High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus
High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus
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DOI：
10.1242/dev.152967
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发表时间：
2017-08-01
期刊：
影响因子：
4.6
通讯作者：
Cha, Sang-Wook
中科院分区：
文献类型：
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作者：
Aslan, Yetki;Tadjuidje, Emmanuel;Cha, Sang-Wook
CRISPR介导的基因组编辑的革命使得几乎任何DNA序列的突变和插入成为可能，特别是在细胞培养中，可以使用选择来恢复相对罕见的同源重组事件。这项技术在动物模型中的有效使用仍然存在许多挑战，包括建立突变株系的时间，创始动物中的嵌合基因编辑以及低同源重组率。在这里，我们报告了一种在非洲爪蟾卵母细胞中进行CRISPR介导的基因组编辑的方法，该方法具有同源定向修复（HDR），该方法在4.4-25.7%的F0蝌蚪中提供了小DNA片段（40-50个核苷酸）的有效非嵌合靶向插入，具有种系传递。对于CRISPR/Cas9介导的HDR基因编辑和插入缺失突变，基因编辑的FO胚胎是均匀杂合的，与仅在母体基因组中的突变一致。除了在体内有效标记蛋白质外，这种HDR方法还将使研究人员能够在非洲爪蟾中创建用于人类疾病建模的患者特异性突变。
The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.