Central Institute for Experimental Animals, Kawasaki-shi, Kanagawa, 210-0821, Japan.
Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita-shi, Osaka, 565-0871, Japan.
Sci Rep. 2019 Sep 3;9(1):12719. doi: 10.1038/s41598-019-49110-3.
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.
基因修饰的非人灵长类动物（NHP）是生物医学研究的有用模型。基因编辑技术使靶向基因敲除（KO）NHP 模型的生产成为可能。CRISPR/Cas9 在狨猴中的靶向基因-KO/敲入（KI）效率尚未得到广泛研究。在这项研究中，检查了 CRISPR/mRNA 和 CRISPR/核酸酶在狨猴胚胎中的靶基因修饰效率的最佳条件。CRISPR/核酸酶在避免嵌合遗传改变方面比 CRISPR/mRNA 更有效。此外，还使用 CRISPR/Cas9 和 2 种不同长度（36 个核苷酸和 100 个核苷酸）的每条正义或反义单链寡核苷酸（ssODN）研究了生成 KI 狨猴胚胎的最佳条件。当将 CRISPR/核酸酶和 36 个核苷酸的正义或反义 ssODN 注入胚胎时，观察到 KI。所有胚胎均表现出 c-kit 的 KI 和 KO 或不精确的 KI 的嵌合突变。尽管需要进一步改进 KI 策略，但这些结果表明 CRISPR/Cas9 可用于通过基因编辑产生 KO/KI 狨猴。
