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Multiplex Gene Editing via CRISPR/Cas9 System in Sheep

通过CRISPR/Cas9系统在绵羊中进行多重基因编辑

YN Yiyuan Niu
YD Yi Ding
XW Xiaolong Wang
YC Yulin Chen

发布: 2017年07月05日第7卷第13期 DOI: 10.21769/BioProtoc.2385 浏览次数: 11955

评审: Anonymous reviewer(s)

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Abstract

Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified sheep. Procedure described sgRNA design, construction of gRNA-Cas9 plasmid, efficient detection in fibroblast, embryos and sheep, and some manipulative technologies. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by targeting multiple genes that are in charge of economically significant traits simultaneously.

Keywords: Cas9 (Cas9) search Sheep (绵羊) search MSTN (MSTN) search BCO2 (BCO2) search ASIP (ASIP) search

Background

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been used to modify many cell lines and organisms in the past. The recent CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) technology development provides an efficient tool for genome modifying of targeting loci. This system has an advantage of modifying multiple loci at the same time. Sheep with specific gene modifications may contribute to breeding. The results demonstrate the first detailed evidence of large animal modification in sheep.

Materials and Reagents

  1. Pipette tips
  2. Centrifuge tubes
  3. 6-well culture plate
  4. HiBindTM DNA Mini Column
  5. Sheep (Healthy ewes [3-5 years old] were selected about 160)
  6. PGL3-U6-gRNA vector (Addgene, catalog number: 51133 )
  7. pUC57-T7-gRNA vector (Addgene, catalog number: 51132 )
  8. E. coli DH5α competent cells
  9. Cas9 mRNA in vitro transcription vector (Addgene, catalog number: 44758 )
  10. T vector (Takara Bio, catalog number: D103A )
  11. Bsal enzyme(New England Biolabs, catalog number: R3535 )
  12. DNA Gel Extraction Kit (Corning, catalog number: D205-04 )
  13. T4 ligase (New England Biolabs)
  14. Ampicillin
  15. EndoFree Plasmid Maxi Kit (QIAGEN, catalog number: 12362 )
  16. Primers
    1. M13R (-47) primer
      5’-CGCCAGGGTTTTCCCAGTCACGAC-3’
    2. Primers for amplifying the selected potential off-target loci, designed by Primer5 software.


  17. Ethanol
  18. Ice acetic acid (Guangzhou Chemical Reagent Factory, catalog number: CB39-GR-0.5L )
  19. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM)
  20. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM)
  21. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM)
  22. Lipofectamine3000 reagent (Thermo Fisher Scientific, InvitrogenTM)
  23. Opti-MEM medium (Thermo Fisher Scientific, GibcoTM)
  24. Blasticidine S hydrochloride
  25. Universal Genomic DNA Kit (CWBIO, catalog number: CW2298M )
  26. KOD-Plus-Neo
  27. PCR cleanup kit (AppliChem, catalog number: A7089,1000 )
  28. T7EI
  29. Agarose
  30. TCM199 medium (Thermo Fisher Scientific, GibcoTM)
  31. Quinn’s Advantage Cleavage Medium and Blastocyst Medium (Sage Biopharma)
  32. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific)
  33. REPLI-g Single Cell Kit (QIAGEN, catalog number: 150343 )
  34. D2000 DNA marker
  35. Tris
  36. Ethylenediaminetetraacetate acid (EDTA)
  37. Agar powder
  38. Tryptone
  39. Yeast extract
  40. Sodium chloride (NaCl)
  41. 50x TAE (see Recipes)
  42. LB medium (see Recipes)

Equipment

  1. Pipettes
  2. 37 °C water bath
  3. Thermal cycler (Bio-Rad thermocycler)
  4. DNA electrophoresis apparatus
  5. Microcentrifuge
  6. Eppendorf FemtoJect system
  7. Olympus micromanipulation system ON3 (Olympus, model: ON3 SERIES )
  8. Microinjection device
  9. Biopsy forceps (Olympus, model: FB-11K-1 )

Software

  1. Primer5 software

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Niu, Y., Ding, Y., Wang, X. and Chen, Y. (2017). Multiplex Gene Editing via CRISPR/Cas9 System in Sheep. Bio-protocol 7(13): e2385. DOI: 10.21769/BioProtoc.2385.

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分类

分子生物学 > DNA > 诱/突变

分子生物学 > RNA > 转染

细胞生物学 > 细胞工程 > CRISPR-cas9

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