiprep DNA (Addgene, cat. no. 71484)
  • CBH‐Cas9‐BlastR plasmid maxiprep DNA (Addgene, cat. no. 71489)
  • Cells and appropriate cell culture media and reagents
  • 0.25% trypsin‐EDTA (for mouse ESCs) or
  • 0.05% trypsin‐EDTA (for human ESCs)
  • EmbryoMax electroporation buffer (EMD Millipore)
  • Y‐27632 ROCK inhibitor (optional)
  • Hygromycin
  • Blasticidin
  • PCR thermal cycler
  • Cell electroporator and cuvettes
  • Vacuum concentrator
  • Centrifuge
  • 12‐well culture plates
  • Additional reagents and equipment for the polymerase chain reaction (PCR; Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), and electroporation (Potter and Heller, )

Basic Protocol 2: scCRISPR Gene Insertion by Homologous Recombination

  Materials
  • sgRNA oligos (see steps below)
  • Primers (see steps below)
  • GFP knock‐in template (see steps below)
  • Dimethylsulfoxide (DMSO)
  • Proofreading PCR polymerase (NEBNext 2× Master Mix)
  • MinElute DNA purification kit (Qiagen)
  • CBH‐Cas9‐BlastR plasmid maxiprep DNA (Addgene, cat. no. 71489)
  • sgPal7‐HygR plasmid maxiprep DNA (Addgene cat. no. 71484)
  • Cells and appropriate cell culture media and reagents
  • 0.25% trypsin‐EDTA (for mouse ESCs) or
  • 0.05% trypsin‐EDTA (for human ESCs)
  • EmbryoMax electroporation buffer (EMD Millipore)
  • Y‐27632 ROCK inhibitor (optional)
  • Hygromycin
  • Blasticidin
  • OneTaq 2× Master Mix with standard buffer
  • Bulk isolated genomic DNA (isolated using Purelink Genomic DNA Mini Kit)
  • Genomic DNA lysis buffer (see recipe)
  • 100% and 70% ethanol
  • 5 M NaCl
  • PCR thermal cycler
  • Cell electroporator and cuvettes
  • Vacuum concentrator
  • Centrifuge
  • 6‐well culture plates
  • Humidified staining chamber, e.g., plastic slide box containing a wet Kimwipe
  • Additional reagents and equipment for the polymerase chain reaction (PCR; Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), electroporation (Potter and Heller, ), qPCR (Bookout et al., ), and enzymatic dissociation of cells with trypsin/EDTA (Phelan and May, )
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Figures

Videos

Literature Cited

Literature Cited
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