Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.
Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.
复制标题
将CRISPR/CAS9相关的mRNA直接注射到孤肠遗传激活的猪卵母细胞中，导致频繁的镶嵌物引起诱因突变。
DOI：
10.3390/ijms160817838
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发表时间：
2015-08-03
影响因子：
5.6
通讯作者：
Miyoshi K
中科院分区：
文献类型：
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作者：
Sato M;Koriyama M;Watanabe S;Ohtsuka M;Sakurai T;Inada E;Saitoh I;Nakamura S;Miyoshi K
一些报道表明，通过一步法向受精卵显微注射CRISPR/Cas9相关成分的mRNA，成功地在猪身上进行了基因组编辑。考虑到妊娠期相对较长和住房成本较高，需要建立一种基于囊胚的单一检测方法来快速优化上述系统。作为概念验证，我们试图使用孤雌激活的猪卵母细胞来干扰编码α-1，3-半乳糖基转移酶的基因(GGTA1)，该基因合成α-Gal表位。α-Gal表位的缺失可以通过荧光标记的异凝素BS-I-B4(IB4)的染色来监测，它与α-Gal表位特异结合。当将GGTA1特异的引导RNA与增强型绿色荧光蛋白(EGFP)和人Cas9mRNAs一起注射到卵母细胞中时，65%(24/37)的发育囊胚显示绿色荧光，但几乎所有的囊胚(96%，23/24)都显示出镶嵌的荧光图案。IB4染色显示，绿色荧光区与IB4的结合活性往往降低。在测试的16个样本中，有6个(5个荧光囊胚和1个非荧光囊胚)具有INDEL突变，这表明EGFP表达与突变诱导之间存在相关性。此外，受精卵微量注射mRNAs可能会导致仔猪产生具有不同突变类型的细胞。
Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.