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Int. J. Mol. Sci. 2017, 18(12), 2610; https://doi.org/10.3390/ijms18122610

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

1
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan
2
Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan
3
Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan
4
Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan
5
The Institute of Medical Sciences, Tokai University, Kanagawa 259-1193, Japan
6
Basic Research Division for Next-Generation Disease Models and Fundamental Technology, Research Center for Next Generation Medicine, Shinshu University, Nagano 390-8621, Japan
7
Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan
8
Department of Hygiene and Health Promotion Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-0065, Japan
9
Department of Pathology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-0065, Japan
*
Author to whom correspondence should be addressed.
Received: 25 September 2017 / Revised: 30 November 2017 / Accepted: 1 December 2017 / Published: 4 December 2017
(This article belongs to the Special Issue Genome Editing 2018)
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Abstract

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 α-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B4 isolectin for 2 h at 37 °C to eliminate α-Gal epitope-expressing cells, the surviving clones lacked α-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these α-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. View Full-Text
Keywords: α-Gal epitope; α-1,3-galactosyltransferase; CRISPR/Cas9; targeted toxin; genome editing; isolectin BS-I-B4; LDLR; PTEN; p53 α-Gal epitope; α-1,3-galactosyltransferase; CRISPR/Cas9; targeted toxin; genome editing; isolectin BS-I-B4; LDLR; PTEN; p53
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Sato, M.; Miyoshi, K.; Nakamura, S.; Ohtsuka, M.; Sakurai, T.; Watanabe, S.; Kawaguchi, H.; Tanimoto, A. 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. Int. J. Mol. Sci. 2017, 18, 2610.

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