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Sci Rep. 2019 Sep 3;9(1):12719. doi: 10.1038/s41598-019-49110-3.

Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing.

Author information

1
Central Institute for Experimental Animals, Kawasaki-shi, Kanagawa, 210-0821, Japan.
2
Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita-shi, Osaka, 565-0871, Japan.
3
Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
4
McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA.
5
Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Wako-shi, Saitama, 351-0198, Japan.
6
Department of Biosciences and Informatics, Keio University, Yokohama-shi, Kanagawa, 223-8522, Japan.
7
Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo, 160-8582, Japan.
8
Central Institute for Experimental Animals, Kawasaki-shi, Kanagawa, 210-0821, Japan. esasaki@ciea.or.jp.
9
Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Wako-shi, Saitama, 351-0198, Japan. esasaki@ciea.or.jp.
10
Advanced Research Center, Keio University, Shinjuku-ku, Tokyo, 160-8582, Japan. esasaki@ciea.or.jp.

Abstract

Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.

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