通过CRISPR/Cas9系统在绵羊中进行多重基因编辑

Yiyuan  Niu; Yi  Ding; Xiaolong  Wang; Yulin Chen

doi:10.21769/BioProtoc.2385

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Peer-reviewed

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 email

发布: 2017年07月05日第7卷第13期 DOI: 10.21769/BioProtoc.2385 浏览次数: 10927

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)

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

Pipette tips
Centrifuge tubes
6-well culture plate
HiBindTM DNA Mini Column
Sheep (Healthy ewes [3-5 years old] were selected about 160)
PGL3-U6-gRNA vector (Addgene, catalog number: 51133 )
pUC57-T7-gRNA vector (Addgene, catalog number: 51132 )
E. coli DH5α competent cells
Cas9 mRNA in vitro transcription vector (Addgene, catalog number: 44758 )
T vector (Takara Bio, catalog number: D103A )
Bsal enzyme(New England Biolabs, catalog number: R3535 )
DNA Gel Extraction Kit (Corning, catalog number: D205-04 )
T4 ligase (New England Biolabs)
Ampicillin
EndoFree Plasmid Maxi Kit (QIAGEN, catalog number: 12362 )
Primers

M13R (-47) primer
5’-CGCCAGGGTTTTCCCAGTCACGAC-3’
Primers for amplifying the selected potential off-target loci, designed by Primer5 software.

Ethanol
Ice acetic acid (Guangzhou Chemical Reagent Factory, catalog number: CB39-GR-0.5L )
Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Gibco^TM)
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM)
Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM)
Lipofectamine3000 reagent (Thermo Fisher Scientific, Invitrogen^TM)
Opti-MEM medium (Thermo Fisher Scientific, Gibco^TM)
Blasticidine S hydrochloride
Universal Genomic DNA Kit (CWBIO, catalog number: CW2298M )
KOD-Plus-Neo
PCR cleanup kit (AppliChem, catalog number: A7089,1000 )
T7EI
Agarose
TCM199 medium (Thermo Fisher Scientific, Gibco^TM)
Quinn’s Advantage Cleavage Medium and Blastocyst Medium (Sage Biopharma)
Phosphate-buffered saline (PBS) (Thermo Fisher Scientific)
REPLI-g Single Cell Kit (QIAGEN, catalog number: 150343 )
D2000 DNA marker
Tris
Ethylenediaminetetraacetate acid (EDTA)
Agar powder
Tryptone
Yeast extract
Sodium chloride (NaCl)
50x TAE (see Recipes)
LB medium (see Recipes)

Equipment

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

Software

Primer5 software

Procedure

English

中文翻译

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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.