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

Wakako Kumita, Kenya Sato, Yasuhiro Suzuki, Yoko Kurotaki, Takeshi Harada, Yang Zhou, Noriyuki Kishi, Kengo Sato, Atsu Aiba, Yasubumi Sakakibara, Guoping Feng, Hideyuki Okano, Erika Sasaki

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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.

Original languageEnglish
Article number12719
JournalScientific reports
Volume9
Issue number1
DOIs
Publication statusPublished - 2019 Dec 1

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Kumita, W., Sato, K., Suzuki, Y., Kurotaki, Y., Harada, T., Zhou, Y., Kishi, N., Sato, K., Aiba, A., Sakakibara, Y., Feng, G., Okano, H., & Sasaki, E. (2019). Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing. Scientific reports, 9(1), [12719]. https://doi.org/10.1038/s41598-019-49110-3