Complexity of Detecting CRISPR/Cas9-Mediated Homologous Recombination in Zebrafish

文献类型： 外文期刊

第一作者： Pi, Y.

作者： Pi, Y.;He, K. Z.;Zhang, W. Q.;Pi, Y.;Dong, Z. Q.;Guo, S.;Pi, Y.;Dong, Z. Q.;Guo, S.;Dong, Z. Q.;Jiang, F. G.;Jiang, K. J.

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关键词： CRISPR; genome modification; complexity; zebrafish

期刊名称：MOLECULAR BIOLOGY （ 影响因子：1.374； 五年影响因子：1.266 ）

ISSN： 0026-8933

年卷期： 2020 年 54 卷 3 期

页码：

收录情况： SCI

摘要： Homology-directed (HD) genome modification offers an opportunity to precisely modify the genome. Despite reported successful cases, for many loci, precise genome editing remains challenging and inefficient in vivo. Here we report an effort to precisely knock-in a GFP reporter intogadlocus mediated by CRISPR/Cas9 system in the zebrafishDanio rerio. PCR artifact was detected in testing for homologous recombination (HR), but was mitigated by optimizing PCR condition and decreasing the injected targeting plasmid concentration. Under this optimized condition, time course analysis revealed a decline of the HR-positive embryos at embryogenesis progressed. GFP signals also diminished at later developmental stages. The GFP signals were consistent with PCR detection, both of which suggested the loss of targeted insertion events at later stages. Such loss of insertion might be one underlying reason for the inability to obtain germ-line transgenic lines with GFP knocked into thegadlocus. Our results suggest that the low HR efficiency associated with CRISPR-mediated knock-in is in part due to loss of insertion after targeted integration into thegadlocus.

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