CRISPR/Cas9‐Directed Gene Editing for the Generation of Loss‐of‐Function Mutants in High‐Throughput Zebrafish F0 Screens

Sunita S. Shankaran1, Timothy J. Dahlem2, Brent W. Bisgrove3, H. Joseph Yost4, Martin Tristani‐Firouzi1

1 Nora Eccles Harrison Cardiovascular Research and Training Institute, and Division of Pediatric Cardiology, University of Utah, Salt Lake City, Utah, 2 Mutation Generation and Detection Core, University of Utah Health Science Center Core Research Facilities, Salt Lake City, Utah, 3 Molecular Medicine Program, University of Utah, Salt Lake City, Utah, 4 Neurobiology & Anatomy Department, Human Genetics Department, University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 31.9
DOI:  10.1002/cpmb.42
Online Posting Date:  July, 2017
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Abstract

The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F0 screens in this organism a reality. This unit describes a detailed protocol for performing an F0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection. Next, two approaches for determining the efficiency of mutation induction by the custom CRISPR/Cas9 reagents that are easily performed using standard molecular biology protocols are detailed. Finally, screening for F0 induced phenotypes using the zebrafish flh gene as an example is discussed. © 2017 by John Wiley & Sons, Inc.

Keywords: CRISPR/Cas9; FO screen; gene editing; loss‐of‐function; recessive mutant; zebrafish

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification of Zebrafish CRISPR/Cas9 Target Site
  • Basic Protocol 2: Preparation of the sgRNA DR274 Plasmid
  • Basic Protocol 3: Zebrafish Microinjections and Scoring Phenotype
  • Basic Protocol 4: Identification of Mutations in CRISPR/Cas9 Injected Embryos by High‐Resolution Melt Analysis (HRMA)
  • Basic Protocol 5: Identification of Mutations in CRISPR/Cas9 Injected Embryos by Mobility Shift Assay
  • Reagents and Solutions
  • Commentary
  • Literaturre Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Identification of Zebrafish CRISPR/Cas9 Target Site

  Materials
  • DR274 is obtained from Addgene, Plasmid #42250
  • LB agar plate containing 50 μg/ml kanamycin
  • LB liquid medium containing 50 μg/ml Kanamycin
  • M13 Forward (−20) universal sequencing primer: 5′‐ GTAAAACGACGGCCAGT‐3′
  • Omega Bio‐tek E.Z.N.A. Plasmid Mini Kit I, V Spin (cat. no. D6943‐02)
  • 60% glycerol, sterile
  • BsaI with Cutsmart buffer (New England Biolabs, cat. no. R0535S)
  • Sterile dH 2O
  • 10 mg/ml Proteinase K
  • 10% Sodium dodecyl sulfate (SDS)
  • Omega Bio‐tek E.Z.N.A. Cycle Pure Kit (cat. no. D6492‐02)
  • Agarose
  • Electrophoresis tracking dye (NEB, cat. no. B7024S)
  • Ethidium bromide (BioRad, cat. no. 161‐0433)
  • 5 M NaCl
  • STE solution (1 mM EDTA, 100 mM NaCl, 10 mM Tris·Cl, pH 8.0)
  • Takara DNA Ligation Kit, Version 2.1 (Clontech, cat. no. 6022)
  • DH5α chemically competent E. coli cells (ThermoFisher, cat. no. 18265017)
  • Ice
  • SOC liquid medium
  • DraI with CutSmart buffer (NEB, cat. no. R0129S)
  • HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, cat. no. E2050S)
  • RNase‐free dH 2O
  • RNeasy RNA purification kit (Qiagen, cat. no. 74104)
  • 100% RNase‐free ethanol
  • Inoculation loop
  • 17 × 100–mm bacterial culture tube, polystyrene, sterile
  • Inoculation stick
  • 37°C incubator‐shaker
  • 15‐ml centrifuge tubes
  • Centrifuge
  • NanoDrop spectrophotometer (http://www.nanodrop.com)
  • Bacterial cryogenic vials
  • 0.2‐ml sterile PCR tubes
  • Vortex mixer
  • PCR machine or 37°C and 50°C water baths
  • Gel electrophoresis equipment
  • Access to Sanger sequencing facility
  • Ape Sequence analysis software: http://biologylabs.utah.edu/jorgensen/wayned/ape/
  • 1.5‐ml microcentrifuge tubes
  • RNase‐free 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Preparation of the sgRNA DR274 Plasmid

  Materials
  • Wild‐type AB or TU zebrafish (Danio rerio)
  • E3 embryo buffer containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl and 0.33 mM MgSO 4
  • Gene‐specific sgRNA RNA and recombinant Cas9 protein from protocol 2
  • Phenol red
  • RNase‐free dH 2O
  • Zebrafish breeding tanks with dividers
  • 9‐cm petri dishes
  • Borosilicate micro capillaries to pull into injection needles using a micropipette puller
  • 28.5°C incubator
  • Bright‐field stereomicroscope with diascopic illumination

Basic Protocol 3: Zebrafish Microinjections and Scoring Phenotype

  Materials
  • Embryos
  • 30 mg/ml Pronase
  • E3 embryo buffer containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl and 0.33 mM MgSO 4
  • Proteinase K lysis buffer (see recipe)
  • Mineral oil (Sigma, cat. no. M5904)
  • BioFire LightScanner MasterMix (ct. no. HRLS‐ASY‐0003)
  • TOPO‐TA cloning kit (Invitrogen, cat. no. 450641)
  • LB liquid medium containing 100 μg/ml ampicillin
  • Omega Bio‐tek E.Z.N.A. Plasmid Mini Kit I, V Spin
  • M13 Reverse (−20) universal sequencing primer: 5′‐ GTAAAACGACGGCCAGT‐3′
  • Ape Sequence analysis software: http://biologylabs.utah.edu/jorgensen/wayned/ape/
  • Access to Sanger sequencing facility
  • 100 × 15‐mm petri dishes
  • 20 μl pipette tips
  • 20‐μl Pipettes
  • Tweezers
  • 96‐well plates
  • Sealing films
  • Vortex mixer
  • 96‐well plate centrifuge
  • PCR machine
  • Primer3Plus or any other primer design program96‐well black shell white well PCR plate (Bio‐Rad, cat. no. HSP9665)
  • Sealing Tape for Optical Assays (Bio‐Rad, cat. no. 2239444)
  • Benchtop centrifuge
  • BioFire LightScanner 96 machine
  • 0.5‐ml PCR tubes or 8‐well PCR strips
  • Kimwipes
  • 37°C incubator‐shaker

Basic Protocol 4: Identification of Mutations in CRISPR/Cas9 Injected Embryos by High‐Resolution Melt Analysis (HRMA)

  Materials
  • GoTaq G2 green PCR mix from Promega
  • Target site specific forward and reverse PCR primers
  • Genomic DNA extracted from control embryos and CRISPR/Cas9 injected embryos
  • 15% Mini‐ Protean TBE gel, 12 well, 20 μl (BIO‐RAD, cat. no. 4565055)
  • Ethidium bromide (EtBR)
  • 0.2‐ml PCR tubes
  • PCR machine
  • ImageJ software
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Figures

Videos

Literature Cited

  Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., & Xi, J. J. (2013). Genome editing with RNA‐guided Cas9 nuclease in zebrafish embryos. Cell Research, 23(4), 465–472. doi:10.1038/cr.2013.45.
  Dahlem, T. J., Hoshijima, K., Jurynec, M. J., Gunther, D., Starker, C. G., Locke, A. S., & Grunwald, D. J. (2012). Simple methods for generating and detecting locus‐specific mutations induced with TALENs in the zebrafish genome. PLoS Genetics, 8(8), e1002861. doi:10.1371/journal.pgen.1002861.
  Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., & Root, D. E. (2016). Optimized sgRNA design to maximize activity and minimize off‐target effects of CRISPR‐Cas9. Nature Biotechnology, 34(2), 184–191. doi:10.1038/nbt.3437.
  Halpern, M. E., Thisse, C., Ho, R. K., Thisse, B., Riggleman, B., Trevarrow, B., & Kimmel, C. B. (1995). Cell‐autonomous shift from axial to paraxial mesodermal development in zebrafish floating head mutants. Development, 121(12), 4257–4264.
  Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., & Zhang, F. (2013). DNA targeting specificity of RNA‐guided Cas9 nucleases. Nature Biotechnology, 31(9), 827–832. doi:10.1038/nbt.2647.
  Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., & Joung, J. K. (2013). Efficient genome editing in zebrafish using a CRISPR‐Cas system. Nature Biotechnology, 31(3), 227–229. doi:10.1038/nbt.2501.
  Jao, L. E., Wente, S. R., & Chen, W. (2013). Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proceedings of the National Academy of Sciences of the United States of America, 110(34), 13904–13909. doi:10.1073/pnas.1308335110.
  Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. doi:10.1126/science.1225829.
  Moreno‐Mateos, M. A., Vejnar, C. E., Beaudoin, J. D., Fernandez, J. P., Mis, E. K., Khokha, M. K., & Giraldez, A. J. (2015). CRISPRscan: Designing highly efficient sgRNAs for CRISPR‐Cas9 targeting in vivo. Nature Methods, 12(10), 982–988. doi:10.1038/nmeth.3543.
  Nasevicius, A., & Ekker, S. C. (2000). Effective targeted gene 'knockdown' in zebrafish. Nature Genetics, 26(2), 216–220. doi:10.1038/79951.
  Ota, S., Hisano, Y., Muraki, M., Hoshijima, K., Dahlem, T. J., Grunwald, D. J., & Kawahara, A. (2013). Efficient identification of TALEN‐mediated genome modifications using heteroduplex mobility assays. Genes Cells, 18(6), 450–458. doi:10.1111/gtc.12050.
  Parant, J. M., George, S. A., Pryor, R., Wittwer, C. T., & Yost, H. J. (2009). A rapid and efficient method of genotyping zebrafish mutants. Dev Dyn, 238(12), 3168–3174. doi:10.1002/dvdy.22143.
  Xiao, A., Cheng, Z., Kong, L., Zhu, Z., Lin, S., Gao, G., & Zhang, B. (2014). CasOT: A genome‐wide Cas9/gRNA off‐target searching tool. Bioinformatics, 30(8), 1180–1182. doi:10.1093/bioinformatics/btt764.
Internet Resources
  www.zfin.org
  Zebrafish Model Organism database (ZFIN): Check this database for information on expression profiles, available mutants, and publications on gene function and interactions for your gene.
  www.uswest.ensembl.org/Danio_rerio
  ENSEMBL Zebrafish page: Check this database to gain knowledge on of the reference genome sequence, alternate transcripts, and gene architecture for your gene.
  http://portals.broadinstitute.org/gpp/public/analysis‐tools/sgrna‐design
  Broad Institutes sgRNA Designer: This sgRNA target site design tool identifies all the possible CRISPR‐Cas9 target sites in a sequence of interest and ranks them based on predicted on‐target activity and potential off‐target complications.
  http://crispr.mit.edu
  MIT Optimized CRISPR Design: This sgRNA target site design tool identifies all CRISPR‐Cas9 target sites in a sequence and then ranks (0‐100) these sites based on the potential for binding and cutting at off‐target sites.
  http://www.crisprscan.org
  This sgRNA target site design tool is specific to the Zebrafish model organism and identifies CRISPR‐Cas9 target sites for a sequence of interest (or gene of interest) and ranks them based on predicted on‐target activity and potential off‐target complications.
  http://eendb.zfgenetics.org/casot/
  CRISPR/Cas system (Cas9/gRNA) Off‐Targeter (CasOT): This tool will create a file listing all potential off‐target sites for each individual CRISPR‐Cas9 sgRNA target site identified with the design tools listed above.
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