Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing
Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing
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DOI：
10.1038/s41598-019-49110-3
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发表时间：
2019-09-03
影响因子：
4.6
通讯作者：
Sasaki, Erika
中科院分区：
文献类型：
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作者：
Kumita, Wakako;Sato, Kenya;Sasaki, Erika
转基因非人类灵长类动物 (NHP) 是生物医学研究的有用模型。基因编辑技术已经能够生产靶基因敲除 (KO) NHP 模型。 CRISPR/Cas9 的目标基因 KO/敲入 (KI) 效率尚未在狨猴中进行广泛研究。在本研究中，检查了狨猴胚胎中 CRISPR/mRNA 和 CRISPR/核酸酶靶基因修饰效果的最佳条件。 CRISPR/核酸酶在避免嵌合遗传改变方面比 CRISPR/mRNA 更有效。此外，使用 CRISPR/Cas9 和 2 种不同长度（36 nt 和 100 nt）的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 nt 有义或反义 ssODN 注射到胚胎中时，观察到 Kls。所有胚胎均表现出带有 KI 和 KO 的嵌合突变，或 c-kit 的不精确 KI。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。
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. Targetgene-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). Kls 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.