Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes
Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes
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DOI：
10.1016/j.theriogenology.2017.11.030
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发表时间：
2018-03-01
期刊：
影响因子：
2.8
通讯作者：
Miyoshi, Kazuchika
中科院分区：
文献类型：
--
作者：
Sato, Masahiro;Kosuke, Maeda;Miyoshi, Kazuchika
最近，已经描述了通过将CRISPR/Cas9相关mRNA组分显微注射到猪受精卵中来成功地进行一步基因组编辑。鉴于猪的妊娠期相对较长，饲养成本较高，迫切需要建立一种有效的基于显微注射的猪基因组编辑方法。之前，我们已经尝试通过在电激活后立即将CRISPR/Cas9相关核酸和增强的绿色荧光蛋白（EGFP）mRNA显微注射到猪卵母细胞中，来破坏编码α-1，3-半乳糖基转移酶（GGTA 1）的基因，该基因合成α-Gal表位。我们发现基因组编辑确实被诱导了，尽管由此产生的囊胚是镶嵌的，并且修饰细胞的频率似乎很低（50%）。为了提高猪卵母细胞中的基因组编辑效率，在电激活后6小时进行细胞质注射，这是形成原核的阶段。发育中的囊胚表现出更高水平的EGFP。此外，T7核酸内切酶1测定和随后的测序表明，这些胚胎表现出增加的基因组编辑效率（69%），尽管仍然观察到诱导突变的高度嵌合性。用荧光标记的同种凝集素BS-I-B-4进行的基于单个胚泡的细胞化学染色也证实了这种嵌合现象。因此，开发一种避免或减少这种镶嵌现象的技术将是通过显微注射有效敲除仔猪生产的关键因素。(C)2017爱思唯尔公司All rights reserved.
Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding alpha-1,3-galactosyltransferase (GGTA1), which synthesizes the alpha-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B-4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection. (C) 2017 Elsevier Inc. All rights reserved.