Abstract

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 into gad locus mediated by CRISPR/Cas9 system in the zebrafish Danio 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 the gad locus. 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 the gad locus.

中文翻译：

---

在斑马鱼中检测CRISPR / Cas9介导的同源重组的复杂性

摘要

同源（HD）基因组修饰提供了精确修饰基因组的机会。尽管报道了成功的病例，但是对于许多基因座，精确的基因组编辑在体内仍然具有挑战性且效率低下。在这里，我们报告了将GFP报告基因准确敲入斑马鱼Danio rerio的CRISPR / Cas9系统介导的性腺基因座的努力。在检测同源重组（HR）时检测到了PCR伪影，但可以通过优化PCR条件并降低注入的靶向质粒浓度来缓解。在这种优化的条件下，时程分析显示在胚胎发生过程中HR阳性胚胎的数量下降。GFP信号在以后的发育阶段也会减弱。GFP信号与PCR检测一致，这两者均表明在以后的阶段丢失了目标插入事件。这种插入的丢失可能是无法获得将GFP敲入性腺基因座的种系转基因品系的根本原因之一。我们的结果表明，与CRISPR介导的敲入相关的低HR效率部分归因于靶向整合入性腺后的插入损失 轨迹。