Generating Mouse Models Using CRISPR‐Cas9‐Mediated Genome Editing

Wenning Qin1, Peter M. Kutny1, Richard S. Maser1, Stephanie L. Dion1, Jeffrey D. Lamont1, Yingfan Zhang1, Greggory A. Perry1, Haoyi Wang2

1 The Jackson Laboratory, Bar Harbor, Maine, 2 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo150178
Online Posting Date:  March, 2016
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Abstract

The CRISPR‐Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including mice. The CRISPR‐Cas9 reagents can be delivered directly into the mouse zygote to derive a mutant animal carrying targeted genetic modifications. The major components of the system include the guide RNA, which provides target specificity, the Cas9 nuclease that creates the DNA double‐strand break, and the donor oligonucleotide or plasmid carrying the intended mutation flanked by sequences homologous to the target site. Here we describe the general considerations and experimental protocols for creating genetically modified mice using the CRISPR‐Cas9 system. © 2016 by John Wiley & Sons, Inc.

Keywords: CRISPR; genome editing; knockout; knock‐in; mouse model

     
 
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Table of Contents

  • Introduction
  • General Considerations for Generating a Mouse Model Using CRISPR‐Cas9
  • Basic Protocol 1: Prepare Microinjection Mixture
  • Support Protocol 1: Guide Sequence Selection
  • Support Protocol 2: Single Guide RNA Synthesis
  • Support Protocol 3: Cas9 mRNA Synthesis
  • Support Protocol 4: Donor Oligonucleotide Synthesis
  • Support Protocol 5: Donor Plasmid Assembly
  • Basic Protocol 2: Microinjection to Deliver the CRISPR‐Cas9 Reagents into Mouse Zygotes
  • Basic Protocol 3: Genotyping by Conventional PCR and Sequencing of PCR Product
  • Support Protocol 6: Long‐Range PCR Analysis
  • Support Protocol 7: Southern Blot Analysis
  • Basic Protocol 4: Crispr‐Cas9 Delivery by Electroporation
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Prepare Microinjection Mixture

  Materials
  • 500 ng/μl Cas9 mRNA ( protocol 4)
  • 500 ng/μl sgRNA (Support Protocols protocol 21 and protocol 32)
  • 500 ng/μl donor oligonucleotide ( protocol 5)
  • 500 ng/μl donor plasmid ( protocol 6)
  • TE buffer, pH 7.5: 10 mM Tris·Cl, pH 7.5/0.1 mM EDTA, DNase‐ and RNase‐free
  • 0.2‐ml RNase‐free PCR tubes (Life Technologies, cat. no. AM12225)
  • 1.5‐ml RNase‐free microcentrifuge tubes (Life Technologies, cat. no. AM12400)
  • Galaxy 20R centrifuge (VWR)
  • BioDrop μLite spectrophotometer (Isogen Life Science)

Support Protocol 1: Guide Sequence Selection

  Materials
  • Computer with Internet access
  • Sequence management software (e.g., Vector NTI, Geneious, ApE)

Support Protocol 2: Single Guide RNA Synthesis

  Materials
  • sgRNA forward primer (Table 15.1.7800, ordered as 4 nmol ultramer from IDT)
  • sgRNA reverse primer (Table 15.1.7800, ordered as 4 nmol ultramer from IDT)
  • Nuclease‐free H 2O
  • dNTP mixture: 2.5 mM each dNTP
  • PrimeSTAR GXL DNA Polymerase (Clontech, cat. no. R050B) and 5× PrimeSTAR GXL buffer
  • QIAquick PCR purification kit (Qiagen, cat. no. 28106)
  • MEGAShortScriptT7 Kit (Life Technologies, cat. no. AM1354)
  • MEGAclear Kit (Life Technologies, cat. no. AM1908)
  • GelRed stain (Biotium, cat. no. 41003)
  • Thermal Cycler (BioRad, cat. no.1851148)
  • BioDrop μLite spectrophotometer (Isogen Life Science)
  • Additional reagents and equipment for PCR (Kramer and Coen, ) and agarose gel electrophoresis (Voytas, )

Support Protocol 3: Cas9 mRNA Synthesis

  Materials
  • Cas9 forward primer (Table 15.1.7800, ordered from IDT)
  • Cas9 reverse primer (Table 15.1.7800, ordered from IDT)
  • pX330 plasmid (plasmid #42230, Addgene)
  • Nuclease‐free H 2O
  • Phusion High‐Fidelity DNA Polymerase (New England Biolabs, cat. no. M0530L) and 5× Phusion HF buffer
  • dNTP mixture (2.5 mM each dNTP)
  • QIAquick PCR purification kit (Qiagen, cat. no. 28106)
  • mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Cat. No. AM1345, Life Technologies)
  • MEGAclear Kit (Life Technologies, cat. no. AM1908)
  • RNase AWAY Decontamination Reagent (Life Technologies, cat. no. 10328‐011)
  • Thermal Cycler (BioRad, cat. no. 1851148)
  • BioDrop μLite spectrophotometer (Isogen Life Science)
  • Additional reagents and equipment for PCR (Kramer and Coen, ) and agarose gel electrophoresis (Voytas, )

Support Protocol 4: Donor Oligonucleotide Synthesis

  Materials
  • NucleoBond Xtra Midi EF kit (Clontech, cat. no. 740420.50)
  • Additional reagents and equipment for standard molecular cloning techniques (Ausubel et al., )

Support Protocol 5: Donor Plasmid Assembly

  Materials
  • Donor female mice (various strains; The Jackson Laboratory)
  • Pregnant mare serum gonadotrophin (PMSG; Prospec, cat. no. HOR‐272)
  • Human chorionic gonadotrophin (hCG; Prospec, cat. no. HOR‐250)
  • Stud male mice (various strains; The Jackson Laboratory)
  • M2 medium (Sigma‐Aldrich, cat. no. M7167‐100ML)
  • Hyaluronidase (Sigma, cat. no. H3506)
  • K‐RVCL‐50 medium (Cook Medical)
  • Mineral oil (Sigma‐Aldrich, cat. no. M8410)
  • Pseudopregnant female mice (CByB6F1/J; The Jackson Laboratory, stock no. 100009; 9 to 11 weeks)
  • Microdissecting forceps
  • MINC Benchtop Incubator (Cook Medical)
  • Microscope slides
  • Zeiss AxioObserver.D1 Microscope (Zeiss)
  • TransferMan NK2 micromanipulator (Eppendorf)
  • Narashige IM‐5A Pneumatic Injector (Tritech Research, Inc.)
  • Thin‐wall glass capillaries (World Precison Instruments, cat. no. TW100F‐4)
  • P‐97 micropipet puller (Sutter Instrument Company)
  • 1.8‐ml cryogenic tube with round bottom (ThermoFisher Scientific, cat. no. 363401)
  • Additional reagents and equipment for injection (Donovan and Brown, ) and euthanasia (Donovan and Brown, ) of rodents, and microinjection (Hogan et al., )

Basic Protocol 2: Microinjection to Deliver the CRISPR‐Cas9 Reagents into Mouse Zygotes

  Materials
  • 2‐mm tail snips or ear punches from mice to be genotyped
  • Alkaline lysis solution: 25 mM NaOH/0.2 mM EDTA, pH 12
  • Neutralization solution: 40 mM Tris·Cl, pH 5.0
  • PrimeSTAR GXL DNA Polymerase (Clontech, cat. no. R050B) and 5× PrimeSTAR GXL buffer
  • dNTP mixture (2.5 mM each dNTP)
  • Forward and reverse PCR primers (designed based on the principles in Fig. ; ordered from IDT and reconstituted to 10 pmol/μl with DNase‐free water)
  • 6× DNA gel loading dye (Life Technologies, cat. no. R0611)
  • HighPrep PCR reagent (MAGBIO, cat. no. AC‐60050)
  • 70% ethanol
  • Elution buffer: H 2O, Tris·Cl, pH 8.0, or TE buffer
  • Sequencing primer (ordered from IDT and reconstituted to 10 pmol/μl with DNase‐free water)
  • 96‐well thermal cycling plate (VWR, cat. no. 89049‐178)
  • MicroAmp Clear Adhesive Film (Life Technologies, cat. no. 4306311)
  • Galaxy 20R centrifuge (VWR)
  • 12.5 μl, 12‐channel VIAFLO II electronic pipet (Integra Biosciences, Part No. 4621)
  • 125 μl, 12‐channel VIAFLO II electronic pipet (Integra Biosciences, Part No. 4622)
  • 1250 μl, 12‐channel VIAFLO II electronic pipet (Integra Biosciences, Part No. 4624)
  • Thermal Cycler with 96‐Well Fast Reaction Module (BioRad, cat. no. 1851196)
  • 96R Magnet Plate (Alpaqua, cat. no. A001219)
  • Additional reagents and equipment for PCR (Kramer and Coen, ) and agarose gel electrophoresis (Voytas, )

Basic Protocol 3: Genotyping by Conventional PCR and Sequencing of PCR Product

  Materials
  • Gentra Puregene Mouse Tail Kit (Qiagen, cat. no. 158267)
  • QIAquick PCR Purification Kit (Cat. No. 28106, Qiagen)
  • 70% ethanol
  • 10 mM Tris·Cl, pH 8.0
  • Additional reagents and equipment for conventional PCR ( protocol 8)

Support Protocol 6: Long‐Range PCR Analysis

  Materials
  • Cas9 mRNA ( protocol 4)
  • sgRNA (Support Protocols protocol 21 and protocol 32)
  • Donor oligonucleotide ( protocol 5)
  • TE buffer, pH 7.5: 10 mM Tris·Cl, pH 7.5/0.1 mM EDTA, DNase‐ and RNase‐free
  • M2 medium (Sigma‐Aldrich, cat. no. M7167‐100ML)
  • EmbryoMax M2 Medium (EMD Millipore, cat. no. MR‐015‐D)
  • Acidic Tyrode's solution (Sigma‐Aldrich, cat. no. T1788)
  • Opti‐MEM medium (Life Technologies, cat. no. 31985)
  • KSOMaa Evolve medium (Zenith Biotech, cat. no. ZEKS‐050)
  • Bovine serum albumin (Sigma‐Aldrich, cat. no. A2153)
  • Electroporation Cuvette Plus, 1 mm gap (Harvard Apparatus, cat. no. 45‐0124)
  • ECM830 Square Wave Electroporation System (UX‐02894‐18, Harvard Apparatus; Fig.  A)
  • 35 × 10 mm Petri dish (Corning, cat. no. 351008)
  • Pseudopregnant female mice (CByB6F1/J; The Jackson Laboratory, stock no. 100009, 9 to 11 weeks)
  • Additional reagents and equipment for harvesting embryos ( protocol 7) and embryo transfer (Hogan et al., )
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Internet Resources
  http://www.ncbi.nlm.nih.gov/
  Download genomic sequence for a gene.
  http://www.ensembl.org/index.html
  Exon/intron structure and sequence analysis.
  http://www.uniprot.org/
  Post‐translational processing and protein functional domains.
  http://crispr.mit.edu/
  Off‐target sequence match analysis.
  https://benchling.com/
  On‐target and off‐target efficiency scores.
  http://www.rgenome.net/mich‐calculator/
  Micro‐homology‐mediated repair.
  http://www.repeatmasker.org/
  Repeat masking.
  https://www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel‐1303
  Sequence divergence among mouse strains.
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