Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System

• Sato, Masahiro

• Miyoshi, Kazuchika

• Nakamura, Shingo

• Ohtsuka, Masato

• Sakurai, Takayuki

• Watanabe, Satoshi

• Kawaguchi, Hiroaki

• Tanimoto, Akihide

抄録

The recent advancement in genome editing such a CRISPR/Cas9 system has enabled isolation of cells with knocked multiple alleles through a one-step transfection. Somatic cell nuclear transfer (SCNT) has been frequently employed as one of the efficient tools for the production of genetically modified (GM) animals. To use GM cells as SCNT donor, efficient isolation of transfectants with mutations at multiple target loci is often required. The methods for the isolation of such GM cells largely rely on the use of drug selection-based approach using selectable genes; however, it is often difficult to isolate cells with mutations at multiple target loci. In this study, we used a novel approach for the efficient isolation of porcine cells with at least two target loci mutations by one-step introduction of CRISPR/Cas9-related components. A single guide (sg) RNA targeted to GGTA1 gene, involved in the synthesis of cell-surface alpha-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B-4 isolectin for 2 h at 37 degrees C to eliminate alpha-Gal epitope-expressing cells, the surviving clones lacked alpha-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these alpha-Gal epitope-negative surviving cells demonstrated a 100% occurrence of genome editing at target loci. SCNT using these cells as donors resulted in the production of cloned blastocysts with the genotype similar to that of the donor cells used. Thus, this novel system will be useful for SCNT-mediated acquisition of GM cloned piglets, in which multiple target loci may be mutated.

Article

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES.18(12):2610(2017)

収録刊行物

• INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 18 (12), 2610-, 2017-12-04

  MDPI

キーワード

• alpha-Gal epitope
• alpha-1,3-galactosyltransferase
• CRISPR/Cas9
• targeted toxin
• genome editing
• isolectin BS-I-B-4
• LDLR
• PTEN
• p53

詳細情報

• CRID: 1050001338939956608
• NII論文ID: 120007099791
• NII書誌ID: AA12038549
• DOI: 10.3390/ijms18122610
• ISSN: 14220067
• HANDLE: 10091/00021603
• Web Site: https://soar-ir.repo.nii.ac.jp/records/20846
• Web Site: http://www.mdpi.com/1422-0067/18/12/2610/pdf
• 本文言語コード: en
• 資料種別: journal article
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